Note: Descriptions are shown in the official language in which they were submitted.
W095/13084 2 ~ 7~6~ PCIIUS94~07043
.
CATHEPSIN D IS AN AMYLOIDOGENIC PROTEASE
IN A I .7~ IMFR'S DISEASE
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of PCT/US93/10889 filed
November 12, 1993, which is, in turn, a crmtinllAtir~n-in-part of U.S. Serial No.
07/995,660 filed December 16, 1992, which is, in turn, a rrlntinllAtir~nin-part of
U.S. Serial No. 07t880,914 filed May 11,1992.
1. Field of the Invention
The invention relates to methods for identifying proteolytic enzymes with
specificity for processing the precursor to the Alzheimer's Disease (hereinafter"AD") beta-amyloid protein; methods for identifying inhibitors of proteases
specific for the precursor to the beta-amyloid protein; and methods for regulating
formation of beta-amyloid protein with inhibitors of proteases specific for the
precursor to the beta-amyloid protein, such as inhibitors of aspartic protease,
cathepsin D, and a chymotryptic-like serine protease.
2. Description of the Related Art
The present assays have utility in the ir~r-ntifirAti~n of the proteases which
control the rate of formation of amyloidic peptides in the brains of AD patients.
As such, they can be used to isolate such proteases, and can also be used to
identify protease inhibitors which can be used as therapeutics for AD. Describedhereinbelow is the application of the assays to identify the aspartic protease,
cathepsin D as a major amyloidogenic protease for processing Amyloid Precursor
Protein (hereinafter "APP"). Also provided is a partial characterization of a
second, serine protease which can form amyloidic precursors for the APP
holoprotein.
AD is a progressive, degenerative disorder of the brain, characterized by
`Uy,lt:s~iV~ atrophy, usually in the frontal, parietal and occipital cortices. The
PCT/US94/07043
WO 95113084 ' ~i
21 755~f
clinical m~ni~ct~tinn9 of AD include p~ s~iv~ memory impairments, loss of
language and visuospatial skills, and behavioral deficits (McKhan et al., 1986,
Neurology, 34: 939). Overall cognitive impairment is attributed to ~g~om~rAtinn
of neuronal cells located throughout the cerebral hemispheres (Price, 1986,
Annu. Rev. Neuro6ci., 9: 489).
Pathologically, the primary distinguishing features of the post-mortem
brain of an AD patient are: (1) pathological lesions comprised of neuronal
perikarya containing accumulations of neurofibrillary tangles; (2)
cerebrovascular amyloid deposits; and (3) neuritic plaques. Both the
cerebrovascular amyloid (Wong et al., 1985, PNAS, 82: 8729) and the neuritic
plaques (Masters et al., 1985, PNAS, 82: 4249) contain a distinctive peptide simply
~l~ci~n~t.~c~ "A4" or "beta-amyloid".
Beta-amyloid is an insoluble, highly aggregating, small polypeptide of
relative molecular mass 4,500, and is composed of 39 to 42 amino acids. Several
lines of evidence support a role of beta-amyloid in the pathogenesis of AD
lesions. For instance, beta-amyloid and related fragments have been shown to be
toxic for PC-12 cell lines (Yanker et al., 1989, Science, 245: 417); toxic for primary
cultures of neurons (Yanker et al., 1990, Science, 250: 279); and cause neuronaldegeneration in rodent brains and corresponding amnestic response in the
rodents (Flood et al., 1991, PNAS, 88: 3363; Kowall et al., 1991, PNAS, 88: 7247).
The strongest evidence however comes from the sites within holo-amyloid
precursor protein (hereinafter when referring to the protein, then "APP") of
amino acid substitutions that co-segregate with certain forms of Familial
Alzheimer's disease (FAD). These point mutations, located N- (Mullan et al.,
1992, Natur~ Genetics, 1: 345), or C- terminal (Goate et al., 1991, Nature, 349: 704;
Yoshioka et al., 1991, Biochem Biophys. Res. Comm., 178: 1141; Murrell et al.,
1991, Science, 254: 97; and Chartier-Harlin et al., 1991, Nature, 353: 844) to the
beta-amyloid peptide sequence within APP are suggested to cause FAD by altering
the rate of endoproteolytic release of beta-amyloid containing fragments (Mullanet al. and Chartier-Harlin et al., both supra).
Kang et al., 1987, Nature, 325: 733, described the beta-amyloid protein as
nri~in~tin~ from and as a part of a larger precursor protein. To identify this
precursor, a full-length complementary DNA clone coding for the protein was
isolated and sequenced, using oligonucleotide probes designed from the known
~ wo 95113084 2 ~ 7 5 5 6 4 PCI/US94/01043
beta-amyloid sequence. The predicted precursor contained 695 residues and is
currently l1~hig..~ 1 "APP 695" (Amyloid Precursor Protein 695).
Subsequent cloning of the gene encoding the APP protein revealed that
the A4 region was encoded on two adjacent exons (Lemaire et al. 1989, Nucleic
Acids Res., 17: 517), ruling out the possibility that A4 Acrl~m~ tirln is the result
of a direct expression of an alternatively spliced mRNA. This implied that A4
accumulation must result from abnormal proteolytic degradation of the APP at
sites both N- and C-terminal to the peptide region within the APP.
APP 695 is the most abundant form of APP found in the human brain, but
three other forms exists, APP 714, APP 751 and APP 770 (Tanzi et al, 1988,
Nature, 351: 528; Ponte et al., 1988, Nature, 331: 525; and Kitaguchi et al., 1988,
Nature, 331: 530). The different length isoforms arise from alternative splicingfrom a single APP gene located on human chromosome 21 (Goldgaber et al.,
1987, Science, 235: 877; and Tanzi et al., 1987, Science, 235: 880).
APP 751 and APP 770 contain a 56 amino acid Kunitz inhibitor domain,
which shares 40% homology with Bovine Pancreatic Trypsin Inhibitor. Both
these forms of APP have protease inhibitory activity (Kitaguchi et al., 1988,
Nature, 311: 530; and Smith et al., 1990, Science, 248: 1126), and at least one of
these forms is probably what was previously identified as Protease Nexin Il
(Oltersdorf et al., 1989, Nature, 341: 144; Van Nostrand et al., 1989, Nature, 341:
546).
The physiological role for the amyloid precursor proteins has not yet been
rrlnfirmr~ It has been proposed to be a cell surface receptor (Kang et al., 1987,
Nature, 325: 733); an adhesion molecule (Schubert et al., 1989, Neuron, 3: 689); a
growth or trophic factor (Saitoh et al., 1989, Cell, 58: 615; Araki et al., 1991,
Biochem. Biop~s. Res. Comm., 181: 265; and Milward et al., 1992, Neuron, 9:
129); a regulator of wound healing (Van Nostrand et al., 1990, Science, 248: 745;
and Smith et al., 1990, Science, 248: 1126); or play a role in the cytoskeletal system
(Refolo et al., 1991, J. Neuroscience, 11: 3888).
Many studies have been performed to examine the role of altered APP
expression in AD, but the results have been cr~nflirtin~ (for example, see review
article: Unterbeck et al., 1990, Review of Biological Researc~l in Agin~, Wiley-Liss,
WO 95113084 ' PCT/US94107043
21 75564
Inc., 4: 139).
Studies have also been performed to examine if changes in the relative
amounts of the different forms of APP are responsible for amyloid ~rrllmlll~tionThe results of such studies have been equally confusing, but have generally
supported the conclusion that the relative expression levels of the Kunitz
domain rnnt~;n;n~ APP's are elevated in AD aohnson et al., 1990, Science, 248
854). Accordingly, transgenic animals expressing elevated APP 751 have been
found to display cortical and hippocampal beta-amyloid reactive deposits (Quon
et al., 1991, NQture, 352: 239).
Recent studies have shown that APP fragments extending from the N-
terminus of A4 to the C-terminus of the full length APP (referred to hereinafteras the "C-100 fragment", because it is comprised of approximately 100 amino
acids) are also capable of aggregation both in vitro (Dyrks et al., 1988, EMBO 1., 7:
949), and in tr~n.cf~ct.o~i cells (Wolf et al., 1990, EMBO J., 9: 2079; and Maruyama et
al., 1990, Nature, 347: 566). C~ver-expression of the C-100 fragment in transfected
P19 cells has been shown to cause cellular toxicity (Fuckuchi et al., 1992, Biochem.
Biophys. ~es. Comm., 182: 165).
Furthermore, C-terminal fragments containing both the beta-amyloid and
the C-terminal domains have been shown to exist in human brain (Estus et al.,
1992, Science, 255: 726), and studies in tr~ncfrctrc~ cell lines suggest that these
fragments may be produced in the r-nr~r~om~l-lysosomal pathway (Golde et al.,
1992, Science, 255: 728).
Collectively, the above reports suggest that a single proteolytic cleavage of
APP at the N-terminus of the A4 region is sufficient to initiate the
pathophysiology associated with AD. Recent studies have shown that cultures of
primary cells and cell lines (including AD transfectants) secrete 3 to 4 kDa
peptides which possess the same N-terminus as beta-amyloid (1-42 amino acids),
and could conceivably comprise full length beta-amyloid (Haas et al., 1992,
NQture, 349: 322; and Shoji et al., 1992, Science, 258: 126). Such peptides have also
been found in the cerebral spinal fluid (h~ ar~ "CSF") of AD and non-AD
patients (Seubert et al., 1992, Nnture, 359: 325; and Shoji et al., 1992, Id.).
APP is also cleaved at a site within the A4 region in the physiological
~ WO 9S/13084 2 ~ 7 5 5 ~ 4 PCT/US94107043
pathway for secretion of the APP extracellular domain (Esch et al., 1990, Science,
248: 1122; and Wang et al., 1991, J. Biol. Chem., 266: 16960). This pathway is
operative in several cell lines and necessarily results in the destruction of the A4,
amyloidic region of the precursor. Evidence that such a pathway is also
operative in the human brain has been obtained. (Palmert et al., 1989, Biochem.
Biophys. ~es. Comm., 165: 182).
The enzymes responsible for the normal, non-pathological processing of
APP have been termed "secretases". C-terminal fragments resulting from
secretase action are smaller than the C-100 fragments (defined above) by 17
amino acids, and will l~ L~l be referred to as the "physiological C-terminal
fragment."
It has been postulated that the net pathological accumulation of A4 is
controlled by the relative activities of the p~th,.l.,gi~ and physiologic pathways of
APP degradation.
Thus, several possibilities exist to explain the accumulation of beta-
amyloid in the brain of persons afflicted with AD, as follows-
(1) a deficiency in the activity or levels of the secretase(s) involved inthe destruction of the amyloidogenic region;
(2) altered cellular sorting of APP such that it might become exposed to
proteases of the pathologic pathway;
(3) an elevation in the levels of the pathologic protease(s);
(4) a deficiency in the levels of degradative enzymes which otherwise
degrade amyloid as fast as it is produced; or
(5) an increased susceptibility of APP to pathologic proteolytic
~gr~ ti~-n caused by mutations in the APP amino acid sequence.
Relatively little is known about the regulation of APP sorting in the cell.
A growing hypothesis is that altered phosphorylation at least in part due to
altered protein Kinase C activity causes altered APP trafficking, ultimately
leading to changes in APP processing (Buxbaum et al., 1990, Proc. N~tl. Acnd. Sci.
LISA, 87: 6003). Thus, treatments designed to alter cellular phosphorylation have
caused both qualitative and quantitative changes in the pattern of APP C-
terminal fragments.
WO 9i/13084 PCT/US94/07043
2~ 755~4
While amyloidogenic APP processing was initially suggested to be an
r-n~ crlm~l-lysosomal event (Golde et al., 1992, Science, 255: 728; and C. Haas et
al., 1992, Nature, 357: 500), there is recent evidence that beta-amyloid is released
by cultured cells (C. Haas et al., lg92, Nature, 359: 322; and Shoji et al., lg92,
Science, 258: 126), along with an alternatively processed form of secreted APP
(Suebert et al. 1993, Nature, 361: 260), consistent with participation of protease
within the secretory pathway or at the plasma membrane in beta-amyloid
formation. Recently, trAncff~rtf~cl cell lines expressing the APP 695 ~cct-ri~h~cl with
the Swedish form of FAD were shown to release beta-amyloid like fr~gmr-ntc 6-8
times faster than cells ~ f ~ 1 with wild type APP (Citron et al., 1992, Nature,360: 672; and Cai et al., 1993, Science, 259: 514; and 1992, Neuroscience Lett., 144:
42), although similar studies of the effect of the London (V to I) mutation
showed no effect on amyloid release (See, Cai et al., Id.). In some cases, the
amyloid released by cultured cells contains an unusual form of beta-amyloid
with an N-terminus starting at valine 594 (numbering according to reference 1)
of the APP precursor (C. Haas et al., 1992, Nature, 359: 322; Busciglio et al., Proc.
Natl. Acad. Sci. USA, 90: 2092), the significance Df which is not understood. The
effect of inhibitors overwhelmingl~l support participation of an acidic cellularcompartment in beta-amyloid production in these systems (Shoji et al., 1992,
Science, 258: 126; Busciglio et al., Proc. Natl. Acad. Sci. USA, 90: 2092; and Haas et
al., 1993, J. Biol. Cllem., 268: 3021) and suggest a lack of involvement of certain
cysteine or serine proteases (Shoji et al., 1992, Science, 258:126; Busciglio et al.,
Proc. NatZ. Acad. Sci. USA, 90: 2092; and Haas et al., 1993,1. Biol. Chem., 268:3021).
Recently, Nitsch et al., 1992, Science, 258: 304, have shown that transfection
of cell lines with certain acetylcholine receptor types followed by receptor
activation caused an increase in APP processing and secretion, in a process
rr~nrl~r-(1 to arise by changes in protein kinase activity. Beside implicating a role
for altered phosphorylation, this latter study provides a link between plaque
pathology and the established perturbations in cholinergic nerve function
rh~r~rt~rictic of AD.
Despite the above observations, there is currently insufficient knowledge
of APP sorting to enable the design of a selective and specific therapeutic agent
that could restore balance to any underlying alterations of cellular sorting.
~ WO 9S/13084 2 1 7 5 ~ 6 4 PCTIUS94107043
Beta-amyloid must be formed by the direct action of protease(s). The
i~ir-ntifirAtirln of the so-called "pathologicn brain protease(s) responsible for the C-
100 or beta-amyloid formation is an essential step in an effort to develop
therapeutic protease inhibitors designed to block amyloid accumulation.
ntifirAtir~n of such enzymes requires the development of specific assays for theactivity of such proteases which would al~ow one to specifically measure the
activity of the proteases in the presence of other brain proteolytic enzymes which
are present in brain extracts.
Such assays are then used to detect the protease during protease
p1lrifirAtir~n Finally, the assays can be used to measure the effect of potential
inhibitors of the enzyme such as is required in phArmAr~11tirAI screening for lead
therapeutic compounds.
Several studies have undertaken the pllrifirAtinn and charArtr-ri7Ati--n of
both the secretases and purported pathologic proteases. Initial studies utilizedassays featuring synthetic peptide ~ub~LlaL~ that only mimirkr-d the expected
cleavage sites within APP. While such assays are useful for mr-Ac1lrin~ the in
vitro activity of a purified protease, they rarely possess sufficient specificity to
allow detection of one protease in a mixture of proteases such as would be
required to monitor a protease pllrifirAtirn. Thus, these peptidase assays failed to
provide the necessary protease specificity, and the peptidase activities thus
quantified were used without success to pursue the pllrifirAtir>n of candidate APP
processing enzyme activities from human brain tissue. Prior to the present
disclosure, no credible candidate protease(s) for either process have emerged, and
the results of the various studies have been rrmflirtin~.
For example, the numerous available studies have proposed thât the
pathologic protease is: Iysosomal in origin (Cataldo et al., 1990, Proc. Natl. Ac~d.
Sci. USA, 87: 3861; and Haas et al., 1992, Nature, 357: 500); a calcium dependent
cathepsin G-like serine protease or a metal dependent cysteine protease
(Razzaboni et al., 1992, Brnin Res., 58g: 207; and Abrahams et al., 1991, An. N.Y.
Acad. Sci., 640: 161); Calpain I (Siman et al., 1990, J. Neurosc*nce, 10: 2400); a
m11ltirAtAIytic protease (Ishiura et al., 1989, FEBS. Lett., 257: 388); a serine protease
(Nelson et al., 1990, J. Biol. Chem., 265: 3836); thrombin (Igarashi et al., 1992,
Biochem. Biop~s. Res. Com~n., 185: 1000); or a zinc metallo-peptidase (WIPO
WO95113084 2 ~ 755t~-4 PCTIUS94/07043 ~
application, WO 92/07068 by Athena Neurosciences, Inc.).
Similar in~nn~ict~ c have arisen in efforts to identify the secretase,
which has been claimed to be: a metallo-peptidase (McDermott et al., 1991,
Biochem. Biophys. Res. Comm., 179: 1148); an acetylcholinerase associated
protease (Small et al., 1991, Biochemistry, 30: 10795); Cathepsin B (Tagawa et al.,
Biochem. Biophys. Res. Comm., 177: 377); or a plasma membrane associated
protease of broad sub-site specificity (Sisodia, 1992, Proc. ~atl. Acad. Sci. USA, 89:
6075); and Maruyama et al., 1991, Biochem. Biophys. Res. Comm., 179:1670).
The general lack of success of past and current efforts to identify the nature
of the APP ~ g enzymes have stemmed from poor specificity of the assays
employed, and from the complex heterogeneity of proteases ACcoriAtP~i with the
cerebral tissue.
SUMMARY OF THE INVENTION
The present disclosure describes a method which identifies some of the
APP processing enzymes with specific assays based on the proteolytic ~I.ogrA~lAtir1n
of recombinant APP in combination with immunochemical detection of the
reaction products. The assays of the present invention identify human brain
proteases that possess the correct specificity and appropriate localization to play a
role in the formation of beta-amyloid from the APP.
The format of the presently disclosed assays in conjunction with the
identified proteases afford the capacity to process reasonably large numbers of
samples and yields good sensitivity due to the immunochemical method of
detection. Furthermore, the simplicity of the assay allows for ready adaption for
routine use by lab technicians and yields ~onsict~nt, reproducible results. These
and other improvements are described hereinbelow.
One goal of the presently disclosed invention is to provide a method for
discovering drugs that can be used to treat AD patients. As stated previously, the
proteolytic degradation of APP to yield the 39 to 4~ amino acid peptide beta-
amyloid is the first step in the pathophysiological process of amyloid plaque
WO 95/13084 PCT/U 4107043
~ 2~7~564 sg
formation. Several lines of evidence point to a causative role of beta-amyloid
and the amyloid plaques in the neuro-degeneration characteristics found in the
AD brain. These include:
(i) the co-localization of plaque material with degenerating neurons
and dystrophic neurites (reviewed in Price et al., 1989, BioEssays, 10: 69);
`' (ii) evidence that beta-amyloid can be toxic to neurons in culture
(Yankner et al., 1990, Science, 250: 270);
(iii) evidence that beta-amyloid is associated with neuronal
degeneration and altered memory when tested in certain animal models (Flood
et al., 1991, PNAS, 88: 3363; and Kowall et al., 1991, PNAS, 88: 7247); and
(iv) co-segregation of certain forms of inherited AD with point
mutations in the APP (Goate et al., 1991, Nnture, 349: 704; Yoshioka et al., 1991,
Biochem. Biophys. Res. Comm., 178: 1141; Chartier-Harlin et al., 1991, Nature,
353: 844; Murrell et al., 1991, Science, 254: 97; and Mullan et al., 199~, Nature
Ger~et*s, 1: 345).
Thus, proteolytic conversion of APP to beta-amyloid appears to be an
essential step in the pathogenesis of AD and, as such, an i~ oi~dn~ target for
therapeutic intervention. T11~ntifi~tinn of the relevant protease activities, aswell as the development of suitable in vitro screening assays, are therefore
essential prerequisites for the development of therapeutic protease inhibitors
that could be used as treatments to block amyloid plaque formation in AD
patients.
The present invention relates to two developments which can be used to
discover inhibitors of proteolytic beta-amyloid fnrm:~tinn
(1) An in vitro assay comprising a holo-APP substrate and either a
highly purified protease that degrades APP or a crude biological extract
ni~ ntifi~d proteases that can degrade APP; and
.
(2) The identification and purification of specific proteases from
human brain that can form amyloidic or pre-amyloidic APP C-terminal
fragments when used in conjunction with the in vitro assay system described in
(1), above.
WO95113084 2 ~ 7 ~5 6 4 PCTIUS94/07043
The assay enables the detection of in vitro APP (lP~r~ tion activity to
yield C-terminal APP fr~mPntc ~Vhen used with crude biological extracts, the
assay can be used to monitor the p1lrifirAtir~n of, or to rh~r~rtPri7P the protease
pul~ible for the detected activity.
Additionally, when used with either a purified protease or a crude
biological extract containing llnirlPntifiPci APP tlP~r~rlin~ enzyme activities, the
assay can be used to measure the inhibition of the APP processing activity by
chemical or biological compounds that are co-incubated in the assay mixture.
Inhibitory compounds thereby identified can have application as therapeutic
inhibitors of the in vivo amyloid plaque formation rh~r~ctPristic of AD patients.
Proteases identified according to (2) above, include the aspartic protease,
cathepsin D and a chymotryptic-like serine protease distinct from cathepsin G
and inhibited by N-tosyl-L-phenylalanine-chloromethylketone (aTPCK") and
alpha-2 antiplasmin and chymotrypsin inhibitor II from potato. The
identification of cathepsin D is particularly ci~nifiri~nt We show that cathepsin
D is able to form C-100-like and beta-amyloid-like fragments of 10.0 kDa and 5.6kDa size, ~ Jt.Lively~
This discovery enables the use of any purified or isolated cathepsin D to
perform a search for inhibitors of its activity using either the in vitro assay
described in (1), above or simpler high Lluvu~ ,vu~ peptidase assays such as those
described in the present invention.
Furthermore, since much is known about the specificity of cathepsin D as
well as the design of specific aspartic protease inhibitors, i~lPntifir~tir~n ofcathepsin D as an amyloidogenic protease enables both the development of
specific cathepsin D inhibitors using established methods, as well as the
ili7~tjr~n of established cathepsin D inhibitors.
Also shown below is that catllepsin D, unexpectedly, hydrolyzes APP at the
peptide bond between Glu(593)-Val(594) (numbering according to Kang et al.,
supr~). The preferred specificity of cathepsin D is, ordinarily, between
hydrophobic residues. This information can be used further in the design of
cathepsin D inhibitors.
~ wo 9~113084 2 1 7 ~ 5 6 4 PCTIUS9410~043
As mentioned above, inhibitory compounds thereby identified have
application as therapeutic inhibitors of the in vivo amyloid plaque formation
rh~r~rt-~rictir of AD patients.
APP ~l~gr~lin~ enzymes identified by the use of the present invention can
be purified and used to:
=.~
(i) develop immllnnrhrnnical reagents necessary to further correlate
the co-!nr~ tinn of protease with AD brain pathology; and
(ii) isolate the corresponding protease cDNA. The cloned cDNA can
then be used to construct transgenic animal modçls for AD in which the effect ofprotease uv~ ion can be assessed.
APP degrading enzyme inhibitors identified by the use of the present
invention are also useful, for example, as ligands in the purification of the APP
degrading enzymes by affinity chromatography. The column will normally be
packed with an inert matrix, e.g., agarose, to which the enzyme inhibitors have
been attached, if necessary indirectly through a hydrocarbon spacer arm. T'ne
composition rnnt~inin~ the enzyme is then applied to the column, and the
enzyme is trapped by the inhibitors while all other proteins pass through and are
discarded. The enzyme can then be liberated from the column either by eluting
with a deforming buffer at a pH which changes the rh~r;2ctr~rictirc of the enzyme
and no longer allows it to bind to the inhibitor, or by the use of a competitivecounter-ligand, which displaces the inhibitor. In both cases, the enzyme passes
through the column and can be collected, now free of other proteins. For furtherdetails, see, e.g., T. Palmer, UnderstQnding Etlzymes, 1991, Ellis Horwood, New
York, 3rd Edition, the disclosure of which is ill~,lp~,ldL~d herein by reference.
Assays il,.ol~uld~ing synthetic peptide substrates are useful for in vitro
enzymological studies of highly purified protease prrr~r~tinns, but are generally
of inc11ffirir-nt specificity to enable the selective detection of a desired protease
activity in crude biologic extracts containing a plethora of proteases. For instance,
brain tissue is abundant with a wide and varied range of peptide processing and
~r-gr~in~ enzymes, which may explain why efforts to isolate specific brain APP
degrading proteases with synthetic peptide SU~L1dL~S have been unsuccessful
(see, Background section, above).
11
WO 95/13084 PCTIUS94/07043
2 1 7 55~4
A~uldil~gly, in Example 3, belûw, it is shown that synthetic peptide assays
lead to the iL1~ntifiL-Atif~n of several peptidases which are unable to degrade APP
to yield C-terminal fragments under the specified assay rr)nt1itiL~nc, and that the
pattern of APP degrading proteases does not resemble in any way the
L ull~byollLlil~g pattern of brain peptidases.
A more definitive approach to this problem is the lltili7Ati~ln of holo-APP
as a substrate, in L~L~njl1nrtiL~n with a method of assessing its specific L1L~gr~flAti~n
following inL~1lhAtiL~n with protease contAinin~ fractions. To this end, the present
disclosure describes such a metl~od, wherein the enzymic degradation of
recombinant APP by brain protease fractiorls is ll,ulLi~ul~d by immunoblot usingantibodies to the C-terminal region of APP.
Our assay procedure focuses on the fL~rmAtiL~n of C-terminal frAgmL~ntc
from APP of size sufficient to include the full length beta-amyloid peptide (a
process requiring endululuL~olybis~ N-terminal to the A4 region).
Human brain tissue (non-AD control or AD) is homogenized and then
sub-fractionated into a soluble fraction (hereinafter "S"), a post 15,000 g pellet
(hereinafter "P-2"), and a microsomal fraction (hereinafter "M") using
conventional ultracentrifugation. The membranous M and P-2 fractions are
solubilized with a Triton X-100 preparation. The resulting soluble fractions from
M and P-2, as well as the S fraction, are then separately subjected to
chromatography on a Mono-Q strong anion exchange column which results in
c,~rArAtil~n of different brain proteases.
Using a synthetic peptide that mimics the amino acid se,~uence
surrounding the N-terminus of beta-amyloid, the peptidase activity of
individual mono-Q fractions from the purification of M, soluble and P-2
fractions is assessed. Contiguous pools of column fractions are made based on
the recovery of discrete peaks of peptidase activity.
The pools of peptidase activity are used to establish assay L~onfliti~ns for
the detection of proteolytic ~gr~ tion of highly pure l~-ùll.bilLdlll APP purified
from a transfected CHO cell line. An immlln~hlL~t assay is developed in which
antibodies directed either to the APP C-terminal domain or the beta-amyloid
12
~ WO95/13084 2 ~ 75564 PcrruS9410~043
region are used to locate C-terminal APP frA~mPntc The assay is used to identifysix potentially different proteolytic activities capable of forming APP C-terminal
frAgTn~nt~ of a size large enough to potentially contain full length beta-amyloid.
The recovery of APP rlP~r~lin~ activity amongst the mono-Q pools is not found
to correlate well with the peptidase activity profiles established in step 2.
Inhibitor studies reveal that the APP llPgrA~in~ activities include both serine and
aspartic protease activities.
The use of the peptidase assay for mr>nitr)rin~ enzyme pl]rific~ti~n is
abandoned. Larger supplies of l~u)ll,bil~ APP are obtained by ex~ iul, in a
baculovirus directed insect cell system, enabling use of the APP ~PgrAfiAti(7n assay
as the primary method to monitor APP degrading activity during protein
p11rifi~Ation. A major aspartic protease activity is identified in fractions from the
mono-Q pllrifirAtil~n of the P-2 fraction.
Further purification and charactPri7Ation experiments demonstrate that
the enzyme is cathepsin D. The cathepsin D is shown to hydrolyze holo-APP
forming a beta-amyloid-like fragment of 5.6 kDa.
Aprotinin sepharose affinity chromatography is used to attempt to isolate
aprotinin sensitive APP degrading activities identified above. A chymotrypsin-
like serine protease activity is partially purified that can degrade APP to formspecific C-terminal fragments of ll, 14 and 18 kDa, that are shown by
immunochemical means to contain full length beta-amyloid.
Through this procedure, we have identified several brain protease
activities that play, with high probability, a role in amyloidogenic riP~rA~Ati(~n of
APP. Each of the identified or 1lni~iPntifiPd activities described herein can inconjunction with the APP degradation assay be used to screen for selective
protease inhibitors of therapeutic value.
As used herein, "APP substrate" shall mean full length APP, whether
derived by isolation or p11rifi~Ation from a biological source or by expression of a
cloned gene encoding APP or its analogs, and fragments of any such protein,
including fragments obtained by digestion of the protein or a portion thereof,
fra~mPnt~ obtained by expression of a gene coding for a portion of the APP
protein, and synthetic peptides having amino acid sequences corresponding to a
13
WO95/13084 2 l 7 5 5 .6 4 PCT/US94/07043 1
portion of the APP
APP substrates for the assays of the present invention can be provided as a
test reagent in a variety of forms. Although preferably derived from, or
ondil g at least in part with the amino acid sequence of, APP 695,
derivatives or analogs of other APP isoforms (supra) are contemplated for use inthe present method as well. APP 695 can be obtained by biochemical isolation or
pllrifirAti--n from natural sources s~ch as desaibed in Schubert et al., 1989, Proc.
NQtl. Acad. Sci. USA, 86: 2066; or by expression of recombinant DNA clones
encoding the protein or a functional portion thereof (Knops et al., 1991, J. Biol.
Chem., 266: 7285; and Bhasin et al., 1991, Proc. Natl. Acad. Sci. USA, 88:10307).
The fragments of the APP protein will comprise a sequence of amino acids
sufficient for r~ccgnitinn and cleavage by the pertinent proteolytic test sampleactivity (supra). Isolation of APP from biological material usually will involvepurification by ~:u~-v~l~Liollal techniques such as chromatography, particularlyaffinity chromatography. Purified APP or fragments thereof can be used to
prepare monoclonal or polyclonal antibodies which can then be used in affinity
purification according to conventional procedures. Resulting purified APP
material can be further processed, e.g., frAgm~nt~l, by chemical or enzymatic
digestion. Useful fragments will be idf~ntifie~l by screening for desired
susceptibility to the pertinent proteolytic test sample activity (supra).
As previously stated, the APP substrate can also be prepared by exE,i~s~io
of recombinant DNA clones coding for APP or a portion thereof. The cloned
APP gene may itself be natural or synthetic, with the natural gene obtainable
from cDNA or genomic libraries using riPgGn~qrAt~ probes based on known amino
acid sequences (Kang et al., 1987, Nature, 32~: 733). Other techniques for
obtaining suitable recombinant DNA clones, as well as methods for expressing
the cloned gene, will be evident to the worker in the field.
A variety of convenient methods are applicable to the detection of
proteolytic cleavage of the APP substrate in the presence of the test sample.
Several of the presently more preferred methods are desaibed below, however, it
will be recognized by the skilled ~orker in the field that many other methods can
be applied to this step without departing from the inventive features hereof. Ingeneral, any method can be used for this purpose which is capable of detecting
14
~ WO 95/U084 2 ~ 7 5 5 6 4 PCTIUS94107043
the occurrence of proteolytic deavage of the APP substrate. Sudh can be affordedby d~lv~l;a~ design of the APP substrate such that cleavage produces a signal
producing species, e.g., an optically l~yun~iv~ product such as a colored or
nuOl~ l dye.
Another principal approach involves the sensitive detection of one or
more cleavage products such as by immunoassay. Presently, such cleavage
product is preferentially a C-terminal fragment of the APP substrate; however,
any fragment which appears upon incubation with samples can be the object of
detection.
The detection of one or more cleavage products characteristic of the
pathologic proteolytic activity can be accomplished in many ways. One such
method, which is further exemplified in the examples which follow, involves
the procedure commonly knûwn as Western blût. Typically, after the in-llh~ti~-n
of APP with test sample, gel electrophoresis is performed to separate the
components resulting in the reaction mixture. The separated protein
cvll.luol~l.L~ are then transferred to a solid matrix such as a nitrocellulose
membrane.
An antibody specific to a fragment characteristic of APP degradation is
then reacted with the components fixed to the membrane and detected by
addition of a secondary enzyme-labeled antibody conjugate. The location of the
resulting bound conjugate is developed with a chromogenic substrate for the
enzyme label.
A variety of imm~lno~c6~y formats which are amenable to currently
available test systems can also be applied to the detection of APP fr~mf~ntc
Typically, the APP substrate will be incubated with the test sample and resulting
intact APP rendered immobilized (such as by capture onto a solid phase), or
alternatively, the test sample is incubated with an imrnobilized form of the APPsubstrate. Proteolytic cleavage is then detected by reacting the immobilized APPsubstrate with an antibody reagent directed to a portion of the APP substrate
which is cleaved from the APP substrate or which defines the cleavage site.
The antibody reagent can comprise whole antibody or an antibody
fragment ~r)mrrisin~ an antigen combining site such as Fab or Fab', and can be of
WO 95/13084 2 1 7 ~ 5 6 ~ PCT/US94/07043
the mnnnrlnn~l or polyclonal type. The detection of antibody reagent bound to
the immobilized phase is indicative of the absence of the characteristic
proteolytic cleavage. Conversely, loss of antibody binding to the immobilized
phase is indicative of APP cleavage. The detection of binding of the antibody
reagent will generally involve use of a labeled form of such antibody reagent orthe use of a second, or anti-(antibody), antibody which is labeled.
Capture or immobilization of APP can be accomplished in many ways. An
antibody can be generated specific to an epitope of APP which is not on the
cleavable fragment. Such an antibody can be immobilized and used to capture or
immobilize intact APP. Alternatively, a ligand or hapten can be covalently
attached to APP and a corresponding immnhili7rcl receptor or antibody can be
used to capture or immohil;7r- APP. A typical ligand.re.~ ul pair useful for this
purpose is biotin:avidin. Examples of haptens useful for this purpose are
fluorescein and ~ itn~rigr-nin
The solid phase on which the APP substrate is immobilized or captured
can be composed of a variety of materials including microtiter plate wells, testtubes, strips, beads, particles, and the like. A particularly useful solid phase is
magnetic or F~r~m~gnr-tir particles. Such particles can be derivatized to contain
chemically active groups that can be coupled to a variety of compounds by simplechemical reactions. The particles can be cleared from suspension by bringing a
magnet close to a vessel containing the particles. Thus, the particles can be
washed repeatedly without cumbersome centrifugation or filtration, providing
the basis for fully ~l1tom~tin~ the assay procedure.
Labels for the primary or secondary antibody reagent can be selected from
those well known in the art. Some such labels are fluorescent or
chr-mill-minrcrr-nt labels, radioisotopes, and, more preferably, enzymes for this
purpose are alkaline phosphatase, peroxidase, and ~-galactosidase. These
enzymes are stable under a variety of rnn~litinnC, have a high catalytic turnover
rate, and can be detected using simple chromogenic substrates.
Proteolytic cleavage of the APP substrate can also be detected by
chromatographic techniques ~hich will separate and then detect the APP
fragments. High pressure liquid chromatography (HPLC) is particularly useful in
this regard. In applying this technique,~a fluorescently tagged APP substrate is
16
~ wo 95/13084 2 ~ 7 5 5 6 4 PCTIUS94107043
prepared. After inrllhAtirm with the test sample, the reaction mixture is applied
to the chromatographic column and the differential rate of migration of
nuL~ ,L~lll hrA~m-ontc versus intact APP is observed.
The present invention is also directed to a method of treating a patient
suffering from AD comprising administering to such patient an amount
effective therefor of an inhibitor of an aspartic protease alone or in admixturewith a non-toxic, inert phArmArrllhrAlly acceptable excipient.
The present invention further relates to pharmaceutical formulations
which contain such inhibitors of an aspartic protease in admixture with a non-
toxic, inert phArmArrllhrAlly acceptable excipient.
The present invention also includes such phArmArr--hrAI fl~rmlllAtir~n~c in
dosage units. This means that the fr~rml]lAtir~ns are present in the form of
individual parts, for example, tablets, dragees, capsules, caplets, pills,
suppositories and ampoules, the inhibitor content of which corresponds to a
fraction or a multiple of an individual dose. The dosage units can contain, for
example, 1, 2, 3 or 4 individual doses or 1/2,1/3 or 1/4 of an individual dose. An
individual dose preferably contains the amount of active compound which is
given in one A~minictrAtion and which usually corresponds to a whole, 1/2, 1/3
or 1/4 of a daily dose.
By non-toxic, inert phArmArrlltirAlly acceptable excipients there are to be
understood solid, semi-solid or liquid diluents, fillers and formulation
All~iliAri~c of all types.
Preferred pharmArr--ltirAI form-llAtirnc which may be mentioned are
tablets, dragees, capsules, caplets, pills, granules, suppositories, solutions,
suspensions and rm1llcir~ns, pastes, r~intmrntc, gels, creams, lotions, dusting
powders and sprays. Tablets, dragees, capsules, caplets, pills and granules can
contain the inhibitor in addition to the customary excipients, such as (a) fillers
and extenders, for example, starches, lactose, sucrose, glucose, mannitol and
silicic acid, (b) binders, for example, carboxymethylcellulose, alginates, gelatin
and polyvinylpyrrolidone, (c) humectants, for example, glycerol, (d)
disintegrating agents, for example, agar-agar, calcium carbonate and sodium
carbonate, (e) solution retarders, for example, paraffin and (f) absorption
WO 95/13084 ~2 1 7 5 5 6 4 PCTIUS94/07043 ~I
, for example, quaternary ~ lll compounds, (g) wetting agents,
for example, cetyl alcohol and glyceIol ~I.",~ lte, (h) al,~ , for example,
kaolin and bentonite and (i) lubricants, for example, talc, calcium stearate,
m~n~cillm stearate and solid polyethylene glycols, or mixtures of the substanceslisted under (a) to (i) directly hereinabove.
The tablets, dragees, capsules, caplets, pills and granules can be provided
with the ~ lldly coatings and shells, optionally ront~inin~ opacifying agents
and can also be of such composition that they can release the inhibitor only or
preferentially in a certain part of the intestinal tract, optionally in a delayed
manner. Examples of embedding compositions which can be used are polymeric
5llhst~n~s and waxes.
The inhibitor can also be present in microencapsulated form, if
appropriate, with one or more of the abovementioned excipients.
Suppositories can contain, in addition to the inhibitor, the customary
water-soluble or water-insoluble excipients, for example, polyethylene glycols,
fats, for example, cacao fat and higher esters (for example, Cl4-alcohol with Cl6-
fatty acid), or mixtures of these substances.
Ointments, pastes, creams and gels can contain, in addition to the
inhibitor, the customary excipients, for example, animal and vegetable fats,
waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols,
silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these
s~lhst~n(~s
Dusting powders and sprays can contain, in addition to the inhibitor, the
customar~ excipients, for example, lactose, talc, silicic acid, ~lllminllm hydroxide,
calcium silicate and polyamide powder, or mixtures of these substances. Sprays
can additionally contain customary propellants, for example,
chlorofluorocarbons .
Solutions and emulsions can contain, in addition to the inhibitor,
customary excipients, such as solvents, solubilizing agents and ~mlllsifi.ors, for
example, water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
18
wo 9S/1308~ 2 1 7 5 5 6 4 PCTIUS94107043
dimethy1fnrm~mirlP, oils, in particular, cottonseed oil, peanut oil, corn germ oil,
olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahy.llurLuru.yl
alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these
SulJ~Ldll~s.
For parental a~ usL-dlio-, the solutions and r-nn1]lcinnc can also be in a
sterile form which is isotonic with blood.
Suspensions can contain, in addition to the inhibitor, customary
excipients, such as liquid diluents, for example, water, ethyl alcohol or propylene
glycol and suspending agents, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose,
~ min~lm metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of
these bUlJbLdll~l b.
The formulation forms mentioned can also contain coloring agents,
preservatives and smell- and taste-improvement additives, for example,
p~Jp~llllillL oil and eucalyptus oil and sweeteners, for example, s~crh~rinr- oraspartame.
The inhibitor should be present in the abovementioned ph~rTn~rPIltirz~l
fnrm~ tinnS in a ( ~ linn of about 0.1 to 99.5%, preferably about 0.5 to 95%
by weight of the total mixture.
The abovrmr-ntinnrcl pharmaceutical formulations can contain multiple
inhibitors, in which case, the total amount of inhibitors in the abov.omr-ntinnr~l
ph~rm~c~lltir~l fnrmlllAtionc is about 0.1 to 99.5%, preferably about 0.5 to 95% by
weight of the total mixture. The inventive fnrmlll~tinnc can contain other active
ingredients in addition to the inventive inhibitors.
The aror~ nPci pharm~rr~ltir~l formulations are prepared in the
customary manner by known methods, for example, by mixing the active
compound or compounds ~ith the excipient or excipients.
The formulations mentioned can be administered orally, rectally,
buccally, parenterally (intravenously, intramuscularly or subcutaneously),
intracis~ernally, intravaginally, intraperitoneally or locally (dusting powder,
19
WO951~3084 2~5564 ~ PCTIUS94107043
ointment or drops). Suitable f~rmlllAti~ns are injection solutions, solutions and
su~e.~sioi~s for oral therapy, gels, pour-on f rmlllAti~ns"~mlllsionc, ointments
or drops. OphthAlmnl~ AI and dermatological formlllAtion~, silver salts and
other salts, ear drops, eye ointments, powders or solutions can be used for local
therapy. It is rul Ll~ possible to use gels, powders, dusting powders, tablets,
sustained release tablets, premixes, ~:OI~C~ la~S, granules, pellets, capsules,
caplets, aerosols, sprays and inhalates. The inhibitor can furthermore be
incorporated into other carrier materials, such as, for example, plastics (e.g.,chains of plastic for local therapy), collagen or bone cement.
Since the site of action is the brain, the inhibitors must pass the blood-
brain barrier. This may require in some cases that the lipophilicity of the
inhibitor be increased, for example, by ~onjll~Ation to a lipophilic carrier or by the
introduction of lipophilic substituents, e.g., hydrocarbons, e.g., long chain alkyl
groups, alkenyl groups, e.g., vinyl, etc. Such modification to increase
lipophilicity is conventional and within the skill of the ordinary practitioner in
the art. See, e.g., R. B. Silverman, "The Organic Che1n*hy of Drug Design and
Drug Action", 1992, Academic Press, San Diego, particularly pages 361-364, the
entire contents of which are incorporated herein by reference. Any conventional
method of accomplishing the increased lipophilicity is contemplated. An
example of a suitable lipophilic carrier is the reversible redox drug delivery
system devised by N. Bodor et al., which is discussed in Silverman, Id., at page362. See also, N. Bodor et al., 1983, Phar1nacol. Ther., 19: 337; and N. Bodor, 1987,
Ann. N.Y. Acad. Sci., 507: 289, the entire contents of both of which are
Ul~OlaLc!d herein by reference.
In general, it is advantageous to administer the inhibitors in total
amounts of about 0.5 to 500, preferably 5 to 100 mg/kg of body weight every 24
hours, if appropriate, in the form of several individual doses, to achieve the
desired results. An individual dose preferably contains the inhibitor in amountsof about 1 to about 80, in particular 3 to 30 mg/kg of body weight. However, it
may be necessary to deviate from the dosages m~nti--n~d and in particular to do
so as a function of the nature and severity of the disease, the nature of the
formulation and of the administration of the medicament and the period or
interval within which A,l".i"i~ n takes place. Thus, in some cases, it may be
sufficient to manage with less that the above-mentioned amount of inhibitor,
while in other cases, the abo~7e-m~ntinnl~l amount of inhibitor can easily be
~ wo 95113084 2 1 7 5 5 6 4 PCrJUS94J07043
tPrminp~l by any expert on the basis of his/her expert knowledge.
DEFlNmONS
The following amino acids may be indicated by the following 3- or 1-letter
codes elsewhere in the spP~ifi~:lti.-n
Aminfl Acid 3-Lett~rCode 1-TPttPrCn~lP
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic Acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic Acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
21
WO 9S/13084 PCTIUS94/07043
21 75564
BRIEF DESCRIPTION OF THE DRAWINGS
Figures la-lf s~how two dimensional contour plots of peptidase activities of
control compared to AD human cortex subfractions.
Subfractions were prepared according to Example 1, by ion-exchange
(mono-Q) separation of P-2, 5 and M fractions. Enzymatic cleavage of N-dansyl-
Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID
NO: 1) by Mono-Q fractions was performed as described in Example 3. Each plot
shows the relative amounts of each fluorescent product (abscissa) obtainable by
incubation of each mono-Q fraction (ordinate) under the same incubation
~ n-litinn.~ The amount of product is represented vertically by contour lines.
Greater numbers of contour lines indicate greater amounts of a particular
product. Mono-Q fractions from control S (a), AD S (b), control M (c), AD M (d),control P-2 (e), AD P-2 (f), were subjected to analysis. The roman numerals on
the right hand ordinate of the three AD plots locate pooled regions described inExample 3, and which were then assayed according to Example 8, and found to
contain significant APP degrading activity.
Figures 2a-2f depict immunoblot analysis of the APP 695 d~ in~ activity
~cgori ~(i with selected Mono-Q pools from the ion-exchange separation of M, S
or P-2 fractions derived from AD cortex.
The pools were made based on their content of peptidase activity as
described in Example 3. Immunoblot assays were performed as described in
Example 8. R~ s~lllaliv~ assays for the following pools are shown:
Figure 2a: Activity ~c50ri~d with P-2 pool V: APP was present in each of
lanes 2 to 6. C-100 from PMTI 73 ~lane 1), no P2-V blank (lane 2), P2-V (lane 3),
P2-V plus EDTA (lane 4), P2-V plus methanol (lane 5), and P2-V plus pepstatin A
in methanol (lane 6).
Figure 2b: Activity ~Cco~ with M pool 111: APP was present in lanes 2
to 7. C-100 from PMTI 73 (lane 1), M-III plus cystatin C (lane 2), M-III plus
aprotinin (lane 3), M-III plus captopril (lane 4), M-III plus EGTA (lane 5), M-III
plus N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-
WO 95113084 ~ ~ ,, 5 ~ ~ ~ PCI~US94107043
Asp (SEQ ID NO: 1) (lane 6), M-III without inhibitor (lane 7), and prestained
molecular weight markers (lane 8).
Figure 2c: Activity Acco~ d with S pool 1: APP was present in each of
lanes 2 through 6. S-I plus N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-
Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) ~lane 2), S-I plus EGTA (lane 3), S-I
plus captopril (lane 4), S-I plus aprotinin (lane 5), S-I plus cystatin C (lane 6), and
C-100 from PMTI 73 (lane 7). Lane 1 contains prestained m~ r1llAr weight
markers.
Figure 2d: Activity recovered in individual mono-Q fractions from the
separation of AD P-2: Mono-Q fractions 38 to 43 corresponding to the
conductance region in which P-2 pool VII is otherwise observed were
individually examined for APP degrading activity. For each fraction, the
inf~lhA~ir)n was carried out both in the absence (-) or presence (+) of recombinant
APP 695. The fraction numbers are located on Figure 2. The C-100 standard used
was from PMTI 100. Mr indicates molecular size markers.
Figure 2e: Cu...~ oll of the position of igr~iQn of C-100 products
directed either by PMTI 73 or PMTI 100: The protein product of PMTI 73 (lanes 1
and 3) and PMTI 100 (lanes 2 and 4) are shown in '""'1'"~~"" with molecular
markers (lane 5).
Figure 2f: Typical time course of product ' ~ion- APP plus M-m were
analyzed at time t= 0 h (lane 1), 5 h (lane 2), 20.5 h (lane 3), 44.5 h (lane 4), and 55
h (lane 5). Molecular weight markers (lane 6), C-100 PMTI 73 (lane 7), and APP
without M-III (lane 8), are also shown.
For each of Figures 2a through 2f, above, migration was from top to
bottom. In 2a-2d and 2f, the upper solid arrow locates the position of migrationof holo-APP, and the lower solid arrow locates the position of migration of C-100.
In Figure 2d, the open arrows locate the positions of migration of putative
oligomers of the enzymatically generated C-100 fragment. The con.~l~L~dLiol~s ofall inhibitors are listed in Table 4, below.
Figures 3a and 3b depict results from further pl1rifi~ ~ion of P-2 VII pool by
gel filtration.
23
WO 95/13084 PCT/US94/07043
2 1 755~4
Figure 3a: P2-pool VII fractions from Mono-Q 10/10 chromatography were
pooled, concentrated to 0.25 ml and applied to a tandem arrangement of two
Superose 6HR 10/30 columns equilibrated in 10 mM tris HCI buffer pH 7.5
containing 150 mM NaCl. Elution was performed at a flow rate of 0.3 ml/min,
and column eluent was monitored at 280 nm. Fractions (0.24 ml) were collected
and subjected to both peptidase activity, and APP degradation assay using the
immunoblot. The arrows locate the region of the chromatogram in which the
APP degrading activity was recovered. A~ Iso shown are the peptidase activities
associated with both K-M (closed circles) and M-D (open circles) bond cleavage.
Chrt~mAtogr~rhy was performed at 22C
Figure 3b: The migration of the APP degrading activity relative to the
indicated standard proteins of known molecular weight was used to calculate an
Mr apparent of the APP degrading protease which is listed in Example 8.
Figure 4 shows Peptide Epitope Mapping of Murine Monoclonal Antibody
C286.8A Raised Against the Beta-amyloid Peptide. Micro-titre plates were coated
with 50 ng of synthetic APP 695 (597-638) (beta-amyloid 1-42), blocked, then
incubated with 100 111 of C286.8A (80 ng of IgG) which had been pr-~incllhAtf~-l (60
min at room temperature) in the presence or absence of the indicated
conL~llllaLi~Jl~ of ~n~ IiLul peptide. Note, that the peptide 1-7 refers to peptide
SEQ ID NO: 1. Following inrllhA~ion for 60 min at room l~lllp~la~ul~, plates
were washed, and the amount of bound antibody l~t~rmin~d by development
with horseradish peroxidase-coupled goat-anti mouse polyclonal antibody
according to standard procedures (Wunderlich et al., 1992, J. of Immllnol.
Met)lods, 147:1). Percent competition (% C) of antibody binding to the plate wascâlculated from the absorbance at 450 nm data using the following equation:
%C= 1.0- O.D. (+ ~Ull~ ol) - O.D. blAnk x 100
O.D. (- competitor) - O.D. blank
Beta-amyloid 1-42, 1-28, 1-16 and N-dansyl-ISEVKMDAEFRHDDDD (rontAinin~1-7) inhibited C286.8A binding dose dependently, whereas 12-28, 25-35 and APP
24
~ wo 95/13084 2 1 7 5 5 6 4 PCTIUS94/07043
645-695 did not, thus localizing the reactive epitope for C286.8A to the N-
terminal 7 amino acids of the A4 region (APP 597-603).
Figure 5: APP 695 ~IO~ activity recovered in ion ~I.a~ fractions
from the purification of human brain P-2 subfraction. A total of 123 fractions
were collected from the column. The first 32 fractions corresponded to the load
and wash phase. The salt gradient started at fraction 33. Screens of fractions 3 to
18 (panel a), 21 to 32 (panel b), 33 to 38 (panel c), and 39 through 44 (panel d), are
shown. For each fraction, the i~ i."l was performed in both the absence (-)
and the presence (+) of APP 695 substrate. Tnrllh~tir~nc of APP 695 for zero or 24
hr is located where d~lu~iial~. Tnrllh~tirlnc were performed as follows: Baculo-derived holo-APP 695 (80 nm) was incubated with 5 1ll of each column fraction
in a total of 15 1ll ront~inin~ 100 mM Mes buffer pH 6.5, 0.008 % (v/v) Triton X-
100,160 mM NaCI, 6.7 mM tris (from the APP stock). Reactions were t~rmin~t~(l
after 24 h by addition of SDS-PAGE sample buffer. Iu~ ul~u~lo~ were developed
using the C-terminal polyclonal antiserum of Example 6, as described in Example
8. The arrows locate the product fragments. Fractions 45 to 86 were also tested
but showed relatively little activity (therefore not shown). Peaks A and B locate
the major activities.
lFigure 6 shows results of purification of P-2 derived APP degraaing
activity on gel filtration: correlation with the elution of cathepsin D. Panel (a), a
280 nm elution profile for the purification of P-2 peak B on a superose 6HR
column. Panels (b) and (c), corresponding APP C-terminal processing activity in
the eluted fractions 49 to 60, fl~tr-rmin~i essentially as described in Example 5.
Arrows locate major product bands. Panels (d) and (e), immunoblot analysis of
eluted fractions using a rabbit polyclonal antibody to cathepsin D (1/300,
dilution). The arrows locate the position of migration of ill~ ul~ul~active bands.
Human liver cathepsin D was also analyzed for . ~ " "~ "~
Figure 7 depicts protease inhibitor specificity of protease activities isolated
from the P-2 subfraction. Reactions (32 1ll) were initiated at 37C by APP addition
to achieve the following initial ~Ulll~)Ul ~ rnnrlir~onc P-2 enzyme (2.54 ,ug/ml)
fraction from the 15-25 kDa region of gel filtration (Figure 6); APP (168 nM), in 96
mM Mes buffer pH 6.5. Reactions were terminated after 26 hr by addition of 15 111
of 3X sample buffer, and subjected to immunoblot analysis using a 1/1000
dilution of the rabbit polyclonal antiserum to the APP C-terminus. The effect of
WO 9S113084 21 7 5 5 6 4 PCTluS94/o7o43 ~
addition of the following inhibitors is shown: No inhibitor (lanes 7 and 20), 1
mM EDTA (lane 5); 400 IlM PMSF~ in ethanol (lane 9); ethanol alone (lane 11); 100
,uM E-64 (lane 13);10 llg/ml aprotinin (lane 22);100 ~M pepstatin A in DMSO
(lane 26); DMSO only (lane 24). The effect of incubation of APP for 0 hr (lanes 2
and 30) and 26 hr (lanes 3 and 28) are also shown. Lanes 4, 8,12,19, 23 and 27
contained the C-100 standard. Prestained molecular weight markers are present
in lanes 1 and 29. The 18 and 28 kDa C-terminal product fragments are located
with arrows.
Figure 8 shows time course of Cathepsin D catalyzed APP cleavage
monitored using an antibody to the APP C-terminal domain. Panel (a) shows
time course of APP proteolysis by cathepsin D in the absence (lanes 10-14) or
presence (lanes 4-8) of 86 IlM pepstatin A. APP was also incubated alone (lanes 1-
3). The numbers indicate the time (hr) after initiation of reactions. Reactions
were initiated at 37C by the addition of APP to achieve the following initial
component ~ L.dtions: APP (82 nM), cathepsin D (9.2 ~Lg/ml), in 89 Mes
buffer pH 6.5. Samples without pepstatin received the same amount of solvent
(1.3% v/v methanol). At t=0, 43, 84, 140 and 215 minutes aliquots (15 ~11) were
removed mixed with 7.5 111 of SDS-PAGE sample buffer and subjected to
immunoblot analysis using the rabbit antiserum to the APP C-terminal domain.
The 18 and 28 kDa reaction products are located with arrows.
Figure 9 depicts pH and ionic strength dependence of APP C-terminal
processing by the P-2 derived enzyme or cathepsin D. Panel (a) shows pH
dependence observed with cathepsin D and the P-2 enzyme (peak B, Figure 5
following gel filtration chromatography, Figure 6). Panel (b) is ionic strength
dependence for both enzymes at pH 6.5. Reactions were initiated at 37C by
enzyme addition to achieve the following initial component concentrations:
cathepsin D (9.2 !lg/ml) or P-2 enzyme from Figure 6 (11.7 llg/ml), 100 mM in
each of sodium acetate, Mes, and Tris-HCI, and purified APP (79 nM). At t=0 and
3 hr, aliquots (15 111) were removed mixed with 7.5 111 of 3X sample buffer and
subjected to immunoblot analysis with the C-terminal polyclonal antiserum
according to ~xample 8. The 28,18 and 14 kDa reaction products are located with
arrows.
Figure 10 shows cleavage of N-Dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-
Glu-Phe-Arg-NH2 (SEQ ID NO: Y) by cathepsin D and the P-2 enzyme (peak B,
26
~ WO95/13084 2 ~ 75 5 6 ~ PCT~US94~0~043
Figure 5) following further purif;~ r n on Superose 6HR. Reactions (30 ~Ll) wereinitiated at 37C by enzyme/inhibitor addition thereby achieving the following
t=0 component c~n.~l-"~ "~c Cathepsin D (2.8 llg/ml), or P-2 enzyme from
Figure 6 (17.5 llg/ml), N-Dansyl-peptide (58 IlM), captopril (0.3 mM) in a cocktail
buffer comprising 130 mM in each of acetate, Mes and Tris pH 5Ø Samples
~nntAin~rl either pepstatin A (213 IlM in 3% v/v final methanol) or an
equivalent final concentration of methanol only. At 0, 2, 4, 8 and 24 hr, reactions
were t~rmin~t~d by addition of 12% (v/v) TFA (10 111) and subjected to HPLC
analysis according to Example 2 and 3. R~ Ld~iv~ traces are shown for: P-2
en2yme, t=0 hr (panel A); P-2 enzyme, t= 24 hr (panel B); P-2 enzyme plus
pepstatin A, t= 24 hr (panel C); cathepsin D, t= 0 hr (panel D); cathepsin D, t= 24
hr (panel E); and cathepsin D plus pepstatin A, 24 hr (panel F).
Figure 11 depicts pH dP~Pn~lPnce of N-Dansyl-Ile-Ser-Glu-Val-Lys-Met-
Asp-Ala-Glu-Phe-Arg-NH2 (SEQ ID NO: 7) cleavage by cathepsin D and the P-2
enzyme (peak B). Reaction rnn-litinns were essentially as described in Figure 10,
except that the cocktail buffer was adjusted to the indicated pH values and the
in-~uh~t;nn times were 23 hours for the P-2 enzyme and five hours for cathepsin
D. (a) Cleavage by cathepsin D, and (b) cleavage by P-2 enzyme. Rates of cleavage
at the -Glu-Val- (closed circles) and -Met-Asp- (open circles) bonds are shown in
each case.
Figure 12 is SDS-PAGE analysis of reaction products from the ~ Jal~
digestion of APP by cathepsin D. Panel (a) is a photograph of the coomassie
stained electroblot prior to excision of bands, panel (b) is the corresponding blot
after band excision, and panel (c) is the corresponding immunoblot analysis
(1/100 dilution of monoclonal 286.8A) of a parallel series of reactions to thosedepicted in panels (a) and (b). Reactions in (a) were initiated at 37C by substrate
addition thereby achieving the following initial W~ Ull~ con.~,.LldLiol~s. APP
(15.6 ,u~I), cathepsin D (0.17 IlM, 7.145 llg/ml), in 40 mM sodium acetate pH 5.0,
nnt:~inin~ 30 mM NaCl. At t= 16 hr the reaction mixture was ~ on~ l~ntr~t~d to 15
111 by speed vac, mixed with 7.5 111 of 3X sample buffer and subjected to SDS-
PAGE. Reactions in (c) were performed in essentially the same manner except
that the APP concentration was decreased to 3.2 IlM. For both experiments (in a
and c), incubations were performed with the complete in~lh~tinn system (lanes
3), and in the absence of cathepsin D (lane 4, cathepsin D added back immf~ tl~ly
after addition of the sample buffer). Lane 5 in each case contained cathepsin D
WO 95/130g4 PCT/US94107043
2 1 75S64
only controls, while lane 6 contailled a purified APP as migration standard.
Prestained molecular weight markers are in lane 1. In (c), the main
immunoreactive products are located with arrows.
Figure 13 shows a time course of cathepsin D catalyzed APP d~r~ inr~
ol~d using a mc lc-1nn~l antibody to the N-terminus of beta-amy~oid.
Reactions were initiated at 37C by APP addition thereby achieving the followinginitial u~ uul~ ul~ Llaliuils. APP (448 nM), cathepsin D (30 nM), and when
included pepstatin A (97.2 IlM) in 83 mM sodium acetate buffer pH 5Ø At the
indicated time points, aliquot (20 1ll), were removed mixed with 10 111 of 3X SDS-
PAGE sample buffer and subjected to immunoblot analysis using monoclonal
antibody 286.8A (l/lO0). Reactions were performed in the absence (-) or presence(+) of pepstatin A (delivered in methanol). All samples received 2.7 % (v/v)
methanol. Lanes 1 and 12 contained prestained Mr markers. Lanes 10 and 18
contained C-100 Mr marker. Lanes 11 and 13 contained APP incubated without
cathepsin D for zero and 21 hr respectively. The main product fragments are
indicated with arrows.
Figure 14 shows the effect of amino âcid s~lhsfifllfion on the time course of
hydrolysis of synthetic peptides by cathepsin D and the P-2 derived enzyme (peakB). Reactions were initiated at 37C by substrate addition to achieve the
following initial ~Ull-pUl el.~ con~l-lldLul-s. Cathepsin D (2.5 llg/ml) or P-2 peak
B enzyme (7.5 llg/ml); N-dansyl-peptide (58 IlM), captopril (0.3 mM), with or
without pepstatin A (213 IlM), sodium chloride (75 mM), in 135 mM buffer in
each of tris, Mes and acetate buffer pH 5Ø At various times, aliquots (30 1ll) were
removed, adjusted to 12.5% (v/v) in TFA and subjected to RP-HPLC analysis.
Time course of hydrolysis for the following substrate/protease combinations are
shown:
(a) N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ
ID NO: 7) by cathepsin D;
(b) N-dansyl-Ile-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ
ID NO: 3) by cathepsin D;
(c) N-dansyl-Ile-Ser-Glu-`ilal-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ
ID NO: 7) by the P-2 enzyme (peak B, Figure 5 following gel filtration, Figure 6);
(d) N-dansyl-Ile-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ
ID NO: 3) by the P-2 enzyme (peak B, Figure 5).
28
WO 95113084 2 1 ~ ~ 5 6 ~ PCTSUS94S07043
Cleavage at the E-V bond (squares, panel A and C), M-D bond (closed diamonds,
A and C), or so as to generate the metabolite at retention time 4.4 min (in B and
D) are shown.
.
Figure 15 shows purification of solubilized P-2 fraction on aprotinin
sepharose. E~pt:.;ll.~l.~ was performed according to Example 1. (a) is typical A280
nm elution profile, and (b) is an immunc~blot assay (rabbit antiserum to C-
terminal domain of APP) for APP processing activity in the eluted fractions. Thearrows indicate the migration of the main APP ~1PgrA~til~n products. Note the
appearance of breakdown products in fractions 8-13 from acid elution.
Figure 16 shows pl]rifi~inn of P-2 derived aprotinin binding protease on
mono-Q. (a) shows A280 nm elution profile, (b), activity of eluted fractions using
a rabbit anti-C-terminal APP antiserum (1/1000 dilution) for detection, and (c),activity of eluted fractions using Mab 286.8A. (1/100 dilution of 1.6 mg~ml pureIgG) for detection. The three arrows indicate the migration of the 11,14 and 18
kDa C-terminal APP ~egrA~lAti~n products.
Figure 17 shows pH and ionic strength dependence of APP ~iegra-l~tinn
catalyzed by the pool Y serine protease. Panel (a) shows pH dependence.
Reactions were initiated at 37C to achieve the following t= 0 component
c--nfl~ntrAtic-n~: Pool Y protease (5 111 of fraction 16 from Figure 16), APP (38 nM),
in a cocktail buffer cnmrricin~ 32 mM in each of acetate, Mes and Tris adjusted to
the indicated pH values. Reaction mixtures ~16 Ill) were t~rminAt~d after 2 hr by
the addition of 7.5 Ill of 3X sample buffer. Immunoblots were developed
essentially as described in Example 8, using a rabbit polyclonal antiserum to the
APP C-terminal domain. Lanes 1 and 12 contain prestained Mr markers. Lane 9
contained C-100 and lanes 10 and 11 contain APP incubated for 0 and 3 hr
e.liv~ly at pH 6.5 in the absence of pool Y. P~anel (b) shows ionic strength
dependence. Reactions were performed essentially as described in (a) except the
buffer was 95 mM Mes pH 6.5 rcmtAinin~ the indicated molar concentrations of
sodium chloride. APP cleavage in the absence (lanes 2 to 7) or presence (lanes 9to 14) of pool Y are shown for each co~ntrAti-n of sodium chloride. Lanes 1
and 8 contain Mr markers and C-100 standard respectively. The arrows indicate
the migration of the 11,14 and 18 kDa APP fragments.
29
wo 95/13084 2 1 7 5 5 6 4 PCT/US94/07043 ~
Figure 18 depicts inhibitor s~l~.l;vily of the pool Y protease. Reactions
were initiated at 37C by enzyme addition to achieve the following initial
component concentrations in a 16 Ill volume: pool Y # 3-5 (14 ~Lg/ml) after
p1lrifi~Atinn on a superdex 75 column, APP (35 nM), in 30 mM Mes buffer pH 6.5.
Reactions were terminated by addition of 7.5 Ill of 3X sample buffer.
Immunoblots were developed using the rabbit antiserum to the APP C-terminal
domain according to Example 8. Data are shown for the effect of the following
inhihitor~- Panel (a) 860 IlM PMSF in methanol (lane 4), 400 IlM pepstatin in
methanol (lane 6), 5 mM bPn7Am;-linP (lane 8), 350 IlM E-64 (lane 9), 7.7 mM
EDTA (lane 10),15 ,uM aprotinin (lane 11), and 0.1% (w/v) deoxycholate (lane 15).
The following controls were also run- no inhibitor (lane 12), ethanol (lane 3),
methanol (lane 5), pool Y only (lane 2), APP only at time zero (lane 13) and 4 hr
(lane 14). Lanes 1 and 7 respectively show p~ ed Mr markers and the C-100
standard. Panel (b) 1.8 IlM alpha-1-antichymotrypsin (lane 2),156 ~LM TLCK (lane3), 46 IlM chymotrypsin inhibitor I (lane 4), 119 IlM chymotrypsin inhibitor 11
(lane 5), 4 IlM alpha-2-antiplasmin (lane 6), 51 ~LM alpha-l-dnLilly~ (lane 7), 98
IlM chymostatin administered in DMSO (lane 10), 153 IlM mPthAnoli~ TPCK
(lane 12). Controls included: no inhibitor (lane 8), DMSO ~lane 11), and
methanol (lane 13). Pre-stained molecular weight markers and C-100 standards
were applied to lanes 1 and 9 respectively. The arrows indicate the migration ofthe 11,14 and 18 kDa APP flp~rArlAti~7n products.
Figure 19 shows 1) that neither DMSO nor DMSO plus 10 uM pepstatin A
effect growth of HEK 293 cells (parlel A), 2) conditioned media harvested from
late ~og phase cultures treated with DMSO plus pepstatin A shows ci~nifi~ntly
lower levels of a 15 kDa APP C-terminal fragment than cultures treated with
DMSO only (panel B). Panel A. HEK 293 cells (ATCC CRL 1573, adapted for
suspension culture) were seeded (1 X 105 /ml final) in roller bottles ~ontAinin~400 ml of MoAb medium (JRH Ri~ iPnrPC, Lenexa, Kansas) plus 0.2 % v/v fetal
calf serum (closed squares). Additional rollers also contained 0.01% v/v DMSO
(open squares) or 0.01% DMSO pl~ 10 uM pepstatin A (closed diamonds). Cell
growth was at 37C in 5 % CO2/95% air. Viable cell numbers in trypan blue
treated samples were quantified with a hemocytometer, and were a constant
percentage (60%) with time. ~on~litil-nPd medium was harvested at the end of
log phase (located by the arrow at day 7), centrifuged at 1500 RPM in a Beckman
Gs-6 bench top centrifuge and subjected to chromatography on columns of
immobilized anti-beta amyloid monoclonal antibody (C286.8A).
~ WO95/13084 2 1 75~4 PCTIUS94107043
Panel B. Immunoblot with Rabbit anti-APP C-terminal antiserum. Lane 1,
prestained mnle~ Ar weight markers; lanes 2-7 inclusive are the respective
analyses of fractions 1-6 inclusive from the pl~rifirAtinn of medium from DMSO
treated cells; lanes 9-11 inclusive are the ~ e-Liv~ analyses of fractions 1-6
inclusive from the pllrifirAtinn of medium from the DMSO/pepstatin A treated
cells. Lane 8 contains C-100 standard (example 5, from PMTI 100), and lane 15
contains recombinant holo-APP695 purified according to example 7, method 2.
Note that fractions 5 and 6 from the DMSO or~y treatment contain a 15 kDa band
that is absent from the corresponding fractions in the DMSO/pepstatin A
treatment. Further details are given in the text to example 12.
Figure 20 s. r^~ri7~C the purification frornL human brain of an aspartic
protease with APP processing activity. a) Elution profile for the pl]rifi(-Atinn of
.~nlllhili7.o~ P-2 fraction (140 mg protein) on a mono-Q HR 10/10 column. Protein
concentration (open circles), ~nnrlllrtAn~ (hatched line) and rates of fnrmAtinn of
N-dansyl-ISE from N-dansyl-ISEVKMDAEFR-NH~ in the absence (closed circles)
or presence (open triangles) of 10 uM pepstatin A, are shown for each collected
fraction. E-V cleaving activity was completely blocked by 10 IlM pepstatin A.
Activities are expressed as the % of the s,ubstrate converted to product by eachfraction. b) APP695-hydrolyzing activity elutes in the same conductance range asthe E-V cleaving peptidase activity in a). Arrows locate fragments of the
indicated sizes (kDa). Cleavage of APP695 was performed with fractions: 5 (lane
2); 11 (lane 3);15 (lane 4); 21 to 26 (lanes 5 to 10); 30 to 36 (lanes 11 to 17).
Incubation of APP695 alone for 0 (lane 18) or 24 h (lane 19) or each of the column
fractions without APP695 (not shown) yielded no fragments. Cleavage by
purified CD (2 ~Lg/ml) was also performed (lane 1). An amount of incubation
mixture containing 0.95 llg of APP695 was applied to each lane. Fractions pooledfor further study are indicated in a).
APP Processing. 95 llg/ml of homogeneous holo-APP 695 (prepared
according to Example 7, method 2) was incubated with 10 Ill of each column
fraction in a total of 15 ~ nntAinin~ 40 mM in each of acetate, Mes and tris pH
4.0, 0.002 % (v/v) triton X-100 and 30 mM exogenous NaCl. Reactions were
t~rminAted after 24 h by addition of SDS-PAGE sample buffer to lX final,
electrophoresed on 10-20 % acrylamide gradient Tricine gels (Novex) at 100 V
constant and then ele~ bl~ d onto Problott membrane (Applied Biosystems).
31
wo 95113084 2 ~ 7 5 5 6 ~ PCI'/US94/07043
Blots were developed with mon()clf)n~l antibody C286.8A at 50 llg/ml final by
standard sandwich procedures using appropriate second antibodies coupled to
alkaline phosphatase.
Peptid~ce: Aliquots of column fractions (20 lal), were added to 10 111 of
reaction mixture to achieve the following initial component concentrations:
synthetic N-dansyl-ISEVKMDAEFR-NH2 (58 IlM), captopril (0.2 mM), methanol
(0.3 v/v) and when included pepstatin A (10 IlM) in methanol, in a cocktail
buffer ~Ulllpl;Dillg 50 mM sodium acetate pH 5.. Reactions were ~ .1 after
24 hr by addition of 10 ~1 of 40 ~LM pepstatin A. Enzymic products were detectedand ql-~ntifi.ot1 by RP-HPLC (Example 2).
Figure 21 shows that immobilized antibodies to human cathepsin D
selectively remove the human brain APP degrading activity from solution. a)
A280 nm elution profiles for the control (solid line) and anti-cathepsin D
(hatched line) columns. Numbered arrow heads refer to 1: initiation of loading;
2, wash with equilibration buffer; 3, elution with 100 mM glycine pH 2.2; 4,
elution with 50 mM ~ th~nnlAmir~ hydrochloride pH 11; 5, elution with 100
mM glycine, 0.5% (v/v) triton X-100. b) APP processing activities are shown for
selected void fractions (1 to 27) from the anti-CD (+) or control (-), as well as for
the applysate (Q-pool), purified cathepsin D (CD) or APP alone. No APP
hydrolyzing activity was detected beyond fractions 40. c) immllnnhlnt reactivityof flow through fractions concentrated 21 fold or pooled glycine/triton eluent
(fractions 80-89, ~ulL~ la~d 8-fold) towards a polydonal antibody to cathepsin
D. Cul~ la~iull was by p~ a~iul~ with 10 % (v/v) TCA.
('hrclm~t~grâ~hy: Control and anti-cathepsin D rabbit antisera were
purified by avid AL chromatography as described (T. T. Ngo et al., 1992,
Chromatograp1ly, 597: 101), and coupled to CnBr activated sepharose 4B
(Pharmacia) by standard methods ( R. Axen et al., 1967, Nature, 214:1302). Equalamounts (4.1 mg protein in 44 ml of 110 mM sodium bicarbonate pH 8.1
c--ntAinin~ 100 mM NaCI) of the pooled fractions from Figure 21b that contained
APP processing activity were applied in paralled to identical sized columns (4.2ml resin) of either immobilized anti-cathepsin D IgG, or immobilized control
IgG. The columns were each consecutively washed with 28 ml of 100 mM
NaHCO3 pH 8.3 containing 500 ml NaCI, 28 ml of 100 mM glycine pH 2.5, 40 ml
of 50 mM fli~th~nl-l~mine hydrochloride pH 11, and finally 30 ml of 100 mM
~ wo 95/13084 2 1 7 5 5 6 4 PC~S94~07043
glycine pH æs ~ g 0.5% (v/v) triton X-100. Fractions recovered in acid or
base were n~lltrAli7l~d with tris. Chr--mAtcgr;~rhy was performed at a flow rate of
0.5 ml/min and 2 ml fractions were collected throughout. All operations were at
4c.
-
APP prot ~ccin~g Homogeneous holo-APP 695 (31 ~Lg/ml final) was
incuba~ed with 5 1ll of each column fraction in a total of 15 ~LI containing 40 mM
in each of sodium acetate, Mes and Tris adjusted to pH 4.0, 40 mM NaCl, and
0.002 % (v/v) exogenous triton X-100. Reactions were 1.~ )A~ after 24 hr by
addition of SDS-PAGE sample buffer, and analyzed by immunoblot developed
with the monoclonal antibody 286.8A. Arrows locate the product fragments.
Highly pure human cathepsin D was present in enzymic incubations at 1.27
g/ml final.
Figure 22 shows immobilized antibodies to cathespin D adsorb a human
brain peptidase that degrades APP mimetic peptides. a) and b), respective
peptidase activities in the flow through fractions and pooled glycine/triton
eluent pool (#80-89, figure 21a, u~ a~d 21 fold) in the degradation of N-
dansyl-ISEVKMDAEFR-NH2. c) and d) corresponding data for N-dansyl-
ISEVNLDAEFR-NH2 hydrolysis. In a) E-V hydrolyzing activity (circles) of flow
through fractions is shown. In c) L-D hydrolysis is shown. Activities are
depicted from fractions recovered from columns rontA;nin~ coupled control
(closed symbols) or anti-cathepsin D IgG (open symbols). Triangles show effect of
10 !lM pepstatin A on amount of ul-~ul-Lluv~ d substrate remaining at the end
of in~l-hati,-nc In b) and d) activities were measured in the absence (open
his~u~;ldllls) or presence (closed l~i~uy,ldllls) of pepstatin A.
~ 5~h~.: Reactions were initiated at 37C by addition of column
fraction (20 1ll) to achieve the following initial component ul~ ldLions in 30
111: peptide substrate (58 IlM), captopril (0.28 mM), methanol (0.1 % v/v) in a
cocktail buffer comprising 40 mM final in each of acetate, Mes and Tris pH 5Ø
When included, pepstatin was at 10 ~LM final. Reactions were terminated after 20hr by addition of 10 IlM final pepstatin A and subject to C-10, RP-HPLC analysisaccording to Example æ
Figure 23 shows an updated summary of sequence ACcignm~n~ of beta-
amyloid fragments formed from the hydrolysis of APP 6g5 by cathepsin D made
33
WO 95113084 2 T 7 5 5 6 4 PCTIUS94/07043 ~
since initial ~cci~nr-- " reported in Table 5. a) N-terminal sequences of BAPP-
derived fragments relative to C286.8A immunoreactivity on immunoblot
(immllnr~blot lanes taken from Figure 12c: Lane 1, BAPP 695 only; Lane 2, BAPP
695 plus CD. Arrows connect immunoblot bands with fragments identified in
corresponding segments of a sequencing blot that were of a sequence and size
sufficient to contain the C286.8A epitope. Lower case letters denote uncertain
sequence ~c~i~nm~ntc APP 695 amino acid numbering is according to Kang et
al., 1987, N~ture, 325: 733. b) BAPP 695 frA~m.ont~tion pattern, including all
recovered fr~m~nt~ that did (hatched) or did not (open) contain the C286.8A
epitope. The length of each fragment is drawn in proportion to the estimated
number of residues per fragment, calculated from the indicated fragment sizes
(from SDS-PAGE) and an assumed average residue mass of 110. Potential effects
of glycosylation on some fragment sizes is not taken into account. The position
of BAP in mature BAPP 695 (shaded segment) is also indicated. In b) the bonds
hydrolyzed ~ ul~ded to BAPP6gs residues: 122-123 (F-V); 303-304 (Y-L); 405-
406 (L-Q); 459-460 (L-R); 532-533 (L-P); 549-550 (F-G); 593-594 (E-V).
Method: Reactions were performed as described in Figure 12.
Segments of the r~)nm~ccif~ stained electroblot that contained fragments specific
to the complete incubation or that co-migrated with CD-dependent
immunoreactive bands in a parallel immunoblot were excised and then
sequenced on an Applied Biosystems model 477A protein sequencer in the gas
phase. Eighty percent of the ~APP was hydrolyzed to smaller fragments. An
amount of BAPP equivalent to that yielding 12.7 pmol of detectable N-terminal
sequence was applied to the gel.
Figure Z4 shows the effect of the ~NL mutation on the hydrolysis of
BAPP695 by cathepsin D. Reactions were initiated at 37C to achieve component
".d~ions of: highly pure human cathepsin D (10 llg/ml), purified ~APP695
or BAPP695~NL (84 llg/ml, expressed according to example 4 method 2 and
purified according to example 7), in 40 mM of each of sodium acetate, Mes and
tris pH 5.0 f~nt~inin~ 0.128% triton X-100. Aliquots (20 111) were removed and
frozen at -80C until analyzed for immunoblot reactivity against the C286.8A
monoclonal antibody according to materials and methods. Immunoreactive
BAPP695 hydrolysis products formed at 0 (lane 1), 5 (lane 2) and 20 hr (lane 3) and
BAPP695~NL hydrolysis products formed at 0 (lane 6), 5 (lane 7) and 20 hr (lane
8) are shown. Incubation of BAPP695 (lane 4) or BAPP695~NL (lane 9) without
34
wo 95/13084 2 1 7 5 5 6 4 PCTtUSg4tO7043
CD for 20 hr is shown. The migration of recombinant C-100 (~APP596-695,expressing using PMTI-100 according to example 5) is shown in lane 5. Arrows
indicate the migration of the 10-12 kDa and 5.5 kDa APP C-terminal fragments.
DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in further detail with reference to the
following non-limiting examples.
Example 1. Human Brain Protease Isolatior~
Two general approaches were taken for protease isolation. In the initial
studies brain protease activities were isolated as described under "Method 1",
below. Cha~ lion of the resultant enzyme activities obtained from ion-
exchange chromatography is described in Examples 3 and 8 and lead to the
i~lPntifir~tion of six different activities which were able to degrade recombinant
CHO cell derived APP (Example 8) but were relatively inactive as peptidases
(Example 3).
One of the six activities was subsequently i~Pn~ifiPd as cathepsin D
(Example 9), and was further ~llala.~ d according to its chromatography on
gel filtration.
In an alternate approach to attempt to affinity purify some of the humân
brain serine proteases described in Example 8 (Table 4), "Method 2" WâS
imrlPmPnte~l This procedure was based on the affinity purification of serine
proteases using aprotinin sepharose at an early step, and lead to the i~lPntifi~ti~n
of a serine protease(s) (Example 10), which also exhibited the capacity for APP C-
terminal processing.
Method 1:
(i) Sub-cellular fractionation. Sections of human frontal cortex
pole 9 region (4.5 g) from four separate, age-matched AD patients were weighed
wo gsll3084 2 1 7 ~ 5 6 4 PCTIUS94107043
out while frozen (-70C), added to 150 m~ of 0.32 M sucrose at 4C and scissor
minced. The suspension was h--m--g~ni7~1 in batches using a lOQ ml Elvehjem
glass teflon potter (lQ return strokes). The combined homogenate was
centrifuged (1000 g x 10 min) in a Sorval SS-34 rotor.
The loose pellet was removed, re-homogenized and centrifuged as
described above. The supernatant for each extraction was combined and
centrifuged at 15,000 g x 30 min in the Sorval SS-34 rotor. The resultant "P-2"
pellet was ~u~ ded in 100 ml of ice cold 0.32 M sucrose by vortexing and
stored at -70C. The supernatant from the last spin was centrifuged at 105,000 g x
60 min to yield the su~ alal-l or soluble fractions ("S"), and the microsomal
fraction ("M") which was resuspended in 60 ml of 0.32 M sucrose. Both S and M
were stored at -70C.
Table 1
Summary of protein l~u~..;es:
Fraction Volume Control AD
(ml) (mg) (mg)
Soluble (S) 250.0 315.0 472.0
P-2 pellet 100.0 436.0 412.0
Microsomal (M) 60.0 105.0 89.4
(ii~ Solubilization. The membranous control or AD subfractions (P-
2 or M) were solubilized by adjusting to the following conditions: 2% (v/v)
Triton X-100 rl~nt~inin~ 50 mM Tris HCI buffer, pH 7.5. After stirring at 4C for
3.5 hrs, the suspensions were centrifuged at 105,000 g x 60 min in a Beckman 70
Ti rotor. The following final protein ~ul~llllalions were used in soluhili7~til-n,
for P-2 (3.9 to 4.0 mg/ml); and for M (1.4 to 1.6 mg/ml). Solubilized su~ ldLdn~were stored at -70C for later use. The soluble fraction was not treated with
detergent but rather was adjusted to 50 mM in Tris HCI, pH 7.5, by the addition of
stock 1 M buffer.
(iii) lon-exchange chromatography. Chromatography was performed
36
WO 95/13084 2 1 7 5 5 6 4 PCT/US94J0~043
using a Gilson gradient liquid chromatograph (model 305 and 306 pumps)
equipped with a 50 ml Rheodyne stainless steel loop injector model 7125, and
connected to a Mono-Q HR 10/10 column (Pharmacia, Piscataway, NJ).
Absorbance of column effluent was monitored at 280 nm using a Pharmacia UV-
M detector and a Kippen-zonen chart recorder.
Protein fractions of P-2, microsomal (M), or soluble (S) were loaded onto
the column and equilibrated with 50 mM Tris HCl, pH 7.5 (conductivity 1.8 mU
at 4C) at a flow rate of 2 ml/min. The column was then washed with
equilibration buffer until the A280 nm in the eluent decreased to zero
whereupon the column flow rate was increased to 4 ml/min.
Proteins were eluted as follows:
Solvents: A = 50 mM Tris HCI pH 7.5
B = 50 mM Tris HCl pH 7.5, containing 1 M NaC1
Program: 0 - 50% B over 70 min
hold 50% B for 10 min
50-100% B over 10 min
hold 100% B for 10 min
re-equilibrate
Four milliliter fractions were collected throughout chromatography. Thefollowing protein loads were applied per column run: P-2, 97 mg (control) and
95 mg (AD); S, 50 mg (control) and 68 mg (AD); and M, 36 mg (control) and 31 mg
(AD).
In the initial studies, eluted fractions were monitored for A280 nm, total
protein (Bradford assay), and peptidase activity (as described in Examples 2 and 3).
Pools made on the basis of peptidase activity were then prepared (Example 3) andthen tested for their capacity to process CHO cell derived APP C-terminally
(described in Example 8).
In all further studies however eluted fractions were also individually
tested for their capacity for C-terminal processing of ~ a-l~ APP derived by
baculo virus directed expression (Example 9).
WO 95/13084 PCTIUS94/07043
2 l 75564
(iv) Gel filtration chron~to~rarhy. A typical example of gel filtrations
is depicted in Figure 3. More generally, chromatography was performed as
described below. Mono-Q fractions from the pllrifi~Ation of P-2, and ~VllLdi~ g
APP C-terminal ~IV~b~ g activity were pooled, ~:ol~ laL~d to less than 0.25 ml
and applied to a tandem ~ of two Superose 6HR columns equilibrated
with 10 mM Tris HCI buffer pH 7.5 and cc ntAinin~ 150 mM NaCI. A flow rate of
0.3 ml/min was used throughout. Fractions were monitored for A280 nm, total
protein (Bradford assay), peptidase activity, and activity for C-terminal processing
of recombinant APP.
Method 2:
(i) Sub-cellular fr~rtic~-tion This was performed essentially as
described in Method 1, above.
(ii) Solubilization. This was pPrformPcl essentially as described in
Method 1, above.
(iii) Aprotinin s~,ha~ e chromalo~;.al,l.y. Soluble (230 mg), P-2
(216 mg) or microsomal fraction (47 mg) described above was applied to a column
of aprotinin sepharose (Sigma, catalog # 42268, 1.5 x 10 cm), previously
equilibrated with 20 mM Tris HCI buffer pH 7Ø Once loaded the column was
washed with equilibration buffer (100 ml~), and then eluted with 60 ml of 50 mM
sodium acetate buffer FH 5.0 rr)n~Ainin~ 500 mM sodium chloride. The flow rate
was 1.0 ml/min throughout. Eluted fractions (4 ml) were Illo~ d at 280 nm,
analyzed using the Bradford protein assay, and examined for APP C-terminal
processing activity as described in Example 8, using recombinant APP derived by
expression in a baculo virus system. Active fractions were capable of forming 11,
14 and 18 kDa (approx.) fragments which were detectable on immunoblots using
an anti-APP C-terminal antibody (see Example 6 for method of antibody
production).
(iv) lon-exchange .I~ y. Active fractions from the
purification of the P-2 fraction on aprotinin-sepharose were pooled, dialyzed
against 50 mM Tris-HCI pH 7.5 and applied to a mono-Q column (HR 5/5),
38
wo 95/13084 PCTIUSg4107043
~1 7';5~4
previously equilibrated with dialysis buffer. Once loaded, the column was elutedessentially as described in Method 1, above. Active fractions (2 ml), were
monitored for A 280 nm, total protein (Bradford assay), and for their capacity for
baculo virus derived APP C-terminal processing. A broad peak of APP-rlPgrA~iin~
activity was observed, which was capable of forming 11,14 and 18 kDa APP C-
terminal fragments which reacted with both the APP C-terminal antibody as well
as a mr-nn~ n~l antibody directed to the N-terminus of the beta-amyloid peptide
(see Example 6 for antibody production).
Based on the A 280 nm profile across the region ~ont~inin~ active
fractions, three pools of activity were prepared each overlapping with a
distinctive A 280 nm peak. The pools comprised the following conductance
ranges: pool X (12.2 to 14.4 mmho), pool Y (14.9 to 18.9 mmho) and pool Z (20.2
to 22.9 mmho).
(v) Gel filtration .I~ o~ -hy. Pools X, Y and Z were each
concentrated to either 2 ml (pool X and Y) or 3.6 ml (pool Z) and separately
applied to a Superdex 75 column (Pharmacia, Piscataway, NJ) previously
equilibrated with 50 mM Tris-HCI pH 7.5, containing 150 mM sodium chloride.
Once loaded, the column was eluted with equilibration buffer. Chromatography
was performed at a flow rate of 1.0 ml/min throughput. Fractions (1 ml) were
monitored for A 280 nm, total protein (Bradford assay) and for C-terminal
processing of baculo expressed APP. The gel filtration was calibrated by
~hlull~dlo~,laphy of each of the following standard proteins: Thyroglobulin (670kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17.5
kDa) and vitamin Bl2 (1.35 kDa).
Example 2. Peptidase Assay D~.lo~ ..L
A peptidase assay was developed to enable the high throughput detection
of endoproteases in human brain tissues which might possess a specificity
appropriate for APP hydrolysis at the junction between the "extracellular"
domain(s) and the N-terminus of the beta-amyloid peptide region. The
technology selected utilized dansylated peptide substrates, in conjunction with
subsequent detection of fluorescent peptide products by RP-HPLC separation, and
post column fluorescent detection.
39
WO 95113084 2 1 7 5 5 6 4 PCT/US94/07043
Evolution of peptide substrate sequence: A fluorescently labelled
dodeca-peptide substrate containing the same amino acid sequence as observed
surrounding the N-terminal region of the beta-amyloid peptide sequence of
human APP was prepared by solid phase peptide synthesis. Peptides were
synthesized on an Applied Biosystems model 430A peptide synthesizer using
Fmoc/NMP-HoBt chemistry (Fields et al., 1990, Int. J. Peptide Protein Res., 3~:
161; Knorr et al., 1989, Tetrahedron Letters, 30: 1927). Usually, the peptides were
cleaved and deprotected in 90/o trifluoroacetic acid, 4% thio~nicnl~, 2%
ethanedithiol, and 4% liquefied phenol for 2 h at room temperature.
However the peptide: N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-
Phe-Arg-His (SEQ ID NO: 2) was found to undergo unwanted carboxy peptidase
digestion when incubated with crude tissue fractions. To attenuate carboxy
peptidase digestion, the following, modified substrates were designed: N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ ID NO: 7) and N-
dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp
(SEQ ID NO: 1).
This latter peptide was relatively ills~.,siliv~ to carboxy peptidase digestion
even in the presence of crude tissue fractions and was used in the peptidase
profiling studies of Example 3. Tlhe C-terminal alpha amide substrate (SEQ ID
NO: 7) was used in the peptidase studies of Example 9 using more purified
enzyme fractions. The degradation of either of the peptides was Illol~i~oltd using
the HPLC protocol of Example 3, below.
xample 3. D~t~rmin~ion of Peptidase Activities in Subfractions of Normal-
Control and AD Brai~s
(i) Tnrl~ha~inn~ Aliquots (20 !ll) of column fractions
described in Example 1 were incubated with 10 Ill of a reaction mixture so as toachieve the following component concentrations: N-dansyl-Ile-Ser-Glu-Val-Lys-
Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) (50 IlM),
captopril (300 IlM), in a cocktail buffer comprising 100 mM in each of: MES, Tris,
and acetate, pH 6.5.
Tm llh~tinn with ion-exchange fractions was performed at 37C for 24 hrs,
WO 95/13084 2 ~ 7 5 5 6 4 PCTiUS94~07043
after which reactions were t~rminAt~d by adjusting to 3% (v/v) final in TFA.
(ii) HPLC q-~-ntifi~.~'inn of proteolytic products. HPLC analysis was
performed using a Hewlet-Packard HP1090 complete with binary solvent
delivery, heated column compartment, and auto injector. Fluorescence
detection (post column) was performed with an in-line Gilson model 121 filter
fluorometer (excitation at 310-410 nm, emission at 480-520 nm) in conjunction
with an HPLC chem-station (DOS series) and suitable software for data analysis.
Aliquots (usually 10 1ll) of the above acidified incubation mixtures were
injected onto a Hypersil 5 IlM C18 column (100 x 4.6 mm) fitted with a guard C185 ~LM guard (20 x 4.6 mm). Isocratic s~ald~io-l was achieved using 100 mM
sodium acetate buffer, pH 6.5, cnntAinin~ 27% (v/v) Ar~tl~nitri~l~ T~ ntifirAtil~n
and ~ a~isoll with the migration of synthetic peptide products, the structure
of which were (~i)nfirm~l by PTC-amino acid analysis and FAB-MS (See Table 2,
below).
Table 2
HPLC retention times of known synthetic peptide standards:
Peptide HPLC retention time Cleavage Site
(N-dansyl-) (minutes)
ISEVKMDAEFRHDDDD 2.228 + 0.024 Substrate
ISEVKM 5.398 + 0.019 M-D
ISEVK 3.413 + 0.004 K-M
ISEV 2.692 + 0.003 V-K
ISE 7135 + 0.002 E-V
IS ~ 4.142 + 0.019 S-E
Also note, the retention time of certain metabolites listed in Table 2,
above, differ to those quoted in Example 2 for the cleavage of N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His (SEQ ID NO: 2) due to variables in
the HPLC set up. For example, the studies which reflect data listed in Table 2
have relatively longer retention times because chromatography was performed
41
WO 95/13084 PCT/US94/07043 ~
~1 75~64
using a guard column in line with the HPLC column.
In all experiments the HPLC column was calibrated for day to day
variation in the retention times of the enzymatically generated products by
analysis of synthetic product standards in parallel with the experimental
samples. Data for the proteolytic metabolite profile of individual ion-exchange
fractions was collected using the HP CHEM station data ~rqllicition software.
The area under the curve for each of the six cleavage products and their
retention times are stored in a peak table file. All peak tables are collated and
transferred to a (6 x n) area array in EXCEL (using a custom utility written in
pro~rAmmin~ language C) where n = the number of Mono-Q fractions. Each row
of the array represents a single peptidase analysis from a Mono-Q fraction. Zeros
are inserted between each column of data to artificially establish a gradient ofvalues in the row direction.
SpyGlass takes this array and transforms it into a three--lim~ncionAl
surface in which Mono-Q fractiorl number, cleavage site and area % for the
product formed are the three axes. Contours are defined according to the
following criteria set manually within the SpyGlass Program: the first contour
line connects contiguous regions of the plot where 1.5% substrate conversion to
the particular product was observed. Similarly successive contour lines
rr~nnr-rtin~ regions of 5%, 10%, 20%, 30%, 40% and 50%, substrate conversion
were also displayed. The resulting contour plots represent brain peptidase maps
in which fraction numbers span the ordinate, and peptide bond cleavage sites areon the abscissa, and the amount of product formed is represented by the
contours.
Results of peptidase profiling of control and AD brain subfractions: Figure
1 shows a comparison of the peptidase profiles obtained for the cleavage of the
N-dansyl peptide substrate by both control and AD P-2, S and M fractions
subjected to further subfractionation by ion-exchange chromatography. The
analysis enables the i~l~ntifi~Ation of a high number of potentially different
peptidase activities throughout the subfractions of control and AD brain.
For each analysis (a to f in Figure 1), the amount of activity for cleavage at
each peptide bond decreased through the series: V-K > K-M > M-D > S-E > E-V,
42
WO 95/13084 2 1 7 5 5 S 4 PCTIUS941071)43
however the K-M and M-D cleavages are of greatest interest because of their
greater likl-lih~Q:l of l~lul~s~ g the site of APP hydrolysis leading to C-100
formation. The metabolite recovered at 1.6 min is probably due to m~thioninP
oxidation to the sulfoxide in the substrate, as treatment of substrate with
hydrogen peroxide generates a product of the same retention time (rt). The
mf~t~ht~lit~c formed with rt at 1.0 and 1.3 and 3.3 min are llniri~ntif~ at present.
For the K-M cleavage, overall levels of activity both in terms of the
spectrum of enzymes and peak activity both for control and AD descended
through the series P-2 > S = M. This was also true for the M-D cleavages in AD,
whereas for the control fractions the order was S > P-Z = M.
Regarding the most abundant peptidase peaks found in AD (only those
bounded by more than one contour line), the following number of obviously
resolved peptidase peaks could be ~ic~riminAt~fl P-2, three K-M peaks and one
M-D peak; M, three K-M peaks and two M-D peaks; S, no K-M peaks and one M-
D peak. Qualitative .UlllUdlisulls between the levels of the more abundant peaksbetween corresponding control and AD subfractions revealed only one notable
difference. The difference was observed in the microsomal fractions wherein the
control M profile contained a single M-D product around fraction 75, whereas in
the AD profile the same region clearly contained a doublet.
Because of potential variation between peptidase levels in the normal and
disease state populations it is of little point to highlight UUdlLLilaLiV~ di
in peptidase levels between control and AD.
In summary while it cannot be discounted that there may be qualitative or
ci~nifilAnt quantitative differences between control and AD fractians in the
levels of minor peptidase forms, the overall profiles looked quite similar with
only one obvious qualitative difference being apparent amongst the more
abundant peaks of peptidase activity.
(iii) Consolidation of peptidase activities into discrete pools. Based
upon the peptidase activity profiles obtained using the N-dansyl-Ile-Ser-Glu-Val-
Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) substrate,
column fractions from the ion-exchange separation of P-2, S and M fractions
were consolidated into contiguous pools (12 pools for M, 13 pools for P-2, and 14
43
WO 95/13084 2 1 7 5 5 6 4 PCINS94/07043 ~1
pools for S). Care was taken in each case to pool the same regions of the
chromatography profile both for AD and control fractions (even if no protease
was detectable in the corresponding control region), and the precision of the
process was checked by monitoring the conductivity of each pool using a YSI
model 35 conductivity meter. -.
Fractions were mAintAin~d at 4C throughout pooling and then stored at -
70C. Each peptidase pool was concentrated using an Amicon Centriprep-10
membrane. Prior to ~ . "-1 1 " ~ ; "~, each Centriprep membrane was washed in 50mM Tris/HC1, pH 7.5. A 15 ml Centriprep was used for peptidase pools that
contained a volume of 5 ml or more. These pools were concentrated on a Du
Pont Sorval tabletop centrifuge at 2700 rpm for approximately 40 min. Pools thatcontained less than 4 ml were concentrated with a 2 ml Centriprep on a Du Pont
Sorval RC centrifuge (SS34 rotor) at 5000 rpm for 40 min.
After concentrating, each concentrated pool was washed with an equal
volume of 50 mM Tris/HCl, pH 7.5. Pools were mAintAin~d at 4C throughout
the concentrating process and stored at -70C. The pools are listed below in Table
3 with the conductivities and the final ~nrf~ntrAt;t~n volumes.
44
21 7556~
WO95/13084 PcrluS94107043
Table 3
Pooling and concentration of ion-exchange fractions from the separation of
human brain S, M and P2 sub-fractions.
-
Pool ~'r~n~ t:~,nr~ Control AD
(mmho) (ml) fold conc (ml) fold
conc
S I 1.6 0.75 16 0.75 16
S II 1.6 0.75 16 0.75 16
s m 5.0-6.8 1.50 13 0.50 48
S IV 7.1-7.8 0.75 16 0.75 16
S V 8.0-8.4 0.60 13 0.60 13
S VI 8.6-8.8 1.00 16 1.00 8
S VII 9.2-10.8 1.25 10 1.00 24
s vm 11.1-11.6 1.00 16 0.80 15
S l,Y 12.8-13.3 1.50 13 1.25 10
S X 15.1-15.8 1.00 12 1.25 13
S XI 20.2 0.75 5 0.75 5
S XII 26.1 0.75 5 1.00 4
s xm 33.1 0.75 5 0.80 5
S XIV 38.4 0.75 5 0.60 7
P2 I 1.6 13.00 4 6.00 9
P2 II 1.6 lD0 24 1.00 28
P2 m 1.6 1.00 36 0.60 53
P2 IV 1.6 1.00 46 2.00 24
P2 V 1.6 1.00 32 0.50 64
P2 VI 1.6-1.9 1.00 20 0.50 40
P2 VII 2.5-5.2 1.00 56 0.50 72
WOg~/13084 PcrlUss4/07043 ~
21 7 5564
Table 3 (con~in~
Pool ('nnrillrt~,nrr Con~rol AD
(mmho) (ml) fold conc (ml) fold
conc
P2 vm 5.5-7.7 1.00 16 0.60 53
P2 IX 8.0-8.6 0.50 16 050 24
P2 X 9.1-10.0 1.00 16 0.80 20
P2 Xl 10.1-13.9 6.00 10 17.00 4
P2 Xll 14.1-17.0 6.00 9 8.00 6
P2 xm 17.1-20.8 1.00 64 11.50 5
M I1.6 2.00 10 1.00 28
M 11 1.8 0.60 25 0.80 15
M m1.8 0.50 8 0.40 10
M IV 1.8 0.50 8 0.50 8
M V9.7-10.2 0.80 5 0.50 24
M VI 10.5 0.50 8 0.50 8
M Vll 10.9-14.5 1.50 40 1.00 56
M Vlll 15.2-15.8 0.80 19 0.80 15
M IX 16.1-16.5 0.80 15 0.80 15
MX20.7 0.50 8 0.50 8
M XI 34.5 0.50 8 0.50 8
MXII 377 0.5~ 1~ 050
46
WO95/13084 2 1 7 55 6 4 PCTlUsg4/07043
Example 4. EA~I~ )r of Recombinant APP 695
This example describes the method for expressing holo-APP 695 which
was then purified as described in Example 7 and then used as the recombinant
substrate for the APP degradation assay described in Example 8 through 10. Two
approaches were used.
Initially, a CHO cell expression system was used to generate APP 695.
Experiments using this source of APP as a substrate included the initial activity
measurements which led to the i~l~ntifi~;~ti/~n of six different protease activities
detectable in contiguous pools of mono-Q fractions from the purification of
human brain P-2, S and M fractions. These studies are described in Example 8.
Subsequently, reromhin~nt APP 695 was obtained by ~A~ aivll using a
baculovirus directed system. The greater amounts of APP 695 thereby generated
made it feasible to perform the more detailed studies outlined in Examples 9 and10, leading to the ifi~ntifi~Ation of certain APP ~ r~in~ enzymes.
Both methods of expression are described below:
ethod 1: Development of a Chinese Hamster Ovary (CHO) cell line
~A~ sil~g holo-APP 695
(i) Vector ~ ,slluclion. A known 2.36 Kb NruI/SpeI fragment of
APP 695 cDNA from FC-4 (Kang et al., supra) was filled in by the large fragment
of E. coli DNA polymerase I and blunt~nd inserted into the SmaI cloning site of
KS Bluescript M13~ (Strahgene Cloning Systems, La Jolla, CA) creating pMTI-5
(APP 695 under the T3 promoter). A new optimal Kozak consensus DNA
sequence was then created using site-specific mutagenesis (Kunkel et al., 1987,
Metl~ods in En~ymology, 154: 367) with the oligo-
5'-CTCTAGAACTAGTGGGTCGACACGATGCTGCCCGG~TTG-3' (SEQ ID NO: 8)
to create PMTI-39. This plasmid was next altered by site specific mutagenesis
(Kunkel et al., Id.) to change the valine at position 614 to a ~ tAm~ (open
reading frame numbering according to Kang et al., Id.) to create PMTI 77.
The full length APP cDNA containing the optimal Kozak consensus
sequence and Val to Glu mutation was then cut out of PMTI-77 with NotI and a
47
WO 95/13084 PCT/US94/07043
21 75564
Hindm partial digest. The 2.36 Kb APP 695 fragment was then gel purified and
ligated into NotI/HindlII cut pcNAINeo (In~ritrogen Corp., San Diego, CA) to
create PMTI 82 in which the APP 695 expression is placed under the control of
the CMV promoter. The Val to Glu mutation was sequence rr~nfirmr-cl and the
vector used to stably transform CH~ cells. -~
(ii) Generation of stable CHO cell lines expressing APP 695 mutenes.
Chinese Hamster Ovary K-1 cells (ATCC CCL 61) were used for ~ r~ , with
the APP 695 construct. Twenty micrograms of an expression plasmid rr~nhinin~
APP 695 and a neomycin drug resistance marker was transfected into 1 x 107 CHO
cells in 0.5 ml PBS by electroporation using a Bio-Rad Gene Apparatus (Bio-Rad
Laboratories, Rirhmr~nrl, CA). A single pulse of 1200 V at 25 llf capacitance was
A,1",;"i~ d to the cells.
Following electroporation, cells were incubated in ice for 10 minutes and
collected by centrifugation. The cell pellet was resuspended in Alpha MEM, 10%
fetal calf serum at a density of 5 x 104 cells/ml, and 1 ml aliquots were distributed
into each well of five 24-well tissue culture cluster plates. After 48 hours
incubation, cells rr~ntAinil~ the neomycin drug resistance marker were selected
by addition of l ml of media containing 1 mg/ml Geneticin (GIBCO-BRL, Grand
Island, NY) and incubation was continued and bi-weekly changes of drug
containing media.
Drug resistant cells were tested for APP 695 expression by Western blotting.
Cells positive for APP 695 expression were cloned by limiting dilution, and
individual clones were isolated and tested for APP 695 expression. A clone
positive for APP 695 expression was subcultured and expanded into roller bottlesfor large scale production of APP 695 expressing cells and subsequent isolation of
recombinant protein.
ethod 2: Expression of Recombinant Holo-APP 695 and Holo-APP 695~NL
using Baculovirus Infected Insect Cells
(i) Construction of Recombinant Vector. For wild type APP695
expression, the Baculovirus vector pVL1392 (Invitrogen) was cut Xba I (in the
polylinker) and ligated with the gel isolated 2.36 Kb Xba I fragment from pMTI-39
(APP 695 NruI/SpeI into KS Bluescript M13+ SmaI site, T3 r~rirntAtir~n, with a
48
~, WO 9S/13084 2 1 7 ~ 5 6 4 PCTIUS94J07043
new Kozak and Xba I site at the SmaI/NruI blunt fusion site). This created pMTI-103, which was ~ldl,srulllled into DH5, selected on Amp, and a lithium prep of
the plasmid DNA made for trAnqf.qrtir)n.
- For ~APP695ANL vector construction, ~APP695 cDNA sequences were
amplified by PCR using the sense primer (5'-agg aga tct ctg aag tga atc tag atg cag-
~ 3') (SEQ ID NO.: 10) and the antisense primer (5'-cat gaa gca tcc ccc atc gat tct taa
agc-3') (SEQ ID NO.: 11) to generate a 0.7 kb ~APP cDNA fragment encoding the
595K, 596M to NL mutation. The PCR fragment was digested with Bgl II/ClaI
and inserted into a vector rontAinin~ the full ~ength 13APP695 which had been
pre-cut with the same enzymes. A 2.5 kb XmaI/SpeI fragment from this vector
~ ntAinin~ full length ~APP695~NT was then inserted into the XmaI/XbaI sites
of baculovirus expression vector pVL1393 (Invitrogen).
(ii) Cells and Virus. Spodoptera fru~iperda (Sf9) cells, purchased from
the American Type Culture Collection (ATCC) were grown as suspension
cultures at 28C in TNMFH media (Summers, M.D., and Smith, G.E., 1987, A
Manual of Methods for Baculov~rus Vectors and Insect Cell Procedures, Bulletin
no. 1555, Texas Agric. Exp. Stn. and Texas A ~ M Univ., University Station, TX)
rontAinin~ 10% fetal calf serum. Wild-type AcMNPV DNA was purchased from
Il~v;llu~ , San Diego, CA.
(iii) DNA Transfection andl Plaque Assays. Foreign DNA was inserted
into the genome of AcMNPV at the polyhedrin gene locus by homologous
recombination by cotrAnsf~ ti~n of purified plasmid DNA (4 llg) and linear viralDNA (1 Irg) into Sf9 cells using the calcium phosphate procedure (Summers et
al., 1987, supr~). Viruses which were released by the transfected cells were
purified by 2 rounds of plaque assay (Summers et al., 1987, supra), where
recombinant viruses were identified by visually screening for polyhedrin-
negative plaques. Purified recombinant viral cultures were subsequently
screened for their ability to produce APP in infected cells by Western blot
analysis.
(iv) Recombinant Protein Production. 5 liter batches of Sf9 cells, grown
as suspension cultures in TMNFH media ( ,-ntAin;n~ 10% fetal calf serum at 1 x
106 cells/ml, were infected with recombinant virus at a M.O.I. of 1. Cel~s were
harvested 24 hours post infection and cell Iysates prepared for purification of
49
WO 95tl30B4 2 1 7 5 ~ 6 4 PCI/US94/07043
recombinant protein.
xample5: Development of Expression Vectors for the Production of
Recombinant C-100 Stamdard by Transient Infection of ~ ~ n
Cells ~~
The C-100 peptide fragment contains the C-terminal portion of APP which
spans from the N-terminus of the A4 peptide to the C-terminus of full length
APP (see above, BACKGROUND section). The C-100 fragment is the purported
initial ~l~gr~rl~ti~n product leading to the ultimate formation of the A4 peptide.
In one embodiment of the present invention, cell Iysates from Hela S3
cells (ATCC CCL 2.2) expressing recombinant C-100 are analyzed in the
innnnlln-)klr~t assay in parallel with tl~e recombinant APP samples that have been
incubated with brain fractions, sub-fractionated by Mono-Q chromatography (See
Example 3). The migration and detection of the C-100 fragments serves both as a
size marker for the genesis of products formed by pathologic proteases as well as
a positive control for the immunodetection of C-terminal APP fragments in
general.
ifi."~ of the size of enzymatically generated products with the size
of the C-100 fragment can provide insights into whether OI not the enzymaticallygenerated fragments result from cleavage close to the N-terminus of the A4
peptide or alternatively within the A4 segment as would be catalyzed by
secretase.
(i) Plasmid construction. Two methods were used to make plasmids
for C-100 e;~ a~iol~ Each plasrnid shall be identified separately as either PMTI73 or PMTI 100.
PMTI 73 construction: The commercially available plasmid PUC-19 was
digested with EcoRI to eliminate its polylinkers. Commercially available PWE16
was then inserted into the digested PUC-19 to create PMTI 2300. PMTI 2301 was
derived from PMTI 2300 following BamHI/Hind III digestion using an
~ligonll~ otide adapter. The EcoRI promoter fragment of APP was inserted into
the Hindm site of PMTI 2301 by blunt end ligation to produce PMTI 2307.
WO 95/13084 ~ ~ 7 5 5 6 4 PCTIIJS94107043
PMTI 2311 was generated by ligating the BamHT fragment from PC-4 (Kang
et al., supra) into the BamHI site of PMTI 2307. The XhoI fragment from FC-4
was inserted into the XhoI site of PMTI 2311 to generate PMTI 2312. PMTI 2323
was generated by insertion of the 2.2 kb BglII/EcoRI fragment from the EcoRI
genomic clone of the mouse m~tAllothi~ninf~-I gene into the ClaI site of PMTI
2312. To generate minigene PMTI 2337, the sequences between the KpnI and
BglII sites of PMTI 2323 were deleted and the clone was ligated using synthetic
oligonucleotide adaptor, sp-spacer-A4.
PMTI 2337 was cut with Bam H1/SpeI and the fragment ligated into the
Bam H1/Xbal restriction sites of Bluescript KS (+) (Stratagene) to create PMTI
2371. PMTI 2371 was cut FIindm/NotI to release a 0.7 kb fragment coding for the
terminal 100 amino acids of APP 695. Also encoded was the sequence of signal
peptide. This insert was ligated into the ~indIII/NotI site of pcDNAINEO
(Invitrogen Corp.) to create the plasmid PMTI 73.
PMTI 100 CemstruLtirn KS bluescript, (M13+) ront~inin~ full length
BAPP under the T7 promoter (PMTI 74) was cut Bgl II, filled in, then cut Eco RV
and religated to generate PMTI 90 (~t-nt~inin~ the entire C-100 segment of BAPP
without a signal peptide. PMTI 90 was cut XbaI/HindIII to release a 0.6 kb
fragment again coding for the terminal 100 amino acids of APP 695 and this was
ligated to the XbaI/Hindm site of pcDNAINEO to create PMTI 100. In each case
vectors, inserts and plasmids were purified by methods known to those skilled inthe art.
(ii~ Tr.9ncf~ction and expression of C-100 fr~m~nt Preparation for
small scale exlJL~iulL of C-100 standard was initiated by seeding 5 x 10~ cell Hela
S1 cells in each well of a 6 well costar cluster (3.5 cm diameter) 24 hours before
use.
Sufficient vaccinia virus vTF7-3 was trypsin treated to infect at a
multiplicity of 20 plaque forming units per cell, mixing an equal volume of
crude virus stock and 0.25 mg/ml trypsin, then vortexed vigorously. The trypsin
treated virus was incubated at 37C for 30 minutes, with vortexing at 10 minute
intervals. Where clumps persisted, the incubation mixture was chilled to 0C
and sonicated for 30 seconds in a coni~tin~ water bath. The chilled sonication
was repeated until no more clumps were detected.
51
2 1 7 5 5 6 4 PCTIUS94107043 ~
The trypsin treated virus was then diluted with sufficient serum free
DMEM for each well with Hela S1 cells to have 0.5 ml of virus. Medium was
aspirated away, then the cells were infected with virus for 30 minutes, with
rocking at 10 minute intervals to distribute the virus. .
Approximately 5 minutes before infection was ceased, fresh tr~ncf~-ti~-n
mixture was prepared as follows: To each well was added 0.015 ml lipofection
reagent (Bethesda Research Labs, Gaithersburg, MD) to 1 ml OPTIMUM
(Bethesda Research Labs, GdiLll~lD~" MD) in a polystyrene tube, mixing gently.
Vortex was avoided. Then, 3 llg CsCl purified DNA was added and mixed gently.
Virus mixture was aspirated from cells, then the trAncf.~ti~n solution was
introduced. The resulting mixture was incubated for three hours at 37C. Each
well was then overlaid with 1 ml of OPTIMUM and incubated at 37C in a CO2
incubator overnight.
Cells were harvested at 20 l~ours post transfection by centrifugation, and
lysates were prepared on ice with the addition of 0.2 ml of a lysis buffer whichcontained 1% Triton X-100, 10 llg/ml BPTI, 10 llg/ml Leupeptin, 200 mM NaCl,
10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mM EDTA, adjusted to pH 7.5.
Complete lysis was monitored by light microscopy, and harvested imm~tliAtf~ly~
Lysis took less than 1 minute to complete, with delay at this step causing lysis of
nuclei resulting in a gelatinous mass.
Recombinant Iysates were stored at -20C for later use. Preferably,
recombinant lysates should be diluted 1:50, in (3X) SDS-PAGE sample buffer
which is devoid of 2-mercaptoethanol prior to freezing.
A comparison of the size of the proteins produced by expression with
either PMTI 73 or PMTI 100 using SDS-PAGE/immunoblot with the APP C-
terminal antibody was performed (Figure 2e). The study showed that PMTI 100
directed the expression of a single immunoreactive band, whereas, PMTI 73
directed the expression of two major bands of similar molecular size. A less
intense band of intermediate size was also evident in PMTI 73 when applied to
gels in higher amounts (Figures 2a-2d).
52
~ WO95/13084 2 1 75~64 PC~IUS94107043
App~ication of the PMTI 100 protein to SDS-PAGE gels in higher amounts
results in the appearance of a series of fainter bands (e.g., Figure 2d). Besides an
intense band of C-100 monomer of apparent Mr 117 kDa, fainter bands are
observed at Mr 25.5 kDa, 35 kDa and 45 kDa, which are attributed to the
frlrm~tir~n of dimeric, trimeric and tPtrAmPrir aggregates, ~ Livelyr of the C-
100 monomer. An ~lflitir,n~l faint band of Mr 18.9 kDa is also observed. SimilarphPn~mPnA have been reported in the literature with similar il~ ations
(Dyrks et al., 1988, EMBO J., 7: 949).
The largest of the three bands produced by PMTI 73 was slightly larger than
the single band observed with PMTI 100. Amino acid sequence analysis of the
largest band from PMTI 73 expression showed that the signal peptide sequence
was cleaved from the initial translation product to yield a C-100 fragment
~r~nt;:~ining, 5 extra amino acids at the N-terminus.
Example 6: Plud~clio.~ ûf 1..~.l,..,1~. I.~l` ir~l Reagents
Three different immunorhPmir~l reagents were used in the studies of the
present invention:
(i) a rabbit polyclonal antiserum which recognized the C-terminus of
APP was obtained and used for immunoblot detection of C-terminal APP
fragments generated by proteolytic processing according to the assay rr,n~liti(~ns
described in Example 8;
(ii) an affinity purified antibody which rPrr,gni7P~ the C-terminus of
APP was prepared and used to synthesize an i"""~ r~r;,-,ly cc~lumn for the
affinity p11rifir~tif~n of APP expressed in a baculo virus directed system (see
Example 7); and
(iii) a mouse monoclonal antibody which l~u~ es the N-terminus of
the beta-amyloid peptide was generated and used in an immunoblot assay to
determine whether C-terminal APP fragments generated by proteolytic digestion
of holo-APP 695 contained the full length beta-amyloid peptide (see Examples 9
and 10 for specific applications).
The method of generation of each of the three immunochemicals is
53
WO 95/13084 PCTNS94/07043 ~
2~ 75564
present below:
(i) Rabbit polyclonal antiserum to the C-terminus of APP. Antisera
were elicited to the C-terminal domain of human APP 695, and were prepared in
accordance with the method as described in Buxbaum et al., 1990, Proc Natl.
Acad. Sci., ~7: 6003. A synthetic peptide (hereinafter ",B APP 645-694")
s~ollding to the COOH-terminal region of APP 695 was obtained from the
Yale University, Protein and Nucleic Acid Chemistry Facility, New Haven, CT.
,~ APP 645-694 was used to immllni7e rabbits to elicit polyclonal antibodies.
Sera were screened by immllnohl~-t analysis of Iysates of ~. coli that expressed a
fusion protein including the amino acids 19 through 695 of human APP 695.
Sera which were immunoreactive against the recombinant fusion protein were
further screened for immunoprecipitating activity against [35S] methionine-
labeled APP 695, which was produced from ~ APP 645-694 cDNA by successive irl
vitro transcription (kit pu~ ased from Stratagene, La Jolla, CA) and translation(reticulocyte Iysate kit purchased from Promega Corp., Madison, WI).
(ii) Polyclonal antibody a:~finity column for the purification of holo-
APP.
pl~lrifi~Ation of Sy~lthetic APP C-t~rminAl Peptide Jmm~lnog~n: 80-90 mg
of crude synthetic peptide (P-142) spanning the C-terminus of APP (649-695) witha cysteine residue at the N-terminus was purified by HPLC (yield 42%; 34 mg).
Amino acid analysis, N-terminal sequence analysis and Laser Desorption Time
of Flight Mass Spectrometry showed the purified peptide to be a mixture of full
length and N-terminally truncated peptides (2/1 full length to truncated).
lmmuni7Ation of pAhbits with pllrified P-142 jmmlln~en: The HPLC
purified peptide APP (649-695) was used to immllni71~ rabbits. Two rabbits each
received an initial challenge with 125 Irg of peptide in complete Freund's
Adjuvant follo--~ed by subsequent boosts of the same amount of peptide in
incomplete Freund's Adjuvant at three week intervals. Fourteen bleeds were
collected over a 9 month interval and optimal production of Ab was observed for
bleeds at 16 thru 32 weeks (shown by Western analysis with Vaccinia C100 and
CHO APP). Bleeds in this interval were pooled for an approximate volume of
90-100 mls of antisera.
54
WO 95/13084 2 ~ 7 5 5 6 4 PCTIUS94/07043
Pre~aration of an immnh;li7~1 APP 649-695 affinity mAtrix for ~urifirAtinn
of Antic-ora: 9.7 mg of purified peptide APP (649-695) was coupled to mAl.~imi~l~
activated BSA using the Pierce Imject activated Immunogen Conjugation kit
with BSA. About 40% of the peptide (3.88 mg) was coupled to BSA as
d~ ed by Ellman's Reagent. The BSA coupled peptide was separated from
uncoupled peptide by gel filtration (p1lrifi~Ation buffer from kit = 83 mM
NaH2PO4 pH 7.2; 900 mM NaCI). The pooled void volume from the gel
filtration column (2.9 mg P-142 conjugated to BSA/12 5 mls) was coupled to 1 gm
(3.5 mls) of CNBr activated sepharose (>90% peptide conjugate coupled by
standard Pharmacia protocol). R-~mainin~ sites were blocked with
ethAnnlAmin~ The sepharose affinity matrix was packed into a 1.0 x 35 cm glass
column.
Purifi~Atinn of pAhbit polyclonal Antihody ll~j~ the APP(649-695) affinity
~Q~m~l- The combined rabbit anti-sera from bleeds of optimal Ab production
were pooled (100 mls/3.9 gms protein) and diluted 1:1 (v/v) with wash buffer
(100 mM NaHCO3 pH8.3; 750 mM NaCl) and loaded onto the peptide affinity
column at 1.0 ml/min at 4C. After loading (200 mls), the column was washed
with wash buffer (75 ml) until A280 returned to zero~ The IgG was eluted with
100 mM Glycine pH 2.5 (40 ml). One minute fractions were collected into tubes
~nntAinin~ 100 111 of 1.0 M Tris HCl pH 8Ø The nl~lltrAli7~d low pH IgG eluantwas pooled (34 mls; 14.7 mg) and dialyzed against 1.0 Iiters of 100 mM NaHCO3
pH8.3; 500 mM NaCl at 4C.
PrepAration of Tmmllnoaffinity cnlllmn Coupling pllrified RAhbit IgG to
~ephArose. 5.0 gms of CnBr Sepharose was activated with 50 mls of coupling
buffer (100 mM NaHCO3 pH 8.3; 500 mM NaCl) and mixed with dialyzed IgG
pool on an orbitron for 21 hrs at 4C. After coupling, the resin was rinsed lX
with coupling buffer through a sintered glass filter, followed by 3X rinses with100 ml ea of blocking buffer (100 mM NaHCO3 pH8.3; 500 mM NaCl; 1.0 M
.thAnnlAmine). Two successive ~ L~ rinse steps with coupling buffer
(100 mls), then low pH buffer (100 mM NaOAc pH 4.0; 500 mM NaCl (100 ml)
and a final rinse with coupling buffer (100 rnl) completes the resin preparation.
The coupling was 87% for a total of 17.5 mls of resin. (0.727 mg IgG/ml resin).
(iii) Generation and epitope mapping of a monoclonal antibody to the
WO 95/13084 2 1 7 5 5 6 4 PCT/IJS94107043
beta-amyloid peptide.
HybridomA Methr~dology. Balb/c mice were immllni7rd by multiple
injections of a mixture of the following two synthetic peptides: 1) APP amino
acids 597 to 638 of holo-APP 695 (numbering according to Kang et al., I~.)
rr~ntAinin~ beta amyloid, and 2) APP 295 amino acids 645-695 containing the C-
terminal domain. Splenoytes from immllni7~d animals were fused with
X63/Ag 8.653 mouse myeloma cells using standard procedures (IIel ellbel~, et al.,
1978, In: D.M. Weir (Ed.), Handbook of Experimeniol Immlmology, pp 25.1-25.7,
Blackwell Scientific pllhlirAtinn~, Oxford, UK). S~-~èll,alcl-~ from the resultant
hybrids were tested for the presence of anti-peptide specific antibodies using an
EIA in which the beta amyloid peptide immunogen was bound to the microtiter
plate. Cultures secreting antibody which reacted with the synthetic peptide usedas immunogen were cloned twice by limiting dilution, and their isotype
d~tl~rminrci as described (Wunderlich et al., 1992, J. of Immtlnol. Methods, 147:
1). Secreted IgG was purified from the serum free f~rmrntAtion broth of cloned
hybridoma cells by protein-A affinity chromatography of the spent culture fluid.
Epitope MAnping. One of the anti-peptide monoclonal antibodies, an IgG
2b tlrci~nAtr-~l C286.8A, gave good reactivity with synthetic beta-amyloid peptide
both by EIA as well as by immunoblot assay. The epitope reactivity of the
monoclonal was determined using a competitive EIA. Synthetic peptides
rr~ntAinin~ amino acids 597-612, 597-624, 597-638, 608-624, 621-631 and 645-695 of
human APP 695 (numbering according to Kang et al., I~;l ) as well as N-dansyl-Ile-
Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO:
1) were tested for the ability to block binding of C286.8A to APP 597-638.
Peptides which are recognized by the antibody will, if prr-inrllh-Atr-~l with
the antibody in solution, deplete the solution concentration of the antibody
available for subsequent reaction with beta-amyloid peptide bound to a
microtiter plate. The result of such an experiment is shown in Figure 4 and
described herein below.
Only peptides APP 597-612, 597-624~597-638, and N-dansyl-Ile-Ser-Glu-Val-
Lys-Met-Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID NO: 1) were able to
inhibit C286.8A binding in a dose-dependent fashion. These peptides contain
respectively amino acids 1-16, 1-28, 1-42 and 1-7 of the beta-amyloid sequence
~ WO 95/13084 2 1 7 5 5 6 4 PCT/US94)07043
(mlmh.orin~ from the N-terminal aspartate residue). Peptides devoid of any beta-amyloid sequence such as APP 645-695, or ~ont~inin~ the beta-amyloid peptide
sequence 12-28 or 25-35 (APP 608-624 and APP 621-631 respectively) did not
inhibit binding of the monoclonal antibody to the homologous antigen. These
results show that the reactive epitope for this monoclonal antibody resides at
least in part, in the first 7 amino acids of the A4 region of human APP, ie, APP597~01.
Example 7. pllrifil~tion of recombinant holo-~PP 6g5.
Initial studies of APP C-terminal processing were performed using
recombirlant APP 695 expressed in CHO cells as described in Example 4, above,
and purified as described by Method 1, below. CharA~t.~ri7.~til-n experiments
using this substrate are described in Example 8 below and led to the i~1~ntifi~ ti-~n
of six potentially different APP degrading enzymes capable of C-terminal
l~lV-~s~il,g.
Subsequently, a baculovirus expression system was developed (see
Example 4), providing higher APP levels than could be achieved with the CHO
expression system. The purification of holo-APP695 from the baculo virus
system is described in method 2 below. The purified baculo virus derived holo-
APP 695 was used to conduct the protease l,~,,.. I-~,,,.,.li(~n experiments described
in Examples 9 and 10 below.
All steps were performed at 0 to 4C unless indicated otherwise. Holo-APP
695 was detected by immunoblot analysis using an anti-human APP 695 C-
terminal antibody essentially as described in Example 8, below.
Method 1. ptlrifi~ n of holo-APP695 from a Stably Tl...~L~;t~ CHO Cell Line.
(a) Isolation of plasma membranes. Whole cell pellets (179 g) from
continuous culture of CHO cells in roller bottles (See Example 4) were collectedby centrifugation (1500 g X 5 min), and r~osllc~n~ d to a total volume of 600 mlin 50 mM tris-HCl buffer pH 8.0 cnnt~inin~ sodium chloride (30 mM),
m:~gn~cil1m chloride (1 mM), EDTA (10 mM), PMSF (200 ~Lg/ml), E-64 (42
llg/ml) and pepstatin (3.8 llg/ml). The cells were homogenized using a teflon
potter (10 return strokes), then layered (25 ml per centrifuge tube) onto 10 ml of
57
WO 95/13084 PCT/US94/07043 ~D
2~ 755~`4
homogenization buffer containing 41% sucrose and devoid of the protease
inhibitors EDTA, PMSF, E-64 and pepstatin. Following centrifugation (26,800
RPM X 60 min, in a Beckman SW-28 rotor, the int~rfAci~l layer was carefully
removed (approximately 150 ml in combined volume), diluted with an equal
volume of homogenization buffer (minus protease inhibitors), resuspended
with a teflon potter (3 return strokes), and recentrifuged as described above toyield a tightly packed pellet. The supernatant was decanted and the pellet
resuspended in 100 ml total volume with 50 mM tris HCI pH 8.0 (teflon potter 3
return strokes). Recentrifugation (50,000 RPM X 60 min in a Beckman 70 Ti
rotor), yielded a pellet which was resuspended to a total volume of 57 ml in 50
mM tris HCl, pH 8Ø
(b~ Solubilization of Plasma Membranes. Thirty seven milliliters of the
above resuspended CHO plasma m,~mhrAn,~ preparation were added sequentially
to a cocktail of protease inhibitors arld stock 20% (v/v) triton X-100 to achieve the
following component ~ dliOns EDTA (1 mM), E-64 (24 !lg/ml), PMSF (53
llg/ml), pepstatin A (11 llg/ml), and triton X-100 (2.2% v/v, final), in the
homogenization buffer (total solubilization volume of 45 ml) described above.
After gently rocking of the mixture at 4C for 30 min, the non-solubilized
material was removed by centrifugation (50,000 RPM X 40 min in a Beckman 70
Ti rotor). the supernatant containing solubilized holo-APP was filtered through
a 0.45 ~LM disc filter.
(c) Purification of solubilized holo-APP 695 by strong anion exchange
chroma~ y. The above supernatant containing holo-APP 695 was diluted
with an equal volume of distilled water and applied to a Mono-Q HE~ 10/10
column previously equilibrated with 20 mM tris-HCl buffer pH 8.0 c~ntAinin~
0.1% triton X-100. Once loaded the column was eluted in a linear gradient of 0 to
1 M NaCl contained within a total volume of 210 ml of equilibration buffer. The
flow rate was maintained at 3 ml/min throughout. Proteins eluting between a
conductivity range of 17 to 22 mmho (4C) contained the majority of
immunoreactive APP 695, and were combined and dialyzed for 4 hours versus
2L of 5 mM tris-HCl pH 8.0 l ont~inin~ 0.025% triton X-100, and clarified to
remove slight turbidity by centrifugation (26,800 x 60 min in a Beckman SW 28
rotor).
(d) Heparin agarose .1.ll ~to~rAphy. The clarified sample was applied to
58
Wo 95/13084 2 l 7 5 5 6 4 rcT~s94107043
.
a column of heparin agarose (15 x 1.6 cm) previously equilibrated with dialysis
buffer. Upon loading a light brown band formed within the top 1/3 of the
column. Once loaded, 5 min fractions were collected (a flow rate of 1 ml/min
was used throughout). The column was then eluted stepwise with 85 ml of
equilibration buffer in which the sodium chloride successively adjusted to the
following final ~. "~ ,,.l ;. " ,c 0, 150, 300, 600, and 2000 mM. The majority of the
imm~ln~ tectable holo-APP eluted at 600 mM NaCl, with the next quantitative
fraction being recovered at 300 mM. The APP recovered at 300 mM and 600 mM
I~aCl were collected s~d-dl~ly and stored in aliquots at -80C. The APP used in
the following studies were from the 300 m~ fraction. The yield of partially pureAPP from the 300 mM heparin agarose eluent was 5.5 ,ug (Bradford assay) per
gram of wet CHO cell pellet. The APP in the ~ dld~iUIl was judged to be about
25% pure based upon SDS PAGE analysis.
Method 2. Purification of Holo-APP695 and Holo-APP695~NL from
Recombinant Baculo Virus Infected Insect Cells.
(a) Solubilization of cell pellets. The cell pellets harvested from two 5L
r~""~,~I,.I;~n runs were combined (total 8.9 g of detectable protein), added to 160
ml of 0.32 M sucrose containing the following inhibitor: pepstatin A (25 llg/ml);
leupeptin (25 llg/ml); chymostatin (25 llg/ml); antipain (25 llg/ml); aprotinin (25
~Lg/ml), b,~n7ami-1in~ (4 mg/ml), PMSF (0.87 mg/ml), and EDTA (25 mM), and
homogenized by teflon potter (10 return strokes). The homogenate was
centrifuged (105,000 g X 1 h in a Beckman 70 Ti rotor) and the pellet was then
resuspended by teflon potter (10 return strokes) in 160 ml of 10 mM Tris-HCl
buffer pH 7.5 containing 0.5 M NaCI and the same inhibitors and rnnt-~ntrAti,,ncas listed above. After brief sonication (Branson Sonifier Cell, 2 min power level
4), Triton X-100 was then added to a final con~-ontrati--n of 5 % (v/Y), and thesuspension was gently stirred for 20 min at 4C. The mixture was centrifuged
(50,000 RPM X 60 min, in a Beckman Ti 70 rotor), and the first supernatant (574
mg of protein) carefully removed for heparin-agarose chromatography. The
pellet was resuspended by teflon potter (20 return strokes) in 160 ml of 10 mM
tris-HCl buffer pH 7.5 containing 0.5 M NaCl, and each of the inhibitors at the
concentrations listed above. S(~ hili7Atir)n with 5% (v/v) triton X-100, and
subsequent centrifugation was performed as described above to yield a second
solubilized supernatant (683 mg of protein).
59
2 1 75564
WO 9~/13084 PCTIUS94/07043
(b) Radial flow chromatography on heparin-agarose. Both of the
sup~ ala~ obtained above were purified separately on heparin agarose as
follows. The Su~ a~al~s were diluted by addition of purified water and lM
Tris pH 9.5 to a volume of 3.5 L, a ~on~ rtAnrf~ of 1.8 mmho, and a pH of 8.0, and
applied to a Superflow 250 column (Sepragen) ~t~ntAinin~ 250 ml of packed resin
and previously equilibrated with 5 mM Tris-HCI buffer pH 8.0 ~ i-ntAinin~ 0.1 %
triton X-100. Once loaded, the column was washed with 3L of equilibration
buffer and then eluted with equilibration buffer ~ontAinin~ 600 mM NaCl. A
flow rate of 30 ml/min was used throughout. Fractions (45 ml were monitored
for A 280 nm, total protein (Bradford assay), and the levels of immunoreactive
APP detected by immunoblot against the anti APP C-terminal antiserum of
example 6 i). Fractions containing ~i~nifirAnt APP were combined and subjected
to antibody affinity ~lu~ ..o~ y.
(c) Antibody affinity chro~ ' O , hy. The 600 mM elution pool from the
pilrifirAtion of the first (contAining 276 mg of protein) and second supernatant(contAinin~ 113 mg of protein) on heparin-agarose were combined, adjusted to
pH 8.3, and applied to an antibody affinity column (10.5 X 1.5 cm) comprising
affinity purified C-terminâl antibody coupled to sepharose as described in
example 6 ii), and previously equilibrated with 100 mM sodium bicarbonate
buffer pH 8.3 f l~ntAinin~ 500 mM NaCI, 0.1 % triton X-100. Chromatography was
performed at a flow rate of 1 ml/min lluuuoliuu~ Once loaded, the column was
washed with 70 ml of equilibration buffer, and then eluted with 50 ml of 100 mM
glycine, pH 2.4 ,-t)ntAinin~ 0.1% triton X-100. Fractions (5 ml) were collected into
0.5 ml each of ~M tris-HCl pH 8.0, and monitored for A280 nm, total protein
(Bradford assay), and the presence of imrnllno~i.otertAhl~ APP as above. Fractions
l-nntAinin~ significant APP were combined. The combined heparin agarose
eluent was cycled through the affinity purification procedure a total of five times.
The APP pool recovered from eac}l successive pllrifi~Ation was combined for a
total of 9 mg of APP.
(d) Strong anion exchange chromatography. Combined fractions from
antibody affinity chromatography (9.0 mg of protein) were applied to a mono-Q
HR 5/5 column previously equilibrated with 20 mM tris-HCl buffer pH 8.0
containing 0.025 % (v/v) triton X-100, and 150 mM NaCI. Once loaded, the
column was eluted with a linear 0.15 to lM NaCl gradient in a total of 70 ml. A
flow rate of 0.5 ml/min was used throughout. Eluted fractions ~ntAinin~
.
WO 95/13084 2 1 7 5 5 6 4 PCTIUS94107043
c;~nifir~nt imm11nr,rlPtectable APP were combined and stored in aliquot6 at -80C
until used.
BAPP (5.6 mg from two starting 5L fr-rmPnt~tir~n runs) migrated as a single
band on SDS-PAGE (Mr 110 kDa), had an amino acid rt~mrr,~iti~n that showed
86% agreement with the theoretical composition, and the expected N-terminus
(Leu-Glu-Val-Pro-Thr-Asp-Gly-Asn-Gly-Leu-) of the mature protein.
BAPP695~NL purification was essentially as described above. The final
preparation (0.3 mg from two starting fermenter runs) had a composition that
showed 70% agreement with the theoretical value and contained only one
r~ntAmin~nt on SDS-PAGE (Mr 64 kDa) which was not BAPP related. Both
forms of purified BAPP reacted on immunoblots with a rabbit polyclonal
antibody to the ~APP C-terminal domain.
Amino acid analysis was performed essentially as described elsewhere
(Dupont, D.R, Keim, P.S., Chui, A.H., Bello, R, Bozzini, M., and Wilson, K.J., "A
comprehensive approach to amino acid analysisn, in Techniques in Protein
Chemistry, ed. by Tony E. Hugli, Academic press, 284-294 (1989)). Samples were
hydrolyzed under argon in the vapor phase using 6N hydrochloric acid with
2.0% phenol at 160C for 2 h. Phenylthiocarbamoyl-amino acid analysis was
performed on an Applied Biosystems model 420A Derivitizer with on-line
model 130A Separation System and Nelson Analytical model 2600
Chromatography Software.
Example 8. The immunoblot assay for the ~etection of the degradation of APP
695 catalyzed by human brain protease subfractions.
(a) ;ncllh~ n with substrate APP
(i) 5 ~LI aliquots of ion-exchange fractions (obtained from steps as
described in Example 1) or concentrated pools of fractions (Example 3) are
inr1lh~tr~ for 24 hrS at 37C urith recombinant human APP 695 (10.75 1ll), whichwas adjusted to 140 mM final in MES buffer pH 6.5 by the addition of the
required amount of 2M stock buffer. The final buffer concentration in the
incubation was 95 mM, pH 6.5. During the incubation time, proteolytic
~lr-gr~rl~tir~n of some of the APP 695 occurs to yield lower Mr fragments.
61
WO95tl3084 2 1 75 PCTtUS94/07043
(ii) The proteolytic reaction was t~rminAtl~d by addition of 7.5 ~
of the following 3X Laemlie SDS-PAGE sample buffer: 1.5 M Tris HCl, pH 8.45,
~ nt~inin~ 36% (v/v) glycerol and 12% (v/v) SDS, 10% (v/v) 2-mercaptoethanol,
and trace bromophenol blue tracking dye. Samples were heated (100C X 87
min), and then cooled.
(b) SDS PAGE analysis:
The reaction mixtures (15 111) were applied to the wells of a 10 to 20%
acrylamide gradient Tricine gel (routinely a 1.0 mm thick, 15 well Novex precastgel, Novex Experimental Technology, San Diego, CA). The gel was run under
constant voltage conditions, and at 50 V until the sample enters the gel
wl~ u~oll the voltage was raised to 100 V. Electrophoresis was ~icrt-ntinIl~d
when the tracking dye reaches to within 0.5 cm of the gel bottom. The gels were
calibrated using prestained Mr markers ranging in Mr from 3 to 195 kDa
(Bethesda Research Laboratories, Gaithersburg, MD.). Ten microliters each of a
kit l nnt~inin~ high and low molecular weight markers were mixed with 10 1ll of
3X sample buffer, and treated as clescribed in section (a) (ii). The following
molecular weight marker proteins were present in the kit as pre-stained markers:Myosin H-chain (196 kDa); phosphorylase B (106 kDa); bovine serum albumin (71
kDa); ovalbumin (45.3 kDa); carbonic anhydrase (29.1 kDa); betalactoglobulin (18.1
kDa); Iysozyme (14.4 kDa); bovine trypsin inhibitor (5.8 kDa); and insulin A andB chains (3 kDa).
(c) Immunoblotting:
(i) The gel was then transferred to a mini trans-blot
electrophoresis cell (Biorad labs, Ri~hm~nll, CA.). Proteins were electro-blotted
onto a ProBlott (TM) membrane (Applied Biosystems, Foster City, CA.), for 1
hour at 100 V (constant), using the following transfer buffer m~int~inl~d at 4C:
20 mM Tris HCL buffer pH 8.5 containing 150 mM glycine and 20% (v/v)
methanol.
(ii) The ProBlott membrane was removed and placed in 15 ml of
blocking buffer of the following composition for 1 hour at room temperature: 5%
(w/v) non-fat dried milk in 10 mM Tris HC1 buffe~ pH 8.0 (ont~inin~ 150 mM
62
~ WO 95/1308~ 2 1 7 5 5 6 4 PCTIUS94107043
NaCI.
(d) rmm~lr~-d~t-~rti~ n of APP and C-terminal ~ r~ tiun products:
- The membrane was transferred to 15 ml of blocking buffer r~nt~inin~ a
1:1000 dilution of rabbit polyclonal antiserum elicited to a synthetic human APP695 C-terminal peptide immunogen and incubated at 4C overnight.
The membrane was rinsed with three successive 15 ml volumes of
blocking buffer with gentle shaking for 5 minutes. The membrane was then
~dl~sr~ d to 15 ml of blocking buffer ron~-Ainin~ a 1:1000 dilution of alkaline
phosphatase-coupled Goat anti-Rabbit IgG (Fisher Scientific, Pittsburgh, PA.),
and inrllh~tr-cl at room temperature for 90 minutes. The membrane was then
rinsed with three successive 15 ml volumes of blocking buffer with gentle
shaking for 10 minutes.
The membrane was next washed with three consecutive 15 ml volumes of
alkaline phosphatase buffer for 5 minutes each, ~Vlll~li~il`l~,. 100 mM Tris HCIpH 9.5, rontAinin~ 100 mM NaCI and 5 mM MgC12. The gel was next incubated
in the dark with 15 ml of 100 mM Tris HCI pH 9.5, r~nt~inin~ 100 mM NaCl, 5
mM MgC12 and 50 Ill of BCIP substrate (50 mg/ml, Promega, Madison, WI.) and
99 ~LI of NBT substrate (50 mg/ml, Promega). Tnrllh~til~n was rontinll~od until
there was no apparent further intrncifir~tifl~ of low Mr immunoreactive bands
(typically 3 hours at room ~ lp~l.,Lul~). The gel was then rinsed with deionizedwater and dried.
Analysis of the capacity of Mono-Q pools of 5l-1.r..- ~ d human AD cortex to
L"~,y ir illy degrade APP 695 to generate C-terminal ~ ,.. .. Ic
Each of the P-2, S and m pools described in Example 3 were subjected to the
immunoblot assay described above. The specificity of the immllnol~gic detection
method, in combination with the use of the authentic APP substrate molecule
provide a selective method to detect the activity of the APP degrading enzymes
- in ~vll~pal~l~iv~ly crude biologic extracts, avoiding the need to use highly purified
enzyme preparations. Thus, certain of the partially purified pools possessed a
proteolytic activity which was capable of formation of C-terminal APP fr~ml~ntc
in a time dependent manner. Representative examples of the immunoblot
wo 95/13084 2 1 7 5 5 6 4 PCI/US94107043 ~
analysis of human AD brain are shown in Figure 2 for the P-2 V (panel a), M III
(panel b) and S I (panel c), as well as for individual fractions prior to pooling of a
P-2 VII pool (panel d).
Time course e~ L~, for example as depicted in Figure 2f, for pool M
III showed that these fragments were not present in the substrate or enzyme
fractions at time 0. Furthermore, i". I~ of the substrate alone did not result
in their formation (for example see Figure 2a, lane 2, Figure 2d, lane 2, Figure 2f,
lane 8). The size range of the bands varied between Mr approximately 11.5 kDa
and 25 kDa, depending upon the enzyme fraction, but the number of different
products formed in the reactions were ~ul~ y,ly low. At pH 6.5, eight out of a
total of 39 AD pools were found to have such activities. The pools could be
~lisLiilguisl~ed from each other based upon i) brain sub-fraction, ii) ionic strength
of column elution, and iii) qualitative APP cleavage pattern.
Six selected pools (~si~n~trcl "M-III, M-VIII, S-I, S-III, P-2 V, and P-2 VII")
were found to contain si~nifir~nt APP degrading activity. Corresponding control
brain pools also contained some of the above activities, but it was not possible to
11etPrminr- whether the levels of the activities were different or not, between
control and AD pools. Each of tlle above six pools had an enzyme activity
capable of forming an 11. 5 kDa APP C-terminal fragment.
The proteolytic product of MR 11.5 kDa was of particular interest because
in further studies it was usually the major immuno-detectable C-terminal
product, and was found to co-migrate with a recombinant C-terminal fragment
of APP comprising an open readin~ frame that would start with the n-terminal
aspartate of the beta-amyloid peptide and extend to the C-terminus of the full
length molecule (the C-100 fragment). This co-migration is exemplified in
Figure 2d. The implication of this is that the 11.5 kDa band is the product of
endoproteolysis of APP at or near the N-terminus of the A4 region, and that the
above protease activities capable of forming this fragment might play a role in
vivo, in the genesis of amyloidogenic peptides. .
Figure 2d shows that at least in the case of P2 pool VII, the 11.5 kDa C-
terminal enzymatic product of APP proteolysis is capable of aggregation. In
addition to the appearance of the peptide band at Mr 11.5 kDa which rrlmi~r~tr~swith the PMTI 100 driven C-100 standard, and the 18 kDa fragment, there appear
64
WO 95113084 2 1 7 5 5 6 4 PCTIUS94107043
.
other bands at Mr 2g.3, 27.4 and 35.5 kDa. The 24.3 and 35.5 kDa bands are of a Mr
expected for dimers and trimers, respectively, of the C-100 fragment, and roughly
comigrate with the corresponding faint bands in the C-100 which are due to
a~,æl~dLiul. (see Example 5 for details).
.
Figures 2a-2c also help to show that the assay can be used to examine the
effect of classical protease inhibitors. For example, it is apparent from Figure 2a,
that P-2 V is inhibited partially by methanol and completely by ml~th~nnli~
pepstatin A, while M-III (Figure 2b) and S-I (Figure 2c) are both completely
inhibited by aprotinin and cystatin. Thus, the assay, in one embodiment, is
applied to the search for novel in vitro inhibitors of the APP degrading
enzymes. The potent compounds thereby i~l.ontifi~cl are tested for in vivo
efficacy using a suitable animal model such as a transgenic animal desiæ-ned to
(.)V~ SS APP or a beta-amyloid-~ont~inin~ fragment thereof.
Table 4, below, ~.""".~ , some of the properties of the six main pools of
APP degrading activity recovered from the Mono-Q fractions, including peptide
product sizes, apparent pH dependence for product fnrm;~tinn, and the effects ofidlly available protease inhibitors.
WO 95113084 PCTIUS94107043
2~ 7~64
Table 4
Properties of human AD brain fractions active in APP proteolysisl.
pool2 ~~rnrlllrtAnr~ Fragment Size3 Optimum
Trial4 Tnhihitclrc~
(mmho) (kDa) pH (pH 6.5)
M m 1.6 11.5 (6.5) A, B aprotinin
cystatin
>11.5 (5.0) - N.D.
S I 1.6 11.5 (6.5) A, B aprotinin
cystatin
>115 (6.5-8.0) A, B cystatin
P2 V 1.6 11.5 (6.5) A, B pepstatin A
aprotinin
18.0 (8.0) - N.D.
P2 VII 2.5-5.2 11.5 (6.5-8.0) A PMSF
>11.5 (6.5-8.0) A PMSF
18.0 (6.5-8.0) A,B N.I.
s m 5.0-6.8 11.5 (8.0) A N.I.
>11.5 (8.0) A N.I.
18.0 (8.0) A, B N.I.
M vm 15.0-16.0 11.5 (8.0) A,B N.I.
>lL5 (8.0) A, B N.I.
18.0 (8.0) B N.I.
IReactions were F.orformrcl as described in Example 8 using pools prepared
and ~ulL~ Lla~d according to Example 3.
2~ ~ " " .,, ~ ,..1 protease pools as defined in Example 3
3Specific C-terminal APP fragments (products) from proteolysis
4The inhibitors studied were:
Trial A: PMSF (0.8 mM), EDTA (7.7 mM), pepstatin A (400 IlM),
E-64 (260 ~M)
Trial B: EGTA (1 mM), cystatin (20 ,uM), captopril (300 IlM),
aprotinin (15 IlM), N-dansyl-Ile-Ser-Glu-Val-Lys-Met-
Asp-Ala-Glu-Phe-Arg-His-Asp-Asp-Asp-Asp (SEQ ID
NO: 1) (90 IlM)
66
WO 95/13084 2 1 ~ 5 5 ~ ~ PCTIUS94~07043
sCOmpounds causing complete inhibition are listed.
N.D. = not ~letl~rmin~.l due to low or inconsistent levels of activity; N.I. =
no inhibition observed
WO 95/13084 2 i 7 5 5 6 4 ~ PCT/US94107043 ~
Some of the activities possessed pH optima in the alkaline range, and were
unlikely to be due to the actions of Iysosomal cathepsins. This observation is
ci~nifi~nt because several investigators have reported that pathologic APP
processing is performed by protease within the endosomal-lysosomal pathway
(Cataldo et al., 1990, Proc. Natl. Acad. Sci USA, 87: 3861; Benowitz et al., 1989,
Experimental Neurology, 106: 237; Cole et al., 1989, Netlrochem Res, 14: 933).
Enzymes within this pathway would be expected to exhibit acidic pH optima.
Based on the available data, M-III and S-I are highly similar by all the listed
criteria, and probably represent the same enzyme cross ~ each of the
S and M fractions. It is probable, therefore, that human brain contains a
minimum of five different protease activities capable of fl~gr~l1in~ APP to yield a
11.5 kDa C-100-like product fragment.
Table 4 shows that some of tlle activities were insensitive to inhibition by
any of the inhibitors tested, and these enzymes may represent members of an
unusual group. The activities involved in the formation of 11.5 kDa C-100
fragments in M-III and S I, are either of serine or cysteine type, or represent
members of an unusual group. Alternatively, these fractions may contain both a
serine and cysteine protease with both enzymes playing an obligatory (sequential)
role in the production of C-100. P2 V contains both an aspartic protease activity
and a serine protease activity. P2 VII contains a serine protease activity basedupon its sensitivity toward PMSF. However, in subsequent studies pepstatin-
inhibitable activity was also noted, inriir~tin~ the co-localization in P2 of anaspartic protease along with the serine protease activity in Table 4. None of the
enzyme activities in S m or M vm were sensitive to any of the inhibitors tested.In no case was it possible to demonstrate inhibition of APP degradation by co-
incubation of the enzyme pool with the N-dansyl peptide substrate used in
Example 3.
Comparison of the recovery of APP activities (Example 8) with the
peptidase activities of the Mono-Q pools (Example 3 and Figure l) clearly shows
that there is little correlation between the two activities. Thus, the APP
r~-1in~ activities were largely contained in pools that exhibited comparatively
little peptidase activity. This suggests that the APP rl~r~rlin~ activities are poor
peptidases and may require an intact folded APP substrate for activity, or
68
~ WO 95/13084 2 l 7 5 5 ~ 4 PCTJUS94~07043
alternatively (but less likely) the peptides selected represent the wrong locus for
pathologic APP processing. Regardless, this finding explains why other
investigators have been unsuccessful in identifying common APP degrading
enzymes using assays based on peptide substrates.
From the above rr~nci~l~rAtit-n~, it is r~n~ cl that the present assay is of a
sufficient specificity to enable the isolation of specific APP ~ rA~1in~ enzymesfrom human brain.
In further studies, we have used the immunoblot assay to track the
recovery of the P-2 VII associated APPase. Work was focused on this pool
because it ~ s~l.L~d the most abundant of the six ~hArArt.~ri7,~d activities, and
because it generated C-terminal frAgTn~ntc that seemed to be amyloidic (Figure
2d). It represents the major activity recoverable from ion exchange separation of
the P-2 subfraction, and is eluted at a point in the gradient which did not coincide
with the main peaks of peptidase activity.
The P-2 VII fractions displaying APPase were pooled and subjected to size
exclusion chromatography on two tandem Superose 12 columns (Pharmacia).
Peptidase and APPase activities in the eluted fractions were analyzed (Figure 3a).
While the K-M cleavage activity seemed to overlap in part, the peak of M-D
activity once again did not coincide with the peak of APPase. Calibration of thechromatography against known molecular weight markers yielded a median Mr
apparent of 31.6 kDa with an ull~lLdil-Ly of plus or minus 6.5 kDa for the APPase
activity of the P-2 VII fractions (Figure 3b).
Example 9. Identification of Cathepsin D as an APP C-terminal processing
enzyme.
Having obtained greater quantities of holo-APP695 by using the
baculovirus expression system described in Example 4, Method 2, and
purification scheme of Example 7, Method 2, it became possible to track the
recovery of APP degrading enzymes in individual column fractions from the
purification of human brain enzymes, rather than assess the content of APP
~grA~in~ enzymes in pools of fractions made on the basis of peptidase activity
(as had been done in Example 8).
69
WO95~13084 2~75564 PCI/US9~1/07043 ~
The content of APP degrading enzyme activity is shown in Figure 5 for
individual mono-Q fractions from the purification of solubilized P-2 fraction
according to the method of Example 1. When compared with a similar analysis
of soluble and microsomal fractions subjected to Mono-Q chromatography, the
relative staining intensity for enzymatic C-terminal APP fragments was
ly greatest in the P-2 subfraction from Mono-Q. APP rlr-grArlin~ activity
in the P-2 was recovered from Mono-Q as two distinct migration peaks (A and B,
Figure 5).
Peak A eluted in the loading and low ionic strength wash, i.e. in a region
roughly corresponding to the recovery of ~P-2 V, seen in our initial studies (Table
4), whereas peak B overlapped with the pooled region in which P-2 VII activity
was previously observed (Table 4), and shown to comprise both serine and
aspartic protease activities. Similar sized degradation products were observed
with both the peak A and B activities at Mr approx. 28,18 and 14 and <11 kDa,
although the relative staining intensity of the 18 kDa band was much greater in
peak B than in peak A. Peak B was pooled and subjected to pllrifir~tinn on
superose 6HR as described in Example 1, Method 1. Eluted fractions contained
two qualitatively distinct types of activity which overlapped in their elution
profiles. The activity which produced an APP breakdown pattern most closely
resembling that observed with the original peak B fractions (figure 5) was
recovered in fractions 51 through 56 from gel filtration (figure 6 b and c),
consistent with an apparent Mr of 15 to 25 kDa. This elution peak was preceded
by elution of an activity which prerlnmin~ntly formed an 18 kDa breakdown
product, and is presumably catalyzed by a protease of larger Mr apparent. This
latter activity probably corresponds to the serine protease activity previously
described in the P-2 VII pool in Exarnple 8, Table 4. Active fractiorLs from the gel-
filtration pllrifir~tir)n of peak B (and within the 15-25 kDa Mr region) were tested
for inhibition by classical protease inhibitors (Figure 7). These studies r~nfirm~o ~
that peak B activity was largely catalyzed by an aspartic protease as determined by
quantitative inhibition by Pepstatin A.
Comparatively few human aspartic proteases are known. Those that have
been identified include, Cathepsins D and E, Renin, and pepsin. To test the
possibility that the activities that we observed might correspond to some of these
enzymes, commercial preparations of human Renin (Calbiochem, San Diego, CA
catalog # 553864), and human cathepsin D (human liver, Calbiochem, San Diego,
WO 95/13084 ;~ 1 7 ~ ~ ~ 4 PCTIUS9410~043
catalog # 219401) were examined for their capacity to enzymically degrade baculoderived holo-APP.
Human cathepsin D (catalog #219401, Calbiochem, San Diego, CA) was
electrophoretically hr)m--gl~nl~ous on SDS-PAGE developed with silver stain (Mr.
apparent 29 kDa under reducing conditions), exhibiting an amino acid
. composition which showed good (93%) agreement with the theoretical
composition of cathepsin D. All N-termini corresponded to cathepsin D, with
the major c~q~lf,nr~c present in ~q~limol~r amounts corresponding to the light
(GPIPEVLKNY) and heavy (GGVKVERQVF) chains of the mature protease.
Whereas renin was inactive (not shou~n), cathepsin D selectively cleaved
the APP so as to produce a similar pattern of C-terminal degradation products tothose observed with P-2 peak B (Figure 5) described above from Mono-Q. Thus,
commercial cathepsin D preparations degraded holo-APP in a time dependent
fashion to produce major C-terminal products of approximate Mr 18 and 28 kDa.
Inhibition of the activity by pepstatin A confirmed the involvement of cathepsinD in the reaction (Figure 8).
A commercial polyclonal antibody to human cathepsin D was obtained
(Dako Corp, Carpinteria, CA, catalog # A561), and found to be reactive toward
human cathepsin D on immunoblots, generating an immunoreactive band of
Mr 27-28 kDa. The antibody was used in an immunoblot assay to examine if
aL~ aphy fractions from the mono-Q pllrifi~Ati--n of either P-2, soluble or
microsomal fractions contained immunoreactive cathepsin D. The antibody did
not cross-react with human renin on immunoblots.
~ nifi~nt amounts of cathepsin D were observed in Mono-Q fractions of
the P-2 (Figure 20d) and soluble fractions (data not shown) that possessed APP
degrading activity. Interestingly, two chromatographically distinct peaks of
cathepsin D reactivity were coml~timl~c observed in the analysis of P-2 mono-Q
fractions each of which coincided with peaks A and B (not shown). The
immunoreactive aspartic protease, cathepsin D associated with peak A activity
coincided with the region in which P2 V of Example 8 had been previously
irif~ntifi~l This suggested that peaks A and B could be due to multiple forms ofcathepsin D. Multiple forms of cathepsin D have been described elsewhere and
attributed to differences in post-translational modification of a single gene
71
WO 95/13084 2 ~ 7 ~ 5 6 4 PCTIUS94/07043
product. Immunoblot analysis of gel-filtration, column fractions from the
further pl~rifirAtirm of P-2 peak B (Figure 5) showed the presence of a peak of
cathepsin D immunoreactivity exactly co-incident with the peak of APP
degrading activity (Figure 6B).
In addition to co-migrating with cathepsin D immunoreactivity and
~Irgr~1in~ APP similarly to cathepsin D, Peak B protease further purified by gelfiltration exhibited the same pH optima (between pH 4-5) and ionic strength
dependence as cathepsin D for formation of C-terminal APP degradation
products (Figure 9). Finally, the immunoreactive band observed in the P-2
fraction exhibited a similar pl (4-6) to that reported for cathepsin D u~hen
subjected to preparative IEF on Biorad Miniphor chromatography system (not
shown). Collectively, these data strongly support the fact that the pepstatin
sensitive APP protease activities observed following mono-Q fractionation of
human brain P-2 are due to the action of cathepsin D.
The peptidase activity of the peak B protease (purified on gel filtration)
and cathepsin D were then compared using the synthetic peptide N-Dansyl-Ile-
Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ ID NO: 7) as the substrate.
Both enzymes hydrolysed the peptide in a time dependent fashion albeit at quite
low rates. For both enzymes, the major cleavage was observed at the -Glu-Val-
bond and to a lesser extent at the -Met-Asp- bond (Figure 10). Note, that in
Figure 10, most of the Met-Asp cleavage product was further converted to the
Glu-Val product by the 24 hr. time point depicted. As expected, the peptidase
reactions catalyzed by Cathepsin D and the P-2 enzyme preparation were both
inhibited by pepstatin A.
Both enzymes exhibited acidic optima at pH 4 for the hydrolysis at the-
Glu-Val- bond (Figure 11). Hydrolysis at the -Met-Asp- bond also exhibited an
acidic optimum u~ith cathepsin D (< pH 3.0), but with the P-2 enzyme, two
optima were observed (at pH 3.0 and pH 7.0), possibly due to participation of anadditional rl~nt~min~t;n~ P-2 protease in the reaction with a neutral pH
optimum (Figure 11). Cathepsin D usually hydrolyses between hydrophobic
residues. However at acidic pH values, ~lulul~aL~d (neutral) forms of the Asp
and Glu side chains might appear sufficiently hydrophûbic to satisfy the subsitebinding re4ui~ of the protease. The pKa of the Asp side chain is more
acidic than the Glu residue, and would be protonated to a lesser degree than the
72
~ WO95/13084 21 7556.4 PcrlUss4107043
Glu residue throughout the pH range examined in Figure 11. This may exp~ain
the lower cleavage rates at the -Met-Asp- bond with cathepsin D, and the hint at a
lower pH optimum for cleavage (< pH 3) at this site when compared with the-
Glu-Val- bond.
Further studies using monoclonal antibody C286.8A in the immunoblot
assay ronfirmP~l that the P-2 enzyme activity was due to cathepsin D. In our
more recent studies, this activity was recovered as a single peak eluted over a
fairly narrow number of fractions early in the salt gradient from ion-exchange
chromatography of a solubilized P-2 fraction (Figure 20a). The activity
hydrolyzed APP 695 in vitro to form fragments ranging in size from 10 to 38 kDa
(Figure 20b), all of which reacted to a murine monoclonal antibody (C286.8A)
that recognizes the first seven N-terminal residues of beta-amyloid (Figure 4).
The activity of the pooled fractions was inhibited completely by 10 IlM pepstatin
A, an aspartic protease inhibitor, but was unaffected by inhibitors of other
protease classes such as EDTA (1 mM), PMSF (0.4 mM), E-64 (0.1 mM), and
aprotinin (10 ~Lg/ml) (not shown). The APP-degrading activity (Figure 20b)
coincided with the elution of a pepstatin sensitive protease which hydrolyzed the
APP mimetic N-dansyl-ISEVKMDAEFR-NH2 at the -E-V- bond (Figure 20a).
Cathepsin D co-eluted with the holo-APP and peptide degrading activities as
judged by immunoblot detection of the 20 kDa cathepsin D light chain.
Hlghly pure human cathepsin D degraded holo-APP to produce a pattern
of C286.8A-immunoreactive products (Figure 20b and 21b) indistinguishable
from those observed with the P-2 aspartic protease activity pooled from mono Q
chromatography (Figure 21b). An immunoadsorbtion experiment was
performed to examine the possible immunologic identity between the P-2
aspartic protease and cathepsin D. Ion-exchange fractions pooled on the basis oftheir APP degrading activity (Figure 20) were applied in equal amounts to eithera column of immobilized affinity purified antibody to cathepsin D or a control
column containing purified control IgG. The A280 elution profiles for the two
columns were superimposable (Figure 21a). The flow through fractions from the
control column contained the same level of APP degrading activity as the
applied pool after the first void volume (i.e. fraction 5 onwards, Figure 21b). By
contrast APP degrading activity was essentially absent from the flow through
from the anti-cathepsin D column up to fraction 9 (an additional 5.7 void
volumes) and did not reach levels equivalent to that in the applied pool until
73
WO 95/13084 2 1 7 ~ 5 6 4 PCT/US94/07043 ~
fraction 27 (31 void volumes). This loss of APP ~gr~lin~ activity in the flow
through from the anti-cathepsin D column coincided with the depletion of
immunoreactive cathepsin D light (20 kDa) and heavy (27 kDa) chains detected
with the same anti cathepsin D antibody as had been immobilized (Figure 21c).
Cathepsin D immunoreactivity was recovered from the anti-cathepsin D column
but not from the control column by elution with 100 mM glycine pH 2.5 when
0.5 % triton X-100 was included. Notice that no protein bands other than those
corresponding to immunoreactive cathepsin D were detected in this eluent
(Figure 21c). This renders unlikely the possibility that the adsorbed APP
degrading activity resulted from an imml-nnlogir~lly cross reacting protease
other than cathepsin D itself. Unr.,l Lul~aL~ly~ only trace amounts of APP
degrading enzyme activity were recovered from the anti-cathepsin D column
(not shown). This is probably because the activity was inhibited by the
neutralized elution buffer: the composition of the elution buffer also
~lual~LilaLiv~ly inhibited the degradation of holo-APP by purified cathepsin D (not
shown).
An immobilized polyclonal antibody to human cathepsin D also
immunoadsorbed the APP peptide hydrolyzing activity present in the ion-
exchange pool (Figure 22). Under ~)nflitions of partial hydrolysis, the ion-
exchange pool cleaved dansyl-ISEVKMDAEFR-NH2 at both the M-D and E-V
bonds (under prolonged incubation conditions such as in Figure 20, the product
from M-D cleavage was eventually converted to the E-V product by secondary
proteolysis). As with APP degrading activity, the peptidase activity yielding
fluorescent products of E-V (Figure 22a) and M-D (not shown) bond cleavage firstappeared in the flow through from the control IgG column in fraction 5, but
were essentially absent from the anti cathepsin D column until fraction 12 to 13(Figure 22a). Low levels of M-D and E-V peptidase activities were subsequently
recovered from the anti-cathepsin D column but not from the control IgG
column by elution with Glycine/triton pH 2 5 (Figure 22b). Each of the peptidaseactivities recovered either in the fl~w through or by acid elution were completely
inhibited by pepstatin A. Similar results were obtained in side-by- side
e~ using the ~u~ onding peptide mimetic (N-dansyl-ISEVNLDAEFR-
NH2) of the APP locus observed in the so-called Swedish FAD (Figures 22c and
22d). However, with this latter peptide the main fluorescent product to
accumulate resulted from L-D bond cleavage. The rate of ~ ml]l~ti~n of this
peptide was so fast that all of the available substrate for this reaction was depleted
74
~ WO95113084 21 75564 PCIIUS94107043
well within the incubation times, leading to an underestimation of the
dirr~l~lLLidl activities in the flow through fractions of Figure 22c. Again, thepeptidase activities recovered either in the flow through or by elution were
inhibited by pepstatin A. Notice that the peptidase hydrolyzed the L-D bond
present in the Swedish peptide mimetic with a faster apparent rate than observedfor M-D bond cleavage in the wild type APP mimetic substrate.
Further experiments explored the identity of the peptide bonds in APP
that are cleaved by Cathepsin D. Larger amounts of APP were subject to
cathepsin D hydrolysis at pH 5Ø Limited proteolysis under non-denaturing
conditions was employed. Incubation mixtures were analyzed by SDS-PAGE,
immunoblotted, and then the individual product bands located either by
coomassie staining or by immunodetection with the anti-beta-amyloid
monoclonal described in Example 6(iii). The main bands located with r~nm~qqiP
blue were subject to N-terminal sequencing.
Figure 12 shows both a coomassie stained blot as well as an immunoblot
(using the anti-beta-amyloid monoclonal antibody) of such a reaction mixture.
As a control, inrllh~til7nq were also performed in the absence of cathepsin D
(wherein cathepsin D would be added back to the incubation mixture after
addition of SDS-PAGE sample buffer), or in the absence of APP 695 substrate.
Eight main product bands were observed by coomassie staining (Figure 12a) of
the complete inrllh~hl7n mixture, and which were also absent from either of the
controls. Some but not all of those bands also reacted with the A4 m--n~ n~l
(Figure 12c), which recognizes an epitope within the first 5 residues of the beta-
amyloid peptide. N-terminal analysis of the coL~massie stained products yielded
the sequence listed in the following table.
WO 95/13084 PCT/US94/07043 0
2~ 7~5~4
Table 5
N-terminal sequences of major proteolytic products following incubation of
purified cathepsin D with holo-APP 695.
Proteolytic Product Nt-terminal sequence peptide bond
band # Size hydrolyzed
(Fig. 12a) (kDa)
3.9 R-V-I-Y-E-R-M- -L-R-
Q-A-V-P-P-R-P- -L~
2 4.4 Q-A-V-P-P-R-P- -L-E-
R-V-I-Y-E-R-M- -L-R-
3 5.6 V-K-M-D-A-E-F- -E-V-
Q-A-V-P-P-R-P- -L-E-
4 6.3 V-S-D-A-L-L-V- -F-V-
10.0 V-S-D-A-L-L-V- -F-V-
L-E-V-P-T-D-G- -A-L-
V-K-M-D-A-E-F- -E-V-
6 15.8 G-A-D-S-~-P-A- -F-G-
7 24.5 L-E-V-P-T-D-G- -A-L-
8 56.2 L-E-V-P-T-D-G- -A-L-
~ The amino acid sequences were determined with an Applied Biosystems
model 477A Protein Sequencer operated in the gas phase with on-line model
120A Analyzer and Nelson Analytical model 2600 Chromatography Software.
With the exception of band 7, all sequences were assigned for the first ten
cycles. For band 7, sequencing was discontinued after cycle 6.
76
WO 95/13084 PCTIUS9410'~043
21 7556~
As expected several products were observed corresponding to cleavages
that were largely consistent with those reported for cathepsin D hydrolysis of
, other subs~l~.L~s (Moriyama et al., 1980, l. Biockem, 88: 619). Exceptions to the
reported cathepsin D specificity included the -Glu-Val- cleavage to form the
major product of band 3, and the minor product of band 5, as well as the -Leu-
Arg- cleavage products of bands 1 and 2 (Table 5).
The cleavage of the -Glu-Val bond at ~PP 593-594 is ùmsi~ L with the
observed capacity of the cathepsin D to cleave the corresponding bond in the
peptide substrate (as described above) in a pepstatin inhibitable reaction.
Cathepsin D usually hydrolyses between pairs of certain hydrophobic residues.
Cleavage at the Glu-Val bond, though unexpected, probably occurs under acidic
(pH 5) /~nn~1itj~-nc due to p.u~u. alion of the side chain of the glutamate residue
(pKa = 4.25), rendering it neutral.
Indeed, it can be calculated that 18% of the -Glu- side chains should be
protonated at pH 5Ø Such acidic ronfii~ionc occur in Iysozomes and secretory
granules, or could be induced upon tissue damage, or following hypoxia or local
ischaemia.
Most significantly, cathepsin D generated a 5.6 kDa product (band 3, Table
5), by atypical hydrolysis at the -Glu-Val- bond three amino acid residues N-
terminal to the purported N-terminal -Asp- residue of the common form of beta-
amyloid. The fragment was absent in the equivalent sections of the blot taken
from the incubation without cathepsin D. Furthermore, the fragment is of the
right size (5.6 kDa) to contain full length beta-amyloid peptide, and its generation
77
WO 95/13084 PCT/13S94/07043 ~
21 75564
suggests that cathepsin D must also cleave the APP at a second site close to the C-
terminal region of the beta-amyloid peptide.
In fact, a precursor substrate for such a C-terminal cleavage was also
identified in band 5, which exhibited an Mr (10.0 kDa). The size of this fragment
suggests that it contains most if not all of the C-terminal domain and that it arose
by a single -Glu-Val- cleavage at APP 593-594.
APP 695 contains numerous other peptide bonds that would seem to have
been ideal substrates for cathepsin D cleavage yet were not cleaved by cathepsinD. The fact that they were not hydrolyzed reflects the high degree of
sequestration of these sites away from access to cathepsin D within the folded
APP structure: most of the hydrophobic pairs would be expected to locate to the
hydrophobic APP protein core. The same considerations explain why the sites
that were shown to be hydrolysed by cathepsin D (Table 5) did not always containthe optimal cathepsin D recognition motif. To be located on the protein surface,such sites would have to contain a greater degree of polarity or charge than
would be ideal for cathepsin D cata~yzed cleavage. It is noL~ LI~y in this regard
that three of the five internal cleaYage sites contained two proline residues each
within eight residues of the scissile bond Such residues are often associated with
a break in secondary structure or with turns which often are found at the protein
surface.
In a parallel immunoblot (Figure 12c) several of the product peptides
(located with arrows), reacted with the monoclonal antibody C~86.8A to the N-
terminal residues of beta-amyloid. These included a band at Mr 5.6 which
78
WO 95/131~84 2 1 7 5 5 6 4 PCT~us94lo7o43
migrated in the same position as band 3 in Figure 12a (Table 5) a doublet between
Mr 9 to 10 kDa r/~mi~rAtin~ with band 5 in Figure 12a (Table 5), and a doublet at
Mr 14 kDa, a doublet at 16 to 18 kDa comigrating with band 6, Figure 12a, and a
band at Mr 40 kDa. Of the bands sequenced (Table 5), only bands 3, 5 and 6
comigrated with bands detected by immunoblot in Figure 12c. (~oncictPnt with
this, only these same three bands in Table 5 were of the appropriate N-terminal
sequence and size to contain the beta-amyloid epitope.
The time course of formation of the beta-amyloid immunoreactive
degradation products described in Figure 12 was performed under slightly
different molar ratios of cathepsin D and APP (Figure 13), both in the absence
and presence of pepstatin A. In the absence of inhibitor, a time dependent
At~ mlllAtion of low molecular weight fragments was observed, starting initiallywith the formation of bands at Mr approx. 16-18 and 28 kDa respectively. At 2 hr,
a band at Mr d~ o~ al~ly 40 kDa was observed. While the 16-18 and 40 kDa
bands further intPncifiPcl beyond 2 hr., the intensity of the 28 kDa band remained
constant beyond this time point. The intensities of the 16-18 and 40 kDa did notincrease further beyond 8 hr. Between 8 hr. and 21 hr. there was a substantial
increase in the intpnciti~s of detectable bands at Mr approx. 14, 10 and 5.6 kDa.
Since these latter three bands did not intensify in parallel with either the 16-18,
or 40 kDa, it is probable that the 14, 10 and 5.6 kDa bands were derived from
secondary degradation of either or all of the 16-18 or 40 kDa bands. the 16-18,10
and 5.6 kDa bands described in Figure 13 correspond to the same Mr bands listed
in Table S and shown in Figure 12c All of the bands observed in Figure 13 were
inhibited by pepstatin A confirming that they arose by the action of cathepsin D.
79
WO 95/13084 2 i 7 ~ 5 6 ~ PCT/US94/07043 ~
Figure 23 provides an update of the sequences of APP fragments formed by
cathepsin D to include those identified since Table 5 was prepared. It also relates
the sequences to the particular C286.8A immunoreactive bands observed in
Figure 12c. For each C286.8A iu--l-ullul~dctive band, the .~ b~vl~dillg segmentsof a sequencing blot yielded a fragment(s) of a size and sequence sufficient to
contain the C286.8A epitope and thus account for the immunoblot band (Figure
23a). Besides the N-terminus of mature ~3APP, digestion with CD yielded ~APP
N-termini (Figure 23b) resulting from seven different cleavages, which were
largely consistent with the reported specificity of cathepsin D (A. Moriyama et al.,
1990, J. Biochem., 88: 619; J. van Noort et al., 1989, 1. Biol. Chem., 264: 14159; and
M. Tanji et al., 1991, Biochem. Biophys. Res. Comm., 176: 798). The
fra~m~nt~ti~n pattern (Figure 23b) suggests that formation of the 5.5 kDa
fragment occurs by IJlV~l~sbiV~ N- and C-terminal nibbling of a larger precursorsuch as the 38 kDa peptide, or even the un-characterized transient 28 kDa
fragment of Figure 13. It is probable that the 5.5 kDa i,lll.ul.vl~dctive fragment
derives directly from the 10-12 kDa fragment with the same sequence. Both of
these fragments and those of Mr 15-16 and 18-19 seem to be of a size sufficient to
contain a full length copy of ~AP. Obviously, other products such as those
resulting from further processing of the 10-12 kDa immunoreactive fragment
may have gone ~In~ rt.orl, perhaps due to further degradations, or to loss
during electroblotting.
The implication of cathepsin D as a major protease in amyloidosis of
Alzheimer's Disease now explains other observations made concerning the
disease. Firstly, there is growing evidence that APP ~ mlll~t~s in lysozomes,
and is processed there to yield amyloid bearing fragments (Haas et al., 1992,
wo 95/13084 2 1 7 5 5 6 4 PCTIUS94107043
Natt~re, 357: 500). Amyloid ~ citil-n is favored at the acid pH of the Iysosome
(Burdick et al., 1992, J. Biol. Chem., 267: 546). Secondly, while cathepsin D is a
lysosomal protease, it has also been shown by hictorhrmictry to be present in
si~nifir~nt levels associated with amyloid deposits in Alzheimer's brain (Cataldo
et al., 1990, Proc. Natl. Acad. Sci USA 87: 3861).
Thirdly, beta-amyloid released by cells in culture ~lllp~ s a minor N-
terminal sequence starting at residue Val 594 (Haas et al., 1992, Natllre, 35g: 322)
which is three amino acids N-terminal to the more abundant sequence
beginning at the Asp 597 residue commonly seem in beta-amyloid 1-42. The
minor sequence probably arises by direct endoproteolysis at the -Glu-Val bond atposition 593-594, ~ the same site as shown presently to undergo specific
proteolysis by cathepsin D. It is emphasized here that since cathepsin D can
hydrolyse both the -Glu-Val- and -Met-Asp- bonds, it has the necessary specificity
to form both of the beta-amyloid fragments sr-qu~ncecl by Haas et al.
Fourthly, the cysteine preotease inhibitors E-64 and leupeptin were
without effect on the release of beta-amyloid by LC-99 cells while general
lysosomal inhibitors blocked the release (Shoji et al., 1992, Science, 2~8: 126),
showing that beta-amyloid formation by these cells was catalyzed by a Iysosomal
enzyme other than a cysteine protease. A remaining candidate protease for such
a reaction would be lysosomal cathepsin D which is not inhibited by the cysteineprotease inhibitors used in their studies.
Further, APP contains a stretch of hydrophobic residues between the C-
terminus of beta-amyloid and the membrane anchor sequence. Some of the
81
WO 95/~3084 2 1 7 5 5 6 4 PCTIUS94/07043 ~
peptide bonds in this region could be hydrolysed by cathepsin D. Indeed the-
Leu-Val- peptide bond at position 645-646 is highlighted by the PEPTIDESORT
computer program as being a probable cathepsin D recognition site. This site is
close to the position of three of the point mutations shown to co-segregate withcertain forms of Familial Alzheimer's Disease (FAD). Cleavage within this
region as well as the -Glu-Val- bond at positions 593-594 could account for the
size of band 3 in Table 5. The FAD mutations at this site could augment the rates
of APP cleavage within this region by cathepsin D.
Conceivably, the -Asn-Leu- mutation at S2' and S3' sites to the -Glu-Val-
scissile bond could augment cleavage by cathepsin D. To test whether this was
the case, we compared the capacity of both cathepsin D and the P-2 enzyme peak
B to hydrolyze the substrate N-dansyl-Ile-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-
Arg-NH2 (SEQ ID NO: 7), and a similar peptide in which the K-M pair was
replaced with NL thereby mimicking the above described F~D mutation (FiguIe
14). While both enzymes cleaved the wild type peptide at the M-D and E-V
bonds in longer time frames, very little cleavage was observed in the short
inrl1h~tion time shown in Figure 14 By contrast, cleavage of the mutant peptide
by both enzymes occurred with initial velocities between 30 to 50 times faster
than observed with the wild type peptide. The single metabolite thereby
generated exhibited a retention time of 4.4 min, and had not been seen
previously using the wild type peptide. This product was subsequently purified
and identified as N-dansyl-ISEVNL (SEQ ID NO: 9) by a combination of mass
spectrometry [M-H]+ = 907, and amino acid composition analysis The product
must therefore result from hydrolysis between the L-D bond of the substrate.
This cleavage by cathepsin D also liberates a peptide with an N-terminus that is
82
wo 95/13084 2 1 7 5 5 6 4 PCTiUSg4107043
the same as that found in the major form of beta-amyloid, and provides a
m~rh~ni~m by which the -NL- mutation observed in this particular early onset
FAD causes enhanced rates of beta-amyloid formation by providing a site that is
more rapidly cleaved by the amyloidogenic protease cathepsin D. The increased
rates of beta-amyloid ~c~-mlllAtil-n that could result, could trigger the early onset
form of Alzheimer's Disease linked to this APP mutation.
The rates of hydrolysis of wild type holo-~APP695 and BAPP695~NL (the
latter of which carries the 595L596D to NL FAD mutation) by cathepsin D were
compared (Figure 24). At pH 5.0 both substrates were hydrolyzed to form the 18-
19 kDa and 15-16 kDa fragments at comparable rates. By contrast, the 10-12 kDa
and 5.5 kDa fragments formed from wild type BAPP695 were barely detectable by
20 hr u~hereas with ~APP695~NL hydrolysis, fr~gm~ntc possessing these same
Mr values were clearly observed. It was estimated that the 5.5 and 10-12 kDa
bands were formed from BAPP695~NL at rates that were 5 to 10 times greater
than the ~uL~ ol~ding rates with wild type ~APP695. The APP695~NL
fri~gm~nts r~mi~r~ho~ both with wild type ~APP695 fragments as well as with a
re~rlmh;n~nt C-100 fragment, consistent with the notion that these fragments
result from a cleavage close to or at the N-terminus of BAP.
The effect of the ~NL FAD mutation in increasing the rate of cathepsin D
catalysed ~APP hydrolysis is consistent with the effect of the same substitution in
dramatically increasing the rate of CD catalyzed hydrolysis of N-dansyl-~APP591-601-amide at the L-D bond adjacent to the N-terminal residue of ~AP (Figure 14),and suggests that the increased rates of formation of the ~APP695~NL derived 5.5and 10 kDa fragments are also due to a more facile cleavage of the 596L-597D
83
WO95/13084 2 1 7 5 ~ 6 4 PCT/US94/07043 ~
bond relative to cleavage of wild type ~3APP at either the 593E-594V or 596M-597D
bonds. The ~APP695~NL fragments are predicted to contain a full length copy of
~AP and as such could serve as int~rm~1iAtl~c in the beta-amyloid deposition
~llala~ ic of the Swedish form of familial Alzheimer's disease. The effect of
the Swedish mutation in increasing the rate of cathepsin D dependent
amylt~ )g~nic processing of APP further underscores the importance of CD in
the amyloidogenesis of AD.
The i~l~ntifi~Atil-n of cathepsin D as a serious candidate for the primary
amyloidogenic protease of Alzheimer's Disease, significantly aids the effort of
development of therapeutic inhibitors for the disease. For example specific
cathepsin D inhibitors could provide Ill~lalJeulic benefit by inhibiting the toxic
Al c.lmlllAtinn of beta-amyloid. The new information provided herein makes it
comparatively strai~;l-Lr~, ~aid to rationally design tight-binding inhibitors as has
been Arrnmrli~hP~ for the design of novel inhibitors of other aspartic proteasessuch as renin and HIV-protease.
Alternatively cathepsin D can now be adapted for use in a high
throughput screen using an in vitro peptidase assay so as to identify therapeutic
inhibitors through random or semi-random search of chemical libraries. A
suitable assay for such purposes could include the N-Dansyl-peptide assay
described in Examples 2 and 3 of the present invention.
84
wo 9~/~3084 2 ~ 7 ~ 5 6 ~ PcrlUSs4107043
Example 10. Identification of a serine protease with specificity for C-terminal
APP ~.ocæ~
, Table 4, showed that human brain contains serine proteases capable of C-
terminal processing of recombinant APP, and that in some cases these serine
proteases were inhibitable with aprotinin. To attempt a more facile isolation ofsuch proteases, an alternate isolation scheme was devised (Example 1, Method 2)
in~ ali.,g affinity pllrifif~tit-n on aprotinin-sepharose as an early step.
Application of this procedure for the further purification of the P-2
fraction was successful in the isolation of APP ~grA-lin~ activity (Figure 15). The
active fractions recovered from the aprotinin-sepharose column by acid elution
were further purified on a mono-Q column (Figure 16). Active fractions (Figure
16a) exhibited the capacity to form APP C-terminal fragments of 11 kDa, 14 kDa
and 18 kDa, when analyzed by immunoblot with a polyclonal antibody to the
APP C-terminus (Figure 16b). The smallest products co-migrated with the
recombinant C-100 standard. Reassay of APP rlf~r~ tion in the active fractions
using an anti-beta-amyloid monoclonal antibody C286.8A led to the detection of
the same three products bands (Figure 16b). Since the antibody C286.8A
recognizes the first seven amino acid residues of the beta-amyloid peptide, as in
Example 6(iii), this experiment shows that all three products contained full
length beta-amyloid.
-
One or more of these product peptides could be amyloid or give rise tobeta-amyloid by further processing of these peptides C-terminal to the beta-
WO 95113084 PCTII~S94107043
21 75564
amyloid region. The serine protease activity involved in formation of theseproducts could therefore play a role in amyloidosis.
The enzymic activities which formed the 11,14 and 18 kDa product bands
described above eluted as a broad peak from mono-Q and could perhaps have
resulted from the action of more t~an one protease. Based on the recovery of
A280 nm absorbing components from the mono-Q column, three different pools
of proteolytic activity were prepared from the mono-Q column fractions termed
pool X, Y and Z (Method 2, Example 1).
Enzymatic activity was recovered in the void volume during
chrl-mAtogrArhy of each pool on superdex 75 (data not shown), ~ol~is~ l with
an apparent Mr >75 kDa, although possible protein aggregation during
chromatography cannot be ruled out. Pool Y ~ L~d the purest pool when
analyzed on SDS-PAGE, and exhibited a major stained band at Mr of approx 100
kDa. Pool Y was selected for further char~rtPri7Atinn The pH dependence for
APP hydrolysis by pool Y showed an optimum between pH 7 and 9 (Figure 17a),
and the enzyme activity was gradually inhibited by increases in sodium chloride
con~'ntrAtit-n beyond 42 mM (Figure 17b). Studies of the inhibitor sensitivity of
the enzyme (figure 18a), confirmed that it was serine protease, being inhibited by
PMSF and aprotinin but l~nAff~ t~cl by pepstatin A, E-64 or EDTA (Figure 18a).
The serine protease inhibitor b~n7.Amil1inl~ was without effect on the enzyme,
suggesting that it was unlikely to be a trypsin-like endoprotease. More likely the
enzyme is of the chymotryptic family with specificity for cleavage of substratescontaining a neutral hydrophobic residue at the S1 subsite.
86
WO 95/~3084 2 ~ PCTIUS94107043
Acw.dil~gly, further inhibitor studies (Figure 18b) showed that the activity
of the pool Y protease was strongly inhibited by chymotrypsin inhibitor II, alpha-
2-antiplasmin and TPCK. Weak inhibition was also observed with chymostatin
and alpha-l-antichymotrypsin, but TLCK, did not inhibit at all. Cathepsin G has
been suggested by others to play a role in APP processing, however immllnclhlcltanalysis of the pool Y protease fraction using a polyclonal antibody to cathepsin G
failed to detect the presence of this serine protease. N-terminal sequencing of the
11,14 and 18 kDa will identify the cleavage sites and is already ongoing.
Example 11. Design of lll..a~a~ic cathepsin D inhibitors.
This example utilizes the nr~m.onrl~tllre of Schechter et al., 1967, Biochem
Biop~lys Res Comm., 27: 157, to describe peptide specificity, wherein the amino
acids in the substrate which flank the scissile bond are l,ull,b~l~d according to
their position relative to the peptide bond being cleaved by the enzyme. Peptidesubstrate amino acid side chains N-terminal to the scissile bond are numbered
consecutively as Pl to Pn with inaeasing distance from the scissile bond. Peptide
substrate amino acid side chains C-terminal to the scissile bond are numbered
~ul~ u~iv~ly as Pl' to Pn'. The Pl and Pl' amino acid side chains .,~ ond to
the amino acids involved in formation of the peptide bond which is to be
cleaved. The side chains Pl to Pn and Pl' to Pn' are envisioned to form specificintr~r,~ctions with a w~ blldillg series of enzyme subsites Sl to Sn and Sl' to
Sn' respectively. The interactions between the P side chains and ~ulle~ Llil~g
S subsites contribute to the binding energy for stabilization of the protease-
substrate complex, and thus confer specificity to the interaction.
87
wo 95/13084 PcT/US94/07043
21 75564
The approach taken to the development of peptidomimetic inhibitors
could utilize either the n-dansyl peptide substrate assays of Examples 2 and 3, or
the assay of holo-APP ~ gr~ tirln described in Example 8, to make enzymologic
measurements, in conjunction with purified cathepsin D.
Q ~'fir.~i n of optimal peptide length and sequence for ~lut~ulyl;c cleavage by
cathepsin D in vitro. Starting with a dodecapeptide peptide of sequence Drls-Ile-
Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2 (SEQ ID NO: 7), the effect of
shortening of the peptide either from the N-terminus or the C-terminus on the
apparent kinetic parameters for enzymic hydrolysis would be determined at
acidic pH and optimal ionic strength. The effect on Km and Vmax for hydrolysis
of variation in the amino acids at each position in peptide of optimal length
would be fl~tl~rmin~r~
Inhibitor synthesis. Pepti~rlmim~tir compounds would be 5ynth~5izf~d
ront~inin~ essential amino acid sequences necessary for optimal cleavage (from 1a, b above), and the appropriate spacer. The amino acid sequences in these
peptides could be the same as those observed around the cleavage site in the APPsubstrate, e.g. Glu-Ile-Ser-Glu-Val-Lys-Met-Asp (SEQ ID NO: 4) and Trp-His-Ser-
Phe-Gly-Ala-Asp-Ser (SEQ ID NO: 5) or alternatively selected from those
sequences found to confer optimal binding to cathepsin D based on studies of
their potency for in vitro inhibition of cathepsin D. In the case of Glu-Ile-Ser-
Glu-Val-Lys-Met-Asp (SEQ ID NO: 4) and Trp-His-Ser-Phe-Gly-Ala-Asp-Ser (SEQ
ID NO: 5) the P1-P1' bond is E-V, and F-G respectively.
Peptidic inhibitors would be synthesized that contain either the above
88
WO 95/~3084 2 1 7 5 ~ 6 4 PCTIUS94J07~)43
sequences or sequences exhibiting optimal cathepsin D inhibition (including
shorter variants perhaps ~ uullg N- and/or C- substitutions), in which the-
CO-NH- atoms of the peptide bond between P1 and P1' are replaced with any of
the following standard spacer groups and using dy~lv~-;d~ synthetic routes so asto obtain any possible stereo-chemical configuration thereof: reduced amide,
hydroxy isostere, ketone isostere, dihydroxy isostere, statine analogs,
phosphonates or phosphonamides, reversed amides. Most of these inhibitors
would function as transition state analogs.
The potency of these first generation compounds as determined using
either of the in vitro assays of the present invention (N-Dansyl-peptide assay of
holo-APP degradation assay) could be optimized by any or all of the following:
(i) Addition or deletion of flanking amino acid residues;
(ii) Alteration of the type of amino acids side chain (D or L) at each
position in the inhibitor;
(iii) N- and C-terminal ~llhctit~ltil~n with blocking groups such as Boc or
acetyl (N-terminally), or O-Me, O-benzyl, N-benzyl (C-terminally).
Beside the inhibitors rationally developed according to the above
methods, other known cathepsin D inhibitors either in whole or in part could be
used as therapeutic inhibitors for Alzheimer's Disease, or as starting points for
vL~ n of inhibitory potency and the development of new derivatives for
therapy of Alzheimer's Disease. Such inhibitors include: 1-Deoxynojirimicin
(Lemansky et al., 1984, J. Biol Chem., 259: 10129); Diazoacetyl-norlf~ in~ methyl
ester (Keilova et al., 1970, Febs Letf, 9: 348); Gly-Glu-Gly-Phe-Leu-Gly-Asp-Phe-
Leu (SEQ ID NO: 6) (Gubenseck et al., 1976, Fel7s Lett, 71: 42); Pepsin inhibitor
89
WO 95113084 2 1 7 5 5 6 4 PCTIUS94/07043
from Ascaris (Keilova et al., 1972, Biochem Biophys Acta., 284: 461); pepstatin
(Yamamoto et al., 1978, European Journal of Biochernistry, 92: 499).
xample 12. The effect of pepsta~in A, an inhibitor of cathepsin D on the
formation of APP C-terminal ~l " ' by Human Embryonal
Kidney (HEK) 293 cells ~ in culture.
The following example shows that pepstatin A, an inhibitor of cathepsin D
activity in vitro inhibits the capacity of HEK 293 cells to form and release into the
tissue culture medium APP C-terminal fragments of the same size (15 kDa) as
those shown to be formed from APP695 by cathepsin D in vitro (example 9).
HEK cells are known to release beta-amyloid from transfected APP695, and so
contain the proteases necessary for amyl~ g~ni-~ APP pib.~ssillg [C. Haass et al.,
lg92, Nature, 3~9: 322]. These cells therefore provide an accepted cellular model
for the study of beta-amyloid formation. Endogenous levels of APP 751/770
present in these cells served as a substrate for the study outlined below. We grew
HEK 293 cells in 400 ml suspension cultures. Some cultures contained cells
grown in medium ~I~nt~inin~ either DMSO so~vent alone (0.01 % v/v final), or
DMSO plus pepstatin A at 10 uM final. As is evident from figure l9a, neither
DMSO nor DMSO plus pepstatin had an adverse effect on the growth rate of the
HEK 293 cells at the ~ ations of these ~ub~Lal~L~ that were used. Aliquots
(185 ml) of medium taken at late log phase from either the DMSO or DMSO +
pepstatin A treated cells were passed over identical sized columns (1.6 x 5 cm) of
monoclonal C286.8A (example 6) immobilized onto sepharose 4B using CNBr-
activated sepharose 4B (Pharmacia) by a rerl-mml~n-l~cl procedure [Axen R. et al.,
1967, Nature, 214: 1302]. Prior to loading, columns were equilibrated with 100
~ woss/l3084 2~ 75~64 Pcrluss4107043
mM sodium bicarbonate buffer pH 8.3 ~ ont~inin~ 500 mM NaCl. Beta-amyloid
rr~ntAinin~ APP fr~gm~ntc that were released into the tissue culture medium by
the HEK 293 cells bound the immobilized monoclonal antibody and were eluted
from the column subsequently at 1 ml/min by washing with l00 mM Glycine,
pH 2.4 ~nnt~inin~ 0.025 % v/v Triton X-100. Eluted fractions (4 ml) were
subjected to the immllnoblrlt procedure of example 8 in order to detect any APP
fragments that may have been bound to and subsequently eluted from the
columns. Immunoblot detection was performed with the anti-C-terminal
antibody of example 6 method i). Fractions detected by this procedure would
have to contain the N-terminal heptapeptide sequence of beta-amyloid (to
explain binding to immobilized C286.8A) as well as the C-terminal domain or a
portion thereof (to explain reactivity with the anti-C-terminal antibody).
Figure 19 b compares the amounts of C-terminal frA~m,~nt~ recovered in
the elution fractions from a column of imm~hi1i7~cl C286.8A that had been
loaded with the media from cells grown either in the presence of DMSO only or
DMSO plus pepstatin A. Chromatography was performed in parallel under
identical rl~nrliti~-n~. As can be seen, treatment with pepstatin A ci~nifi( Antly
reduced the amount of an eluted 15 to 16 kDa APP-derived fragment that could
be detected by immllnt-hlt~t. This fragment is the same size as the fragment
formed in vitro by cathepsin D with the N-terminal sequence G-A-D-S-V-P-A-
(Table 5 and Figure 23), and could represent an intermediate in the cellular
formation of the smaller 5.6 kDa fragment with an N-terminus corresponding to
a form of beta-amyloid. Other APP fragments that are formed by cathepsin D in
~itro were not detected in this ~ 1. The llntl~t~ t~cl fragments may have
been present below the detection limit or further degraded in the cells by other
91
WO 9!;/13084 2 1 7 ~ ~ ~ 4 PCINS94/07043 ~
proteases. This experiment shows ~hat HEK ~93 cells, an accepted cell line for the
..,;,,.1ion of cellular amyloid formation make and release at least one APP
fragment that resembles the APP695 fragments formed in vitro by cathepsin D
and that formation of this fragment is inhibited by a non toxic dose of a cathepsin
D inhibitor. Thus, peptidic based inhibitors of cathepsin D have utility in
altering cellular APP processing.
Example 13: Inhibition Studies
The in vitro hydrolysis of N-dansyl-ISEVKMDAEFR-NH2 by cathepsin D
was used to screen 250 peptidic compounds selected from the Renin and HIV-
protease inhibitor programs for their capacity to inhibit human cathepsin D. Of
these, the compounds identified in Table 6 below displayed potent cathepsin D
inhi~ito~ potency
~ WO95/13084 2 1 75564 PCr~US941û7043
Table 6: Potency Of ~ Active In The In Vi~ro Inhibition of
Human Cathepsin D
Inhibitor # ICso Inhibitor # ICso
(nM) (nM)
0.6 14 10
2 4.4 15 0.9
3 1.6 16 0.4
4 1.8 17 56
5 o.g 18 0.6
6 1.1 19 14
7 4.1 20 24
8 0.7 21 1.2
9 19 22 2.0
22 23 2.0
11 1.9 24 1.3
12 1.5 25~ >1000
13 30 26" >500
Compounds 25 and 26 are included as negative controls
The structures of the inhibitors corresponding to the numbers given in
Table 6 are presented in Table 7 below:
-
93
WO 95/13084 2 1 7 5 ~ 6 4 PCTNS94/07043 *
Table 7: Inhibitor Structure
Inhibitor #/Structure
~ O ~ "
X~ \-)I\H~
2/ -- OH O
- H~NQ
S/~ S OH /\ O
- H~H$
/~ S
94
W0 951~3084 2 ~ 7 5 5 6 4 PCTJUS94107043
4/
~ O
N~)I\N~N~/\
o_~ CH3 S~S OH A
5/ ~3 ~
X H
~0--lCI--H ICl H ¦Cl H
O O O OH CH3 0
--Lo--c--N C--N C--N-- ~ /~\
¦¦ H ¦¦ H 1I H _ _
O O O OH CH3 0
Wo 95/13084
2 1 7 5 s 6 4 PCT113S94/07043 ~
7/
P
HN~
8/
~I P
~N ~CI HN ~~--N C--N/~~; /~\
9/ ~
BOC Phe--Asn N
H _ "P--~~2Hs)2
96
-
WO 95113084 2 1 7 5 5 6 4 PCTIUS94~07043
10/
,. O
~\ N~ N~
O /~ OH; O
S~S ~
11/
~ ~3
k~J'N ?
O ~ OH
12/
X~ N~ ?
97
WO 95/13084 PCT/US94107043 ~D
21 75564
~0 ~ .
N--C--N C--N Cl--N~N~ /\A
--( S O 0~ ~ OH CH3 O
HO~
~/OH
OH
14/
+ O--C--N IC--NyC--H/~H~ NH2
CONH2
15/
~ O
~ H~H~ ,P--(c2Hsk
H3C
98
WO 95/13084 2 1 7 ~ ~; 6 4 PCTIUS94107043
16/
><O N~ H~ ~--(c2Hsk
O ~ OH O O
17/
H~ _ H)~
O ~ O
18/
OCH3
P
><o N/(
S~5
99
WO 95/13084 PCTIUS94107043
21 75564
19/
CHS(CH2)12CO--Phe--Asn ~ -
HN , "P--(G2H5k
20/
~ - H~N~ ~
S~S OH O
21/
X~/~N~N$ ~;;3
O ~ OH O
.
100
WO 9S/13084 E~crluss4107043
21 75564
22/
.- ~ ~
N\~A N s CH3
23/ ~ ~
C2Hs--O ICI Hlll N~ H~H~¢ CH3
N
OH
24/ ~ ~
+--lCI--HNlll--NXIC--N/(~H NH2
101
PCTIUS94/07043
WO 95~13084
21 75564
25/
2 HCl x H2N ~
H OH o P--(OC2Hs)2
26/
N~ ~
>= F3C
H2N
102
~ WO 95/~3084 2 1 7 ~ 5 6 ~ PCIIUS94~07043
Ln all formulas herein, the abbreviation "BOC" l~ St~L~ tert-butoxy
carbonyl.
The foregoing inhibitors can be prepared as follows: Inhibitors 1, 2, 3, 4, 10
and 20 have been described in German application DE 4,215,874, filed on May 14,
1992, which corresponds to U.S. Serial No. 08/059,488, filed on May 10,1993. Thedisclosures of both of these applications are incorporated herein by reference.
The inhibitors are disclosed to be antiviral agents, specifically HIV protease
inhihitnrc Inhibitor 1 was prepared as follows:
First step: A stirred solution, cooled to 0C, of 614 mg (2.20 mmol) of
(2R)-N-(tert-L,u~o~yedl~bl,yl)-2-amino-2-[2-(1,3-dithiolan-2-yl)]acetic acid (EP 412
350) and 337 mg (2.20 mmol) of 1-hydroxybenzotriazole (HOBT) in 10 ml of
anhydrous dichloromethane is treated with 434 mg (2.10 mmol) of
dicyclohexylcarbodiimide (DDC) and the mixture is stirred for 5 min. A solution
of 1.10 g (2.20 mmol) of 1-{(2R, S, 4S, 5S)-[5-amino-6-cyclohexyl-4-hydroxy-2-(1-
methyl)ethyl-hexanoyl]}-S-isoleucinyl-2-pyridylmethylamide dihydrochloride
[EP 437 729] and 0.88 ml (8.0 mmol) of N-methylmorpholine in 10 ml of
dichloromethane is then added dropwise. The cooling bath is removed and the
reaction mixture can be stirred at room temperature for 2 hr. The end of the
reaction is rl~tl~rmin~d by thin layer chromatography. The resulting urea is
removed by filtration, the filtrate is ~ llLLdL~d in vacuo and the crude productis purified by chromatography on 90 g of silica gel (dichlorometh~ne m~thane
95:5). 1.29 g (88% of theory) of the compound:
103
WO 95~13084 2 1 7 5 5 6 4 PCT/US9~/070~3 ~
27/ O
H~ o \~
S~S
are obtained as a pale powder.
Second stey: A solution of 2.41 g (3.28 mmol) of compound 27, above, in
17 ml of a 4 N solution of gaseous hydrogen chloride in anhydrous dioxane is
stirred at 0C for 30 min. 15 ml of toluene are then added and the mixture is
concentrated in vacuo. This process is:repeated a further two times, and the
residue is then triturated with ether, filtered off with suction and dried in a high
vacuum over potassium hydroxide (KOH) to yield 2.29 g (98% of theory) of the
compound:
28/ O
- H~
S~S
as a colorless powder.
wo 9S/13084 2 1 7 5 5 6 4 PCTIUS94107043
Third ste~: A stirred solution, coole~ to 0C, of 0.80 g (2.40 mmol) of (2S)-
3-tert-butylsulphonyl-2-(1-naphthylmethyl)-propionic acid [prepared according toH. Buhlmayer et al., 1988, J. Med. Chem., 31: 1839] and 0.40 g (2.64 mmol) of
HOBT in 20 ml of anhydrous dichl~,lo~ l-alle is treated with 0.52 g (2.52 mmol)
of DCC and stirred for 5 min. A solution of 1.55 g (2.19 mmol) of compound 28,
above, and 0.96 ml (8.74 mmol) of N-methylmorpholine in 30 ml of
dichloromethane is then added dropwise and the reaction can be stirred at room
temperature for 2 hr. The resulting urea is removed by filtration, the filtrate is
concentrated in vacuo and the crude product is, if desired, purified by
chromatography on 360 g of silica gel (dichlor~-m~th~nP methanol 95:5) to yield
586 mg (28% of theory) of the non-polar (2R)isomer as a colorless powder and
690 mg (33%) of the polar (2S)-isomer also as a colorless powder.
Inhibitors 2, 3, 4, 10 and 20 can be prepared analogously by coupling the
appropriate acids with the appropriate amine hydrochlorides, which are known
or can be prepared by conventional means. In the case of inhibitors 3 and 20, itwill be necessary to start from the compound having the formula:
29/
o
BOC~ N~Q
-
\J
105
WO 95/13084 ; PCT/US94/07043
21 75564
The preparation of compound 29 is analogous to that of compound 27, butstarting from 249 mg (0.89 mmol) of (2R)-N-(tert-butoxycarbonyl)-2-amino-2-[2-
(1,3-dithiolan-2-yl)]acetic acid [EP 412 350] and 440 mg (0.81 mmol) of 1-{(2R, S, 4S,
5S)-[5-amino-6-cyclohexyl-4-hydroxy-2-(2-propenyl)-hexanoyl]}-S-isoleucinyl-2-
pyridylmethylamide dihydrochloride [prepared according to EP 437 729] and by
chromatography of the crude product on 24 g of silica gel
(dichl~ l 9:1). 553 mg (93%) of compound 29 are obtained as a
pale powder. The preparation of the amine hydrochloride is then analogous to
that of compound 28, but starting from 560 mg (0.91 mmol) of compound 29. 452
mg (91%) of the amine hydrochloride are obtained as a colorless powder.
Inhibitor 10 will require the starting material of the formula:
30/ O
BOGNHJI~ ,~ N $
H _ - H
S~~S OH -b
The preparation of compound 30 is analogous to that of compound 27, butstarting from 258 mg (0.92 mmol) of (2R)-N-(tert-butoxycarbonyl)-2-amino-2-[2-
(1,3-dithiolan-2-yl)]acetic acid [EP 412 35D] and 500 mg (0.84 mmol) of 1-{(2R, S, 4S,
5S)-[5-am ino-6-cyclohexyl-4-hydroxy-2-(2-phenyl)-hexanoyl]}-S-isoleucinyl-2-
106
WO 95/~3084 2 1 7 5 ~ 6 4 PCTJUS94107043
pyridylmethylamide dihydrochloride [prepared according to EP 437 72g] and bychromatography of the crude product on 20 g of silica gel
(dichlorometharlP ."~ ".-l g5:5). 593 mg (90%) of compound 30 are obtained as
an amorphous powder. The preparation of the amine hydrochloride is then
analogous to that of compound 28, but starting from 589 mg (0.75 mmol) of
compound Z9. 484 mg of the amine hydrochloride are obtained as a colorless
powder.
Inhibitor 7 is known from published European application EP 0 472 077,
which was published on February 26,1992, and the entire contents of which are
incorporated herein by reference. The inhibitor is disclosed therein as an
inhibitor of HIV protease activity.
Inhibitors 8 and 13 are known from published European application EP 0
441 912, which was published on August 14, 1991, and the entire contents of
which are incorporated herein by reference. The inhibitors are disclosed thereinas inhibitors of renin.
Inhibitors 9,15,16 and 19 are known from published European application
EP 0 472 078, which was published on February 26, 1992, which is equivalent to
U.S. Patent No. 5,147,865, which issued September 15,1992 The entire contents
of both publications are incorporated herein by reference. The inhibitors are
disclosed therein as inhibitors of HIV protease activity.
~ .
Inhibitors 11 and 12 are described in German application DE 41 26 485,
which was filed on August 10, 19g1, and corresponds to U.S. Serial No.
107
WO 95/13084 2 1 7 5 5 6 4 PCT/US94107043 0
07/920,216, filed July 24,1992, still pending, and European application EP 0 528242, which was published on February 24, 1993. The complete disclosures of
these three applications are incorporated herein by reference. The inhibitors are
disclosed therein as inhibitors of HIV protease activity. The inhibitors can both
be prepared as follows: -
First step: A solution of 5.00 g (20.21 mmol) of (s)-2-(tert-buL~cy~dlL
amino-1-phenylbut-3-ene [J.R. Luly et al., 1987, J. Org. Chem., 52:1487] in 100 ml
of a 4 N solution of gaseous hydrogen chloride in anhydrous dioxane is stirred at
room temperature for 30 min. 15 ml of toluene is then added and the mixture is
concentrated in vacuo. This process is repeated a further two times, then the
residue is triturated with a little ether, filtered off with suction and dried in a
high vacuum over KOH to yield 3.69 g (99% of theory) of the compound:
31/
HCI x
as colorless crystals.
Secon~l step: A stirred solution, cooled to 0C, of 4.81 g (22.13 mmol) of N-
(tert-butoxycarbonyl)-L-valine and 3.29 g (24.35 mmol) of HOBT in 40 ml of
anhydrous dichloromethane is treated with 5.29 g (25.65 mmol) of DDC and
stirred for 5 min. A solution of 3.70 g (20.12 mmol) of compound 31 and 8.85
108
WO9~;113084 2 1 7 5 ~ 6 4 l'CTlUS94107043
(80.48 mmol) of N-methylmorpholine in 30 ml of dichloroethane is then added
dropwise. The cooling bath is removed and the reaction mixture is stirred at
room ~ p~ldLLIl~ for 2 hr. The end of the reaction is rl~tPrmin~d by thin layer
chromatography. The resulting urea is removed by filtration, the filtrate is
concentrated in vacuo and the crude product is purified on 450 g of silica gel
(dichlorom~oth~n~ methanol 95:5). 6.07 g (87% of theory) of the compound:
32/
BOC-Val-NH~
is obtained as a colorless foam.
Third step: A solution of 6.08 g (17.53 mmol) of compound 32 in 100 ml
of a 4 N solution of gaseous hydrogen chloride in anhydrous dioxane is stirred at
room temperature for 30 min. 15 ml of toluene is then added and the mixture is
concentrated in vacuo. This process is repeated a further two times, then the
residue is triturated with a little ether, filtered off with suction and dried in a
high vacuum over KOH to yield 4.90 g (99% of theory) of the compound:
33/
HCI x H-Val-NH
109
OWO 95/13084 PCT/US94/07043
21 75564
as a colorless powder.
Fourth step: A stirred solution, cooled to 0C, of 1.50 g (4.47 mmol) of (2S)-
3-tert-butylsulfonyl-2-(1-naphthylmethyl)-propionic acid [prepared according to
H. Buhlmayer et al., 1988, J. Med. Chem., 31: 1839] and 0.66 g (4.92 mmol) of
HOBT in 15 ml of a~ ydluus dichlulu~Lll~l e is treated with 0.97 g (4.69 mmol)
of DCC and stirred for 5 min. A solution of 1.15 g (4.07 mmol) of compound 33
and 1.80 ml (16.27 mmol) of N-methylmorpholine in 10 ml of dichloromethane
is then added dropwise and the reaction is stirred at room temperature for 1 hr.The resulting urea is removed by filtration, the filtrate is concentrated in vacuo
and the crude product is purified by chromatography on 270 g of silica gel
(dichloromethane: methanol 95:5). 2.01 g (88% of theory) of the compound:
34/
k ~ H
o
is obtained as a colorless foam.
Fifth ~tep: A stirred suspension, cooled to 0C, of compound 34 in
dichloromethane is treated with 2 equivalents of m-chloroperbenzoic acid
(MCPBA) (80% strength) and stirred at this temperature for 2 hr. A further 1
equivalent of MCPBA is then added and the mixture is additionally stirred at
110
wo gS/13084 2 1 7 5 5 6 ~ PCTruS94107043
room l~",r~,,.l,.,~ for 1 hr. Ethyl acetate is then added and the reaction mixture
is stirred into a 10% Na2SO3 solution The organic phase is separated off, washedthree times with a NaHCO3 solution and dried over MgSO4. After evaporation
of the solvent in vacuo and titration of the residue with a little ether/pentane,
- the compound:
35/
X~/ H O
O
is obtained as a colorless powder.
Sixth ste~: A solution of u~ uul~d 35 and either (2S)-2-(trifluoromethyl)-
pyrrolidine [cf. G.V. Shustov et al., 1987, Is~est. Akad. Nnuk. SSSR, 1422 (engl)]
(to prepare inhibitor 11) or (2S)-2-(trifluoromethyl)-piperidine (to prepare
inhibitor 13) in n-propanol is stirred in a pressure vessel at elevated temperature.
After cooling, the reaction mixture is concentrated in vacuo and
chromatographed on silica gel. After tritl~rAtin~ with n-pentene, the inhibitor is
obtained.
-
Inhibitors 5 and 6 are within the generic teachings of EP 0 441 912, supra,and can be prepared following the preparation schemes taught therein. Thus,
inhibitor 5 can be prepared as follows:
111
WO 95113084 2 1 7 5 5 6 ~ PCTIUS94/07043 4
First step: 300 g (1.91 mol) of L-phenylalanine are suspended in 360 ml
of dioxane and 360 ml of H~O. 432.9 g (1.98 mol) of di-tert-butyl dicarbonate are
added while stirring at pH 9.8. The pH is m~int~in~ll constant with about 975 mlof 4N NaOH. After 16 hr, the reaction mixture is extracted with ether, and the
aqueous phase is adjusted to pH 3-4 with citric acid and then extracted with ether
2 x and ethyl acetate 2 x. The organic phases are combined and washed 3 x with
water. Con.t~ d~io-- in a rotary ~vdpuldLul and crystallization from diethyl
ether/hexane results in 291.6 g (60.7%) of the compound:
36/ ~
~\
Boc-NH COOH
Secon-l step: 265 g (1.0 mol) of compound 36 are dissolved in 2 l of
methanol and hydrogenated on 20 g of 5% Rh/C under 40 atm for 5 hr. The
catalyst is filtered off through celite with suction and washed with methanol, and
the resulting solution is concentrated. 271 g (100%) of the compound:
37/ f)
Boc~l~H COOH
112
W<l g~ll3084 2 1 7 5 5 6 4 PCTIUS94/07043
are obtained.
Third step: 163.0 g (0.601 mol) of compound 37 and 40.3 g (0.661 mol) of
N,O-dimethylhydroxylamine are dissolved in 2 l of methylene chloride at room
temperature. At 0C, 303.5 g (3.005 mol) of triethylarnine are added dropwise (pH
~ 8). At max. -10C, 390.65 ml of a 50% strength solution (0.601 mol) of n-PPa in
methylene chloride are added dropwise. The mixture is warmed to 25C
overnight and is stirred for 16 hr. The reac~ion is then 1..,..`.-,.l,,.1,-.1, 500 ml of
saturated hirArhl~nAtP solution are added to the residue, and the mixture is stirred
at 25C for 20 min. After three PYtrA( tifln~ with ethyl acetate, the organic phase is
dried over Na2SO4 and ~ ~-"~ Crude yield: 178 g (94.6%). The crude
material is chromatographed on silica gel (mobile phase system CH2Cl2:CH3OH
98:2). 136.6 g of the compound:
38/ ~
Boc-NH CO N--OCH3
CH3
are obtained.
:Fo1lrth step: In a flame-dried apparatus under nitrogen, 63.7 g (0.21 mol) of
compound 38 are dissolved in 1.5 1 of alumina-treated ether and, at 0C, 10 g
(0.263 mol) of LiAlH4 are added in portions, and then the mixture is stirred at 0C
for 20 min. Then a solution of 50 g (0.367 mol) of KHSO4 in 1 1 of H2O is
cautiously added dropwise at 0C. The phases are separated, the aqueous phase is
113
WO 9S/13084 PCTIUS94/07043 0
2 1 75~4 .-
then extracted with 3 x 300 ml of diethyl ether and the combined organic phasesare washed three times with 3N HCl, 3 x with NaHCO3 solution and 2 x with
NaCl solution. The organic phase is dried over Na2SO4 and concentrated. 45 g
(84.1%) of the compound:
39/
Boc-NH CHO
are recovered. /~nmro~ln~l 39 is either further processed immP~1iAt.oly or stored at
-24C for one to two days.
Fifth step: 14.6 g (35 mmol) of "instant ylide" (Fluka 69500) are
suspended in 90 ml of anhydrous tetrahydrofuran. While cooling in ice and at a
reaction temperature between 20 and 25C, a solution of 9.0 g (35 mrnol) of
compound 39 in 45 ml of anhydrous tetrahydrofuran is added dropwise. The
reaction mixture is stirred for 15 min. and then poured into 250 ml of ice and
extracted twice with 150 ml of ethyl acetate/n-hexane 3:1 each time. After drying
over Na2SO4 and ~ L~d~ion, the residue is chrnmAtngrArhed on silica gel
(mobile phase ether:hexane 7:3). 3.2 g (40.0%) of the compound:
40/ ~ ,
Boc-NH
114
WO 95113084 PCTIUS94107043
2~ 7556~
are obtained.
Si~th st~r: 202.4 g (0.8 mol) of compound 40 are dissolved in 1000 ml of
mesitylene and heated to 140C with a water trap. At this ~ lult, a mixture
of 197 g (1.6 mol) of N-benzylhydroxylamine and 1.6 mol of acetaldehyde in 800
ml of ll-~si~yl~ e is added dropwise over the course of 2 hr. After a reaction time
of 4 hr and 8 hr, the same amount of N-ben~ylhydroxylamine and ethylaldehyde
in mesitylene is added dropwise. After a total reaction time of 16 hr, the mixture
is cc~n~ .ontr~h~ diethyl ether is added to the residue, and the mixture is thenwashed with 1 M of KHSO4 solution. After drying over Na2SO4 and
concentration, the residue is chromatographed on silica gel (mobile phase
ether:hexane 3:7). The compound:
41/
boc-NH/~
N
2_~
is obtained.
Seventh ste~: 18.1 g (45 mmol) of compound 41 (diastereomer C) are
dissolved in 300 ml of methanol. After addition of 14.2 g (225 mmol) of
115
WO 95/13084 2 ~ 7 ~ 5 6 4 PCT/US94/07043 ~!
Rmmnnillm formate, the apparatus is thoroughly flushed with N2, and 3.6 g of
palladium/carbon (10%) are added. The mixture is stirred under reflux for 3 hr.
Cooling is followed by removal of the catalyst by filtration, concentration of the
solution, dissolution in ethyl acetate and washing twice with saturated
bicarbonate solution. The organic phase is dried over sodium sulphate, filtered,~rm~ ntrRt~ and dried under high vacuum. 11.36 g of the compound:
42/
Boc~
OH CH3
are obtained.
E~.ghth step: 6.6 g (21 mmol) of compound 42 are dissolved in 500 ml of
methylene chloride. With exclusion of moisture (CaCl2 tube) a solution of
pentanoic anhydride [prepared from 2.16 g (21 mmol) of pentanoic acid and 2.16 g(10.5 mmol) of dicyclohexylcarbodiimide in 50 ml of methylene chloride,
filtration] in methylene chloride is added at room temperature. After 3 hr,
,-nnrPntrati,~n is carried out, followed by taking up in ethyl acetate, washing with
saturated bicarbonate solution and drying over sodium sulphate. Filtration and
concentration are followed by drying under high vacuum. 8.0 g (95.2% of theory)
of the compound:
116
WO 951~3084 2 1 7 5 5 6 4 PCTIUS94/07043
, ~
~ NH- CO~
Boc-NH . ~
OH CH3
are obtained.
Ninth ste~ 7.57 g (19 mmol) of compound 43 are stirred in 70 ml of 4N
hydrochloric acid/dioxane with the exclusion of moisture for 30 min. The
solution is concentrated, mixed with diethyl ether and evaporated to dryness.
After drying under high vacuum, 5.54 g (16.5 mmol) of the ~u~ uul~ding
hydrochloride, 4.46 g (33 mmol) of HOBT and 16.5 mmol of Boc-Val-OH are
dissolved in 500 ml of methylene chloride. After cooling to 0C, the pH is
adjusted to 8.5 with N-methyImorpholine, and 3.57 g (17.3 mmol) of
dicyclohexyl~dll,odiill.ide are added. After 16 hr at 20C, the urea is filtered off,
the solution is - -". .~ 1, taken up in ethyl acetate and washed with saturated
bicarbonate solution. Drying over sodium acetate is followed by u~ Llalion
and drying under high vacuum. The compound:
117
WO 95/13084 ` PCI/US94/07043
21 75564
44/ ~
~~ .
~, NH- CO~\ ,-
Boc--Val- NH ~
OH CH3
is obtained.
TPnthL step- 1.8 mmol of compound 44 are stirred in 11 ml of 4N
hydrochloric acid/dioxane for 30 min. The solution is concentrated, mixed with
diethyl ether and evaporated to dryness. After drying under high vacuum, 1.8
mmol of the resulting hydrochl~ride are dissolved in 50 ml of methylene
chloride and cooled to OC. After addition of 1.8 mmol of Boc-phenylalanine, thepH is adjusted to approximately 8 with triethylamine, and 875.2 mg (1.98 mmol)
of benzo triazolyloxy-tris(dimethylamino)-phosphonium hexafluorophosphate
are added. Reaction at room temperature for 16 hr is followed by l r~nrPntr~tion,
taking up in ethyl acetate and washing 3 x with saturated bicarbonate solution.
Inhibitor 5 is obtained in crude form and then chromatographed on silica gel.
Inhibitor 6 is obtained analogously to inhibitor 5, except that in the eighth
step a solution of 3-methylpentanoic anhydride [prepared from 21 mmol of 3-
methylpentanoic acid and 2.16 g (10.5 mmol) of dicyclohexylcarbodiimide in 50
ml of methylene chloride, filtration] in methylene chloride is used.
Inhibitor 17 is within the generic teachings of EP 0 472 077, sllpra, and can
118
WO gs/13084 ~ 1 7 5 5 6 4 rcT~sg4J/n~43
be prepared following the preparation schemes taught therein. Thus, inhibitor
17 can be prepared as follows:
First step: A solution of 5.07 g (20.00 mmol) of (S)-2-(tert-~uLu,cy~ul.ylamino-1-cyclohexylbut-3-ene U.R. Luly et al., 1987, ~. Org. C}1em., 52: 1487] in 100 ml of a 4
N sûlution of gaseous hydrogen chloride in anhydrous dioxane is stirred at room
temperature for 30 min. 15 ml of toluene is then added and the mixture is
concentrated in vacuo. This process is repeated twice more, then the residue is
tritura~ed with a little ether, filtered off with suction and dried in a high vacuum
over KOH. 3.76 g (99% of theory) of the compound:
45/
HCI x H2N~
are obtained as colorless crystals.
Second step: A stirred solution, cooled to 0C, of 4.63 g (21.3 mol) of N-
(tert-butoxycarbonyl)-L-valine and 3.29 g (24.35 mmol) of HOBT in 40 ml of
anhydrous dichloromethane is treated with 5.29 g (25.65 mmol) of DDC and the
mixture was stirred for 5 min. A solution of 3.60 g (19.00 mmol) of compound 45
and 8.85 ml (80.48 mmol) of N-methylmorpholine in 30 ml of dichloromethane
is then added dropwise. The cooling bath is removed and the reaction mixture
stirred at room temperature for 2 hr. The end of the reaction is ~ t~rmin~d by
119
WO 95/13084 2 1 7 ~ 5 6 4 PCI/US94/07043
thin layer chromatography. The resulting urea is removed by filtration, the
filtrate is concentrated in vacuo and the crude product is purified by
chromatography on 450 g of silica gel (dichloromethane/methanol 95:5). 4.33 g
(65% of theory) of the
46/
Boc~Val- NH~
are obtained as colorless crystals.
Third step: A solution of 4.32 g (12.30 mmol) of compound 46 in 100 ml
of a 4 N solution of gaseous hydrogen chloride in anhydrous dioxane is stirred at
room temperature for 30 min. 15 rnl of toluene is then added and the mixture is
concentrated in vacuo. This process is repeated twice more, then the residue is
triturated with a little ether, filtered off with suction and dried in a high vacuum
over KOH. 3.37 g (g5% of theory) of the compound:
47/
HCI x H-Val--NH~
are obtained as a colorless powder.
120
21 75564
WO 95/13084 PCTllTS9-~107043
FQllrth step: A stirred :~U~ iUlL~ cooled to 0C, of 4.47 mmol of Boc-L-
cyclohexylalanine [M.C. Khosla et al., 1972, J. Med. Chem., Z5: 792] and 0.66 g (4.92
mmol) of HOBT in 15 ml of anhydrous dichloromethane is treated with 0.97 g
(4.69 mmol) of DCC and the mixture is stirred for 5 min. A solution of 4.07
mmol of compound 47 and 1.80 ml (16.27 mmol) of N-methylmorpholine in 10
ml of dichloromethane is then added dropwise and the reaction is stirred at
room temperature for 1 hr. The resulting urea is removed by filtration, the
filtrate is concentrated in vacuo and the crude product is purified by
chromatography on 270 g of silica gel (dichlor--m~th~nf~ mf~thanol 95:5). The
compound:
48/
X J~ ~HJ~
H - H
o
is thus obtained.
Fifth step: A stirred suspension, cooled to 0C, of 0.60 mmol of
compound 48 in 3 ml of dichlorom~th~m~ is treated in portions with 2
equivalents of m-chlulv~lbel~uic acid (80% strength) and the mixture is stirred
at this ~ ul~ for 2 hr. A further 1 equivalent of m-chloroperbenzoic acid is
121
WO 95/13084 2 1 7 5 ~ 6 4 PCT/US94/07043
then added and the mixture is subsequently stirred at room temperature for 1 hr. 10 ml of ethyl acetate is then addecl and the reaction mixture stirred into 20 ml of
a 10% strength NazSO3 solution. The organic phase is separated off, washed 3 x
with 10 ml of NaHCO~ and dried over MgS04. After evaporating the solvent in
vacuo and triturating the residue with a little ether/pentane, inhibitor 17 is
obtained.
Inhibitor 14 is known from published European application EP 0 437 72g,
which was published on July 24, 1991, and corresponds to U.S. Patent No.
5,145,951, which issued on September 8, 1992. The entire contents of both
publications are incorporated herein by reference. The inhibitor is disclosed
therein as an inhibitor of HIV protease activity.
Inhibitor 18 is known from published European application EP 0 403 828,
which was published on December 27, 1990, and corresponds to U.S. Serial No.
07/876,697, filed April 28, 1992, still pending, which is a ~ ontinll~tinn of U.S.
Serial No. 07/524,779, filed May 16, 1990, now abandoned. The complete
disclosures of these three applications are incorporated herein by reference. The
inhibitor is disclosed therein as an inhibitor of HIV protease activity.
Inhibitor 21 can be prepared as follows: A stirred solution, cooled to 0C,
of 227 mg (0.68 mmol) of (2S)-3-tert-butylsulphonyl-2-(1-naphthylmethyl)-
propionic acid [prepared according to H. Buhlmayer et al., J. Med. Chem., 31: 1839
(1988)] and 104 mg (0.68 mmol) of HOBT (1-hydroxybenzotriazole) in 5 ml of
anhydrous dichlor~m~th~nf~ is treated with 134 mg (0.65 mmol) of DDC
(dicyclohexylcarbodiimide) and stirred for 5 min. A solution of 400 mg (0.62
122
~ wo 95/13084 2 1 7 5 5 ~ S94~07043
mmol) of 1-{2R,S,4S,5S)-5-[S-valinyl-amino]-6-cyclohexyl~-hydroxy-2-(1-methyl)-
ethyl-hexanoyl}-S-isoleucinyl-2-pyridylmethylamide ~ hlf~ri~ and 0.27 ml (2.47
mmol) of N-methyl-morpholine in 10 ml of dichloromethane is then added
dropwise and the reaction is stirred at room temperature for 2 hr. The resultingurea is removed by filtration, the filtrate is ~ d~d in vacuo and the crude
product is purified by chromatography on 60 g of silica gel
(dichlor--m~ ""~ l 95:5) 68 mg (11% of theory) of the less polar (2R)-
isomer is obtained as a colorless powder. [~elting point: 187-189C (dec.); Rf =0.15 (dichloromethane:methanol 95:5); MS (FAB): m/z = 890 (M+H)+.]
Furthermore, 75 mg (13% of theory) of the more polar (2S)-isomer is obtained as
a colorless powder. [Melting point: 239-240C (dec.); Rf = 0.13
(dichlorom~ ",f ll.dnol 95:5); MS (FAB): m/z = 890 (M+H)+.]
1-{2R,S,4S,5S)-5-[S-valinyl-amino]-6-cyclohexyl-4-hydroxy-2-(1-methyl)-
ethyl-hexanoyl}-S-isoleucinyl-2-pyridylmethylamide .1iehlr,ril1~ is prepared as
follows: A solution of 505 mg (0.75 mmol) of 1-{2R,S,4S,5S)-5-[N-(tert-butoxy
carbonyl)-S-valinyl-amino]-6-cyclohexyl -4-hydroxy-2-(1-methyl)-ethyl-hexanoyl}-S-isoleucinyl-2-pyridylmethylamide in 4.6 ml of a 4 N solution of gaseous
hydrogen chloride in anhydrous dioxane is stirred at 0C for 1 hr. 10 ml of
toluene are then added and the mixture is ~ulL~ ila~d in vacuo. This process is
repeated a further two times, and the residue is then triturated with ether,
filtered off with suction and dried in a high vacuum over KOH. 405 mg (84% of
theory) of 1-{2R,S,4S,5S)-5-[S-valinyl-amino]-6-cyclohexyl~hydroxy-2-(1-methyl)-ethyl-hexanoyl}-S-isoleucinyl-2-pyridylmethylamide tliehlf,ri~l.o are obtained as a
colorless powder. [Melting point: 177-179C (dec.); Rf = 0.38 (acetonitrile/water
9:1); MS (FAB): m/z = 574 (M+H)+.]
123
WO 95/13084 PCT/US94/07043
2 1 75564
1-{2R,5,4S,5S)-5-[N-(tert-butoxycarbonyl)-S-valinyl-amino]-6-cyclohexyl~-
hydroxy-2-(1-methyl)-ethyl-hexanoyl~-S-isoleucinyl-2-pyridylmethylamide is
obtained as follows: A stirred solution, cooled to 0C, of 239 mg (1.10 mmol) ofN-(tert-bu~o~y~all,ol~yl)-S-valine and 149 mg (1.10 mmol) of HOBT in 10 ml of
anhydrous dichloromethane is treated with 434 mg (1.05 mmol) of DDC and the
mixture is stirred for 5 min. A solution of 0.55 g (1.00 mmol) of 1-~(2R, S, 4S, 5S)-
[5-amino-6-cyclohexyl-4-hydroxy-2-(1 -methyl)ethyl-hexanoyl]}-S-isoleucinyl-2-
pyridylmethylamide dihydrochloride [EP 437 729] and 0.44 ml (4.00 mmol) of N-
methylmorpholine in 10 ml of dichloromethane is then added dropwise. The
cooling bath is removed and the reaction mixture can be stirred at room
temperature for 8 hr. The end of the reaction is ~ieterminP~ by thin layer
chromatography. The resulting urea is removed by filtration, the filtrate is
concentrated in vacuo and the crude product is purified by chromatography on
45 g of silica gel (dichlo~ ,PII~ ]~ l 9:1). 507 mg (75% of theory of 1-
~2R,S,4S,5S)-5-[N-(tert-b utoxycarbonyl)-S-valinyl-amino]-6-syclohexyl-4-hydroxy-
2-(l-methyl)-ethyl-hexanoyl~-s-isoleucinyl-2-pyridylmethylamide are obtained as
a colorless powder. [Melting point: 187C (dec.); Rf = 0.39, 0.44
(dichloromethane/methanol 9:1); MS (FAB): m/z = 674 (M+H)+.]
Inhibitor 22 is prepared as Çollows To a stirred solution of 122 mg (0.22
mmol) of the compound from example XI~I; page 62 in EP 528 242, the disclosure
of which is i~ ol~.L~d herein by reference, and 26 mg (0.22 mmol) of 1-methyl-
lH-tetrazole-5-thiol in 2 ml of dry dichloromethane at 0C was added 28 111(0.22mmol) of boron triflllf)ri~P etherate. The mixture was stirred for 45 min at 0Cand then poured into a mixture of 10 ml of ethylacetate and 10 ml of saturated
124
WO g5/13084 2 1 7 5 5 ~ 4 PCT~US94107043
aqueous NaHCO3. The organic layer was separated, washed with 10 ml of
saturated aqueous NaHCO3 and water and dried over MgSO4. Removal of the
solvent under reduced pressure and ~lu~ aLo~ phy of the residue on 50 g of
silica gel (dichl.,-oll.eLl,al,e:methanol 95:5) afforded 45 mg (31%) of inhibitor 22
as colorless crystals. [Melting point: 178C (dec.); Rf = 0.18
(dichloromethane/methanol 95:5); MS (FAB): m/z = 660 (M+H)+; IH-NMR (250
MHz, CD30D)~ = 3,88 (s, 3H, NCH3), 5.01 (s, 2H, PhCH2O), 7.2 (m, lOH, Ph).]
Inhibitor 23 is prepared as follows: 100 mg (0.11 mmol) of 1-{(3RS, 4S)-4-
[N-(Ethoxycarbonyl)-S-phenylalanyl-S-histidyl-amino]-5-cyclohexyl-3-hydroxy-
pentanoyl~-S-leucyl-3{N-(benzyloxycarbonyl)-aminomethyl~enzyl-amide
[prepared by standard methods of peptide synthesis] in 5 ml of methanol are
stirred with 100 mg of 10% Pd/C and 150 mg (2.4 mmol) of Ammon;l1m formate
at 60C for 4 hr. After filtration and concentration, the residue is taken up indichlorr m~thAn~ washed with brine twice, and dried over Na2SO4. Removal of
the solvent and Iyophilization gave 60 mg (71%) of inhibitor 23 as a white fluffy
powder. [Rf = 0.42, 0.45 (dichloromethane/methanol/conc. aq. ammonia 9:1:0.1);
MS (FAB): m/z = 788 (M+H)+.]
Inhibitor 24 is prepared as follows: 2.60 g (2.7 mmol) of 1-{(3RS, 4S)-4-[N-
(tert.-butoxycarbonyl)-S-tyrosyl-S-isoleucyl-amino] -5-cyclohexyl-3-hydroxy-
pentanoyl~-S-leucyl-3-[N-(benzyloxycarbonyl)-am inomethyl~enzylam ide
[prepared by standard methods of peptide synthesis] in 100 ml of ml~th~nrll,
~ntAinin~ 300 mg of 10% Pd/C, are hydrogenated at room temperature and
atmospheric pressure for 5 hr. After filtration and concentration, the residue is
purified by chromatography on silica gel eluting with
125
WO 95/13084 2 PCT/US94/07043
dichl~,lv~ Lllal~e/methanol/conc. aq. ammonia (15:1:0.1-->9:1:0.1) to give 1.71 g
(76%) of inhibitor 24 as a white solid. [Rf = 0.26
(dichloromethane/methanol/conc. aq. ammonia 9:1:0.1); MS (FAB): m/z = 823
(M+H)~ ]
Compounds 25 and 26 are known from EP 472 078 (equivalent to USP
5,147,865) and EP 528 242, respectively. These compounds are inhibitors of
another aspartic protease (HIV protease), but, as shown in Table 6 above, are not
active (ICso's > 1 IlM) in vitro against cathepsin D nor do they inhibit cellular
amyloid release. Accordingly, these compounds are included as negative
controls.
Example 14. Inhibition of BA4 in cell culture.
Cell ctllt1-re: Transfectants of HEK293 cells were done with
DNA/lipofectin mixtures (Gibco/BRL) of a pCEP4 construct (Invitrogen Corp.,
San Diego, CA) t~ tnt~inin~ the full-length open reading frame of APP695 cDNA
(Kang et al., 1987, Nature, 325: 733-736). Stable cell lines were selected through
vector mediated hygromycin resistance.
Vector construction for CHO ~PIIC A 2.36 kb NruI/SpeI fragment of
APP695 cDNA form FC~ (Kang et al., id.) was filled in by the large fragment of E.
coli DNA polymerase I and blunt-end inserted into the SmaI cloning site of the
KS Bluescript M13+ vector (Stratagene, La Jolla, CA) resulting in pMTI-5. A new
Kozak consensus DNA sequence was then created using site-specific mllt~gf~n~cic
(Kunkel et al., 1987, Metllods in Enzymology, 154: 367) with the oligo: 5'-ctc tag
126
wo 95/13084 2 7 5 5 ~ 4 PCTIUS94107043
aac tag tgg gtc gac acg atg ctg ccc ggt ttg-3' (SEO ID NO.: 8) to create pMTI-39. pMTI-
39 was NotI/HindIII digested and the 2.36 kb APP695 cDNA fragment was then
gel-purified and ligated into NotI/HindIII cut pcDNAlNeor (I~viLlvg~ ) to createpMTI-72 in which the APP695 expression is placed under the control of the CMV
~ promoter. (-~n~r~tinn of stable CHO cell lines is as described in example 4,
method 1, part (ii).
Vector construction for HE~93 cells: A 2.8 kb SmaIlHindIII fragment of
the APP695 cDNA was isolated from the pSP65/APP695 cDNA (prepared by
Dyrks et al., 1988, EMBO J., 7: 949-957). The pCEP4 vector (Invitrogen) was cut
with PvuII/HindIII, and then ligated with the 2.8 kb APP SmaI/HindIII fragment
resulting in pCEP/695. In this plasmid the expression of the APP cDNA is under
the control of the CMV promoter.
M~int~n~n~e of cell lines: HEK293 cells were maintained in DMEM, 10%
fetal calf serum, 50 units Pen/Strep, and 2 mM gl~ rnine (BRL/Gibco) at 37C
under 5% CO2, 95% humidity.
The CHO cell line was maintained in c~MEM, 10% fetal calf serum, 50
units Pen/Strep, and 2 mM glutamine (BRL/Gibco) at 37C under 5% CO2, 95%
humidity.
All media were ~ llas~d from Gibco/BRL and JRH Biosciences.
On the day of the experiment, cells u~ere split into 60 mm dishes to reach a
confluency of d~lv,-i,-,a~ely 80%. Drugs (stock solutions in DMSO) were added 8
127
2 1 7 5 5 ~ 4 PCT/US94107043 o
hr after plating to a final concentration of 10 IlM and incubated for 14 to 16 hr in
the incubator. The next day, the media was removed, cells were washed hwice in
pl~w~ ed PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCI, 2.7 mM KC1,
pH 7.4), starved in DEM mPthi~-ninP minus media for 30 min. and then labeled
with 150 IlCi 35S-methionine (Amersham) for 3 hr (all in the presence of drug).
Cell dishes were placed on ice, the conditioned media was removed (4C),
cellular debris was spun down (4C, 5 min at 15,000 g), the supernatant was
adjusted to 1 x RIPA buffer (150 mM NaCI, 1%NP-40, 0.5% Na deoxycholate, 0.1%
SDS, 50 mM Tris-HCl pH 8.0) with the following protease inhibitors (1 ,ug/ml
leupeptin, 0.1 llg/ml pepstatin A, 1 mM PMSF, 2 llg/ml Aprotinin), and
incubated at 100C for 5 min. and cooled down again.
Imm--n~ ,,tion: 1 ml of conditioned media was preincubated (2 hr
at 4C, rocking) with 15 1ll normal mouse serum and 150 Ill Omnisorb
(Calbiochem). The slurry was cleared by centrifugation (4C, 5 min. at 10,000 g)and incubated with 10 ~1 monc)~ n~l antibody 286.8A (l~O~ epitope '1-7' of
~A4 sequence) (4C, 16 hr). Bound immunocomplexes were precipitated with
Omnisorb (150 Ill, 4C for 2 hr) and spun down (4C, 5 min at 10,000 g).
Imlllullo~l~cipitates were washed h~ice for 5 min with ice-cold wash buffer C (10
mM Tris pH 8.0, 150 mM NaC1). Tmml1n~ mrlexes were resuspended in 2x
Tris/Tricine sample buffer, boiled (100C for 10 min) and separated on reducing
and ~i.on~t1lring 16.5% or L0-20% Tris/Tricine-SDS/PAGE (Schagger and Jagow,
1987, Anal. Biocl~em., 166: 368). Gels were fixed, dried and amplified (Amplify,~mPr~h~m) and subsequently exposed to Fuji Phospo-imagerTM plates and to
Kodak X-ray film (-70C).
128
wo ss/l3084 ~ 1 7 5 5 6 4 PC~IUS94107043
~ esults: Of the HEK293 transfectants, one clcne (293/695.9) sho~red an
even higher expression of APP695, than was reported for the CHO/695 cells, as
demonstrated by Western-blotting with anti-APP monoclonal antibody 22C11
(prepared by Weidemann et al., 1989, Cell, 7: 115-126) and by
immunv~ iL~Ition with the anti-APP C-terminal antibody 91.07 raised against
the cytoplasmic region of ~PP (as described in example 6 (ii)). Both cell lines also
rapidly secreted a major portion of their APP pools into the r-~trArrlll~lAr media
(conditioned media), demonstrated by immunoprecipitation with polyclonal
rabbit antibody 45.7, raised against a bacterial APP fusion protein (prepared byWeidemann et al., i~.).
To detect Lhe released ~A4 in rr,n-litir,n~d media, cells were radioactively
labeled with 35S-mr-thir,ninP and the labeled proteins in the conditioned media
released by the cells were immunoprecipitated with monoclonal antibody
286.8A. Both cell lines showed immunoreactive bands irl the rr~n~itir~nr-d mediaof approximately 4 kDa on the fluorograms of the 16.5% Tris/Tricine
polyacrylamide gels, which were visible after 24 hr exposure time, or about 2 hrexposure on the phosphoimager plate. In addition, mr,nrlrlr,nAI antibody 286.8A
precipitated a protein with an apparent molecular weight of 102 kDa (~Ptrrminr~
for the CHO/695 cell line), which ~ se- Ls the secreted APP cleaved at the C-
terminus by the so-called o!-secretase (APPs). The identity of the 4 kDa band was
rrlnfirmf~d by three additional independent antibodies, two polyclonal rabbit
antibodies raised against the ~A4 sequence '2-43' (rPAb 63122) and "1-40"
(rPAb3572) and a monoclonal antibody against the ~A4 sequence '17-24' (4G8;
Kim et al., 1988, Neur. ~es. Comm., 2: 121-130). FurLhermore, the identity and
approximate size of ~A4 was confirmed b~ running in parallel an in vitro
129
WO95/13084 2 ~ 755b4 PCTIUS94/07043 ~
translated radioactively labeled (35S-m~thi~nin~) BA4 '1-42' peptide. Antibodies4G8, 63122 and R3572 precipitated also a peptide which had approximately the
same intensity as BA4, at around 3 kDa. This signal is derived from a precursor
molecule, which was cleaved by the -secretase (o~-cut; position +16/17 of BA4)
and at the C-terminal end of ~A4 (herein denoted y-cut; after position +39 to 43 to
BA4; see also Figure 25). Previously, this fragment was also referred to as 'p3'(Haass et al., 1992, N~l~ur~, 359: 322-325). Therefore, it can be rr~n~ rl that the
two transfected cell lines tested produce cignifi~Ant levels of BA4 and release it
into the medium within a relatively short time.
The two transfected cell lines were used to evaluate the ability of the
inhibitor compounds (example 13) to inhibit the secretion of ~A4. The cells wereincubated for 16 hr with 10 ~LM of the inhibitor. Next, the cells were washed with
serum-free media and then metabolically labeled with 355-m~thionin~ for 3 hr in
the presence of the inhibitor. The intensities of the BA4 signals were
by phospho-imager analysis.
Six compounds out of the 24 selected cathepsin D in vitro inhibitors were
identified in the cellular assay with HEK293/695 cells, which reduced the levelsof BA4 to 50% or less. The experiments were repeated at least twice and the
values for compounds which produced a .cignifil Ant reduction is given in Table
8. Structurally very similar compounds, such as inhibitors #25 and #26, which
showed no strong activity in the cathepsin D in vitro assay, were also tested for
activity in the cellular assay system. These latter two compounds did not
influence significantly the levels of BA4, as shown in table 8. the amounts of
secreted APP did not change profoundly, as judged by the phospho-imager signal
130
wo 95/13084 2 1 7 5 5 6 4 PCIIUS94107043
intensi~y at about 100 kDa on high-resolution gels. None of the inhibitors tested
inflll~n~cl cell viability, since there was no change of cellular gross morphology.
As a further indication of cell viability, inhibitors which inhibited the
production of ~A4 in cells were tested for their effect on the conversion of the" salt MTT into formazan (CellTiter96TM Assay, Promega). Except for a
slight reduction with inhibitor #10 to 67% of control values, no Ri~nifi~ Rnt
change in the ability of cells to reduce MTT was observed.
In view of the foregoing, it should be clear that six of the inhibitors
~ onfifi~cl in example 6 also show significant effects in these cell-based assaysystems. All six compounds reduced the amount of secreted ~A4 by more than
50% when compared to a control. The reduction in 13A4 was rt~nfirmf~d by an
additional independent anti-~A4 antibody. Moreover, a cell viability tests
showed no measurable difference, except for #10. Therefore, the six identified
compounds, may prove to be therapeutically beneficial by inhibiting the
production/R,^~Iml1lAfi--n of the ~A4 subunits to form amyloid plaques.
131
WO 95113084 PCTIUS94/07043
2 1 75564
Table 8: Potency of Inhibitors of Cathepsin D as Inhibitors of li~A4 Formation
in Cell Culture
Inhibitor # % Reduction of ~A4 ICso in ~M
(at 10 !lM) (where 11Pt.~rTnin,
> 90 nd
2 290 ~2
3 290 -0.4
4 53 nd
290 nd
21 2 90 ,= 0.4
0 nd
26 22 nd
132
wo g5~30W 2 l 7 5 5 6 ~ PCTJUS94107043
.
It will be appreciated that the instant sp~ifil-~ti~)n and claims are set forth
by way of illustration and not limit~ti~n, and that various moflifi~til~nc and
changes may be made without departing from the spirit and scope of the present
invention. Specifically, other inhibitors disclosed in the abov.~ml~nti(-n(~.1
applications will also be useful as described herein. The claims are intended tocover these other embodiments as well.
133
WO 95/13084 2 1 7 5 5 6 4 PCr/US94/07043
r~F- LIJTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT: Tamburini, Paul P.: Benz, Gunter; Habich,
Dieter ; Dreyer , Robert N .; Koenlg , Gerhard
(ii) TITLE OF INVENTION: CATHEPSIN D IS AN AMYLOIDOGENIC
PROTEASE IN Ar~7:HT~TMER ' S DISEASE
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Miles Inc.
(B) STREET: 400 Morgan Lane
(C) CITY: West Haven
(D) STATE: Connecticut
( E ) COUNTRY: USA
(F) ZIP: 06516
(V) O,'~1~U'1'L K R E A n ART E FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch, 800 k~ storage
( B ) ~J. .~u 1 ~K: Sharp PC 4 6 0 0
(C) OPERATING SYSTEM: MS-DOS
( D ) SO FTWARE : WordPer f ect 5 .1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT~US94/07043
(B) FILING DATE: June 21, 1994
(C) CLASSIFICATION: Unassigned
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PC~US93/10889
(B) FILING DATE:: November 12, 1993
(vii) PRIOR APPLICATION DATA:
134
RECrl~IED SHEET (~ULE 91)
~ WO 951130~4 2 1 7 5 5 6 4 PCrlUS94107043
(A) APPLICATION NUMBER: 07/995, 660
(B) FILING DATE: December 16, 1992
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NTJMBER: 07/880, 914
(B) FILING DATl~: May 11, 1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pamela A. Simonton
(B) REGISTRATION NUMBER: 31, 060
(C) REFERENCE/DOCKET NUMBER: MTI 224 3
(iX) TT~'T,T;'('~ lMT(' ~TION INFORMATION:
(A) TELEPHONE: (203 ) 937-2340
(B) TELEFAX: (203 ) 937-2795
( 2 ) INFORMATION FOR SEQ ID NO: 1:
U~:N~:~: CHARACTERISTICS:
(A) LENGTH: 16 ami~o acids:
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(Xi) ~ U~;N~:~: DESCRIPTION: SEQ ID NO: 1:
Ile Ser Glu Val Lys Met Asp Ala Glu Phe
5 10
Arg His Asp Asp Asp Asp
( 2 ) INFORMATION FOR SEQ ID NO : 2:
135
WO 9~/13084 4/07043
2 ~ 7 ~ 5 ~ 4 Pcr/usg ~
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
~B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ile Ser Glu Val Lys Met Asp Ala Glu Phe
5 10
Arg His
( 2 ) INFORMATION FOR SEQ ID NO: 3 -
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
( B ) TYPE: amino acid
( D ) TOPOLOGY: 1 inear - = ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ile Ser Glu Val Asn Leu Asp Ala Glu Phe
5 10
Arg
( 2 ) INFORMATION FOR SEQ ID NO: 4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
( D ) TOPOLOGY: linear ~ -
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Glu Ile Ser Glu Val Lys Met Asp
136
.
-
21 75564
~ WO 95/13084 PCT~Ss4Jn7n43
~ 2 ) INFORMATION FOR SEQ ID NO: 5:
( i ) S~;UU~;NI~; CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) ~:UU~;N~: DESCRIPTION: SEQ ID NO: 5:
Trp His Ser Phe Gly Ala Asp Ser
( 2 ) INFORMATION FOR SEQ ID NO: 6:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
( D ) TOPOLOGY: l inear
(Xi) ~ U~N(:~: DESCRIPTION: SEQ ID NO: 6:
Gly Glu Gly Phe Leu Gly Asp Phe Leu
~ 2 ) INFORMATION FOR SEQ ID NO: 7:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ll amino acids ~
(B) TYPE: amino acid
( D ) TOPOLOGY: linear:
(Xi) S~:UU N~:~: DESCRIPTION: SEQ ID NO: 7:
Ile Ser Glu Val Lys Met Asp Ala Glu Phe
5 l0
Arg
137
WO 95/130~4 2 1 7 5 5 6 4 PCr/US94/07043 ~
( 2 ) INFORMATION FOR SEQ ID NO: 8:
~i) ~il:;~,,)U N(.:~: CHARACTERISTICS:(A) LENGTH: 39 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single = .=.
~D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: cDNA to mRNA
(iii) PUBLICATION INFORMATION:
(A) AUTHORS: Kan~ et al.
(B) JO~RNAL: Nature
(C) VOLUME: 325
(D) PAGE: 733
(E) DATE: lg87
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CTCTAGAACT AGTGGGTCGA CACGATGCTG~ CCCGGTTTG . 39
(2) INFORMATION FOR SEQ ID NO: 9:
(i) ~:i~;UU~:N~: CHARACTERISTICS:
(A) LENGT~: 6 amino acids
(B) TYPE: amino acid
( D ) TOPOLOGY: linear
(Xi) ~:QU~;N~:~: DESCRIPTION: SEQ ID NO: 9:
Ile Ser Glu Val Asn Leu
(2) IN~ORMATION FOR SEQ ID NO: lû:
.)U~:N~:~: CE~ARACTERISTICS:
138
WO95113084 2 1 7 5 5 6 4 PCT~S94/07043
(A) LENGTH: 30 nucleotides
(B) TYPE: nucleic acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
- AGG AGA TCT CTG AAG TGA ATC TAG ATG CAG 30
(2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 nucleotides
(B) TYPE: nucleic acid
( D ) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:
CAT GAA GCA TCC CCC ATC GAT TCT TAA AGC 30
139
