Note: Descriptions are shown in the official language in which they were submitted.
CA 022428~1 1998-07-13
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--1--
PROTEIN FIBRIL ASSFMRLY ASSAY
The invention is a method for in vi tro monitoring of
~ protein fibril assembly at physiologically relevant
concentrations. The invention i5 readily adaptable for
in vitro monitoring of fibril assembly processes
associated with various amyloidosis disorders, such as
Al~hetm~'s disease, multiple myeloma, rheumatoid
arthritis, diabetes, and prion disorders. The invention
has particular utility as a screening assay for potential
inhibitors of fibril formation, which in turn may be
candidates for treatment o~ Alzheimer's disease or other
amyloidosis disorders.
Alzheimer's disease is a neurodegenerative disorder
of the elderly. While neurodegeneration in Al~h~;m~r's
disease begins slowly, its progression follows an
exponential course, and it finally culminates in dementia
and death (Muller-Hill and Beyreuther, 1989). Key
pathological features of Al~h~;m~-'s disease found in the
brains of afflicted patients include the following: a)
senile plaques, which are extracellular amyloid deposits
in close contact with neurons, b) neurofibrillary
tangles, which are intraneuronal deposits of paired
helical filaments, and c) cerebrovascular amyloid, which
are amyloid deposits in the walls of cortical and
meningeal vessels (Selkoe, 1991; Katzman and Saitoh,
1991). The link between the symptoms of Alzheimer's
disease and its pathology arises from the plaques and
neurofibrillary tangles being concentrated around the
hippocampus, a region of the brain responsible for memory
and learning.
.
A major component of senile plaques and
cerebrovascular amyloid is Al~hPtm~r/s ~-amyloid peptide
(A~), a 39-43 residue peptide that folds into a ~-sheet
structure and assembles into fibrils 60-goA in diameter
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--2--
(Fraser et al., 1993). A~ is a fragment of a much larger
integral membrane protein called amyloid precursor
protein (APP). APP possesses one transmembrane segment, a
large extracellular ~om~; n, a small intracellular ~om~; n,
several glycosylation and sulfation sites, and a Kunitz
protease inhibitor ~om~; n . A~' is derived from residues
672 to 715 of APP, and it encompasses the C-terminal
region of the extracellular ~om~; n of APP and the
N-terminal half of the transmembrane ~o~;n of APP.
Other proteins found in senile plaques include: serum
complement proteins (Clq, C3d, and C4d), serum amyloid P,
~1-antichymotrypsin, Apo-E, and heparan sulfate
proteoglycans. The relative amounts of these proteins in
senile plaques are, however, much lower than that of A~.
The other characteristic feature of Alzheimer's
disease are neurofibrillary tangles, which are
intraneuronal deposits of paired helical filaments.
Paired helical filaments are not as well characterized as
senile plaques (Crowther, 1991). The protein components
of paired helical filaments discovered so far include:
neuro~ilament protein, a microtubule associated protein
(MAP) called tau, MAP2, and MAP5. Recent evidence
suggests that paired helical filament formation results,
at least in part, from abnormal phosphorylation of tau.
The molecular pathogenesis of Alzheimer's disease is
not well established. However, there is accumulating
evidence that deposition of A~'-fibrils in senile plaques
may account for much of the neurotoxicity of Alzheimer's
disease (Selkoe, 1993). Neurons which contact A~-fibrils
are dystrophic. Patients that have three copies of the
APP gene (i.e. Down's syndrome patients) produce a
greater number of senile plaques and develop Alzheimer's
disease when they reach their late thirties. Mutations
in the APP gene are often seen in cases of familial
Alzheimer's disease, and related genetic diseases (Goate
CA 022428~1 1998-07-13
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et al., 1991). A~-fibrils are toxic to neurons in
culture (Yankner et al., 1989; Pike et al., 1993).
Alzheimer-type neuropathology is induced in transgenic
mice overexpressing a mutant form of APP (Games et al.,
1995). These findings all point to A~ as the primary
~ neurotoxic agent o~ Alzheimer's disease. The mechanism
of A~ neurotoxicity is also not well understood, but it
appears to be related to calcium ion homeostasis in
neurons (Mattson et al., 1993).
Two experimental findings that must be reconciled by
any proposed model o~ Alzheimer's disease pathogenesis is
that A~ is a product of normal cells (Haass et al.,
1992), and that it is present in cerebrospinal fluid and
blood plasma o~ healthy individuals (Seubert et al.,
1992). Experimental evidence suggests that there may be
multiple primary causes that ultimately lead to A~
deposition in senile plaques and Alzheimer's disease
(Selkoe, 1993). While certain individuals may develop
Alzheimer's disease by producing greater amounts o~ A~,
others might develop the disease by producing a ~orm o~
A~ that is especially prone to fibril formation. Thus,
the concentration of A~ in the brain as well as its
intrinsic tendency to aggregate are both integral to the
process of A~-fibril deposition in Alzheimer's disease.
Given the possibility that fibril formation by A~
may account for the neurotoxicity in Alzheimer's disease,
it follows that molecules that inhibit fibril formation
should be good candidates for therapeutic agents for the
treatment of Alzheimer's disease. One critical research
tool for evaluating therapeutic inhibitors is an assay
~ ~or A~-fibril formation. Current methods that monitor A~
fibril formation employ various biophysical techniques,
~ such as electron microscopy, X-ray diffraction, ~ourier
transform infrared (FTIR) spectroscopy, circular
dichroism (CD) spectroscopy, and nuclear magnetic
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resonance (NMR) spectroscopy. Studies utilizing these
biophysical techniques have shown that A~ fibrils possess
remarkable intrinsic stability. Even after altering much
of the sequence of A~, fibril formation still occurs
(Fraser et al., 1992; Hilbich et al., 1992; Fraser et
al., 1994). Asp23 of A~, however, is a unique residue;
since when it is replaced with Lys a significant
reduction in the stability of the fibril is observed
(Fraser et al., 1994). Two regions of the A~ sequence
show a high propensity to form ~-structure: residues 9
to 28 (Fraser et al., 1991) and residues 34 to 42
(Halverson et al., 1990). One factor which has a major
effect on fibrillogenesis is pH; at low (~ 3) and high
(~10) pH, fibrillogenesis by A~ analogues is both slower
and less stable (Fraser et al., 1991; 1992). It appears
that hydrogen bond, hydrophobic, and electrostatic
interactions all act in concert to stabilize the A~
fibril; however, a high resolution structural model of
the A~ fibril has yet to be developed.
The studies on the mechanism of fibrillogenesis
mentioned have employed methods of measuring fibril
formation which require sample concentrations that are
200,000 to 5,000,000-fold higher than the physiological
concentration. Fibril assembly is a bimolecular
reaction: A~ + (A~)n z (A~)n~l. One distinguishing
feature of bimolecular reactions is that both their
kinetics and thermodynamics are concentration-dependent.
It follows, therefore, that the concentration of A~ in
the brain and cerebrovasculature controls the rate of
amyloid fibril formation as well as the intrinsic
stability of amyloid plaques. Consequently, the
mechanism of fibril assembly at low physiological
concentrations may differ from the mechanism that
operates at the high concentrations required by the above
mentioned biophysical techniques. Any attempt to model
Alzheimer's disease in vitro should take into
.
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--5--
consideration the relatively low concentrations of A~ in
the brain and cerebrovasculature.
- Another limitation of using biophysical techniques,
such as electron microscopy, FTIR, CD, and X-ray
di~fraction, to follow fibril formation is the technical
complexity of such techniques. Specialized training and
involved procedures are required; thus, limiting the
usefulness of these techniques as screening tests for
fibrillogenesis inhibitors.
In contrast to prior techniques, the invention
provides a very sensitive fluorescence technique ~or
monitoring fibrillogenesis by A~ at concentrations close
to the physiological concentration of A~ in the brain.
This fluorescence techni~ue can be adapted into a rapid,
technically simple, standardized assay to simultaneously
screen many compounds that could potentially inhibit
fibril formation.
The skilled person will appreciate that the method
as applied to A~ fibrillogenesis has general application
for the assaying of fibrillogenesis associated with other
amyloidosis disorders as mentioned above.
Accordingly, the invention provides a method for in
vitro monitoring of peptide or protein fibril assembly,
comprising the steps of:
1) attaching a donor ~luorophore to a first
fibrillogenic peptide or protein;
2) attaching an acceptor ~luorophore to a second
fibrillogenic peptide or protein, the donor and acceptor
fluorophores being located on said first and second
peptides or proteins so that they are juxtaposed to
permit energy transfer between them upon fibril
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--6--
formation;
3) mixing the first and second fluorophore-
containing peptides or proteins in solution at an
approximately normal physiological concentration; and
4) monitoring formation of fibrils by observing
the occurrence of fluorescence energy transfer between
the donor and acceptor fluorophores upon excitation of
the donor fluorophores.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows fluorescence emission spectra of
fibrils composed of Trp-A~(9-25) and AEDANS-A~(9-25).
Figure 2 is a graph showing pH dependence of fibril
formation monitored by fluorescent fibrillogenesis assay,
and attenuation of the titration curve in the presence of
0.5 M NaCl.
Figure 3 is a graph of the kinetics of mnnom~r
fibril exchange.
Figure 4 is a graph showing the promotion of fibril
formation by heparin as monitored by the fluorescence
assay of the invention.
The invention will be described in relation to a
specific assay for A~ fibril assembly. The following
description while specific for A~ fibrillogenesis
illustrates the general principles of the invention which
may be used to assay fibril assem-bly processes in other
amyloidosis disorders.
Develop~nent of a fluorescent fibrillogenesis assay.
Fluorescence spectroscopy is a very sensitive
CA 022428~1 1998-07-13
W O 9~J074~2 PCT/CA9~ '5
--7--
technique, and depending on the quantum yield of the
fluorophore, the technique may be sensitive down to the
nAnom~lar concentration range. The physiological
- concentration of A~ in the brain is estimated to be in
the nanomolar range; therefore, the sensitivity of
~1uorescence spectroscopy is in principle sufficient to
detect physiological concentrationS of A~. Unfortunately,
fluorescence cannot be used to ~Am;ne fibril formation
by native A~ because the only intrinsic fluorophore
present in A~ is the phenolic side chain of TyrlO, and
its very low quantum yield is ;nA~e~uate ~or sensitive
measurements. Extrinsic fluorescent groups, however, can
be chemically attached to A~, and the techni~ue of
nonradiative fluoresence energy transfer can be used to
measure fibril assembly by A~ in the nanomolar
concentration range.
The principle of fluore~cence energy transfer is
that when appropriate donor and acceptor fluorophores are
close in space, light energy absorbed by the donor can be
transferred to the acceptor, and the efficiency of energy
transfer depends on the distance separating the donor and
acceptor (see Fairclough and Cantor, 1978). The following
example outlines the general method of energy transfer
measurements. The fluorescent amino acid tryptophan,
absorbs light of 281 nm, and after absorption it emits
light of 337 nm. The fluorescent compound 5-acetylethyl-
di~minonAphthalene-l-sulfonic acid (AEDANS), on the other
hand, absorbs light of 281 nm very poorly but it absorbs
light of 337 nm very strongly; after absorption AEDANS
emits light of 490 nm. Since the absorption band of
AEDANS coincides with the emission band of Trp, Trp and
AEDANS can be used as donor and acceptor, respectively,
in a fluorescence energy transfer experiment. To
~ illustrate the method consider a solution contA;n;ng a
mixture of Trp and AEDANS, if the mixture is irradiated
with light of 281 nm then the wavelength of the emitted
CA 022428~1 1998-07-13
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light depends on the distance separating the Trp and
AEDANS fluorophores. If the two fluorophores are close
in space (~ 30 A) then the emitted light will be of 490
nm pre~om;n~ntly; on the other hand, if the chromophores
are well separated in space the emitted light will be of
337 nm pre~om;n~ntly. Using Trp and AEDANS as the
donor-acceptor pair, a method for monitoring fibril
formation by A~ using nonradiative fluorescence energy
transfer was devised.
The fluorescent fibrillogenesis assay of the
invention uses a fragment of A~ corresponding to residues
9 to 25. This fragment of A~ forms fibrils that display
similar morphology, pH-dependence, X-ray diffraction
pattern, and secondary structure as the full length A~
molecule (Fraser et al., 1994). Adapting the assay
procedure to the full length A~ molecule is very
straightforward and simple.
Two forms of the fibrillogenic fragment A~(9-25),
SEQ ID NO:1, were synthesized: one form had a Trp
residue appended to the N-terminus of A~(9-25) (denoted
Trp-A~(9-25)SEQ ID NO:2), and a second form (denoted
AEDANS-A~(9-25)SEQ ID NO:3) had a cysteine residue
appended to the N-terminus of A~(9-25), and the AEDANS
group chemically linked to the sulfhydryl side chain of
cysteine (Table 1). The basis of the method is that when
Trp-A~(9-25) is mixed with AEDANS-A~(9-25), fibrils
composed of both forms of A~ will assemble, and in the
~ibril state the Trp and AEDANS groups will be closer in
space than in the nonfibril state. Since fluorescence
energy transfer between Trp and AEDANS increases when the
two ~luorophores are close in space, the ef~iciency of
energy transfer between Trp and AEDANS will increase as
Trp-A~ (9-25) and AEDANS-A~ (9-25) assemble into fibrils.
At a separation distance of 22 A energy transfer between
Trp and AEDANS is easily measurable (Fairclough and
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_g _
Cantor, 1978; the upper limit of energy transfer is
~40A).
~ TABLE 1
Peptide Amino acid sequence
A~(9-25) ~Y~v~QKLVFFAEDVG (SEQ ID NO:1)
Trp-A~(9-25) Ac-~W Y~v~QKLVFFAEDVG-CONH2
(SEQ ID NO:2)
AEDANS-A~(9-25) Ac-C(AEDANS)G~Y~v~QKLVFFAEDVG-CONH2
(SEQ ID NO:3)
Trp and AEDANS are relatively large chemical
structures. There is a possibility, therefore, that
introduction of these large groups into A~ may disrupt or
alter fibril formation through steric mechanisms. For
this reason, it was decided to place the fluorescent
groups at the N-terminus of the A~ (9-25) sequence rather
than at an internal position, because placement in the
interior could potentially disrupt certain interactions
which are known to stabilize fibril formation (Halverson
et al., 1990; Hilbich et al., 1992; Fraser et al., 1994).
In addition, a glycine residue was inserted between the
fluorescent group and the rest of the A~ (9-25) sequence.
Glycine is the smallest amino acid, it lacks a side
chain, and it should act like a flexible tether for the
fluorescent group; therefore, as the fluorescent-labeled
A~ (9-25) molecule assembles into fibrils the flexible
glycine tether will act like a hinge and allow the
fluorescent group to be pushed out of the way if steric
clashes occur with other parts of the fibril.
For the fluorescence method of the invention to
operate as intended, the fluorescent-A~ molecules must be
prevented from forming fibrils before they are mixed
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--10--
together. One approach to achieving this objective is to
mix the peptides in solid lyophilized form before
dissolving them in buffer. The shortcoming of this
method, however, is that the relative amounts of each
peptide will have to be determined using dry weights,
which is a very inaccurate method. An alternative
approach is to first dissolve the two peptides in a
solution containing a denaturant that prevents fibril
formation, and then to initiate the fibril assembly
process by diluting out the denaturant. This method will
involve: a) preparing concentrated stock solutions of
Trp-A~ and AEDANS-A~ in denaturant and accurately
determining the peptide concentrations by W absorbance
using the absorption coefficients of Trp and AEDANS, b)
mixing appropriate amounts of each peptide solution to
produce equimolar mixtures of both peptides in denaturant
solution, c) initiate fibril assembly by diluting out the
denaturant, and d) simultaneously starting to monitor the
evolution of fluorescence energy transfer in the
fluorimeter. This method should be very rapid and it
should allow evaluation of the kinetics and thermo-
dynamics of fibril assem.bly. The kinetic measurement will
be limited only by the dead-time required for diluting
out the denaturant, which for m~nl~l mixing is around 10
seconds. If the kinetics of fibril assem.bly occur too
rapidly using the m~nll~l mixing method, then this method
can easily be adapted into a stopped-flow fluorimetric
method which has a dead-time of 30 milliseconds.
As regards the choice of denaturant, one possibility
is to use formic acid which is known to prevent fibril
assembly (Halverson et al., 1990). Initiating fibril
assem~ly, however, would then require neutralization of
the acid, which in turn would cause an increase in ionic
strength of the solution. Thus fibril assembly at low
ionic strengths cannot be explored using an acid as the
denaturant. In addition, by placing the peptides in acid
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--11--
solutions the risks of acid hydrolysis, deamidations, and
formylations exist. Therefore, formic acid is not an
ideal denaturant for the purposes of the invention. The
organic solvent 40~ trifluoroethanol has been shown to
maintain A~ in a soluble monomeric helical state at very
~ high peptide concentrations and at pH values between 1.3
and 10 (Barrow and Zagorski, 1991). Accordingly, this
solvent was chosen as the preferred denaturant because it
does not cause any harmful side reactions, it is
nonviscous, so mixing is e~ficient; and it is volatile
allowing for easy ~ ov~l.
The basic protocol of the fluorescence method of the
invention using the described A~ analogs is: 1)
Trp-A~(9-25) and AEDANS-A~(9-25) are dissolved separately
in 40~ trifluoroethanol at millimolar concentrations.
Under these conditions the A~ analogues will adopt
mono~riC helical conformations. 2) After determining
the concentration of the stock peptide solutions by W
absorbance, an equimolar mixture of the peptides at
micromolar concentration is prepared. 3) 20 to 30 ~l of
the equimolar peptide mixture is delivered into a cuvette
in the fluorimeter which contains 3 ml of aqueous buffer,
after rapid mixing, fluorescence emission between 300 nm
to 550 nm is monitored (excitation ~ = 281 nm).
Induction of energy transfer by fibril formation.
Fibrils composed of Trp-A~(9-25) alone,
AEDANS-A~(9-25) alone, and equimolar mixtures of the two
peptides were formed by diluting concentrated solutions
of the peptides dissolved in 40~ TFE into aqueous buffer.
The fluorescence emission spectrum of these peptide
preparations are shown in Figure 1. The emission of the
Trp fluorophore is significantly quenched in the mixed
~ fibril preparation compared to the Trp-emission of
fibrils composed of Trp-A~(9-25) alone. The
AEDANS-emission, on the other hand, is significantly
CA 022428~l l998-07-l3
WO 97/07402 PCT/CA~Gi'~SC5
-12-
enhanced in the mixed fibril preparation, relative to the
emission of fibrils composed of AEDANS-A~(9-25) alone.
Donor quenching of Trp and the sensitized emission of
AEDANS are typical features of energy transfer.
pH dependence of fibril fo~ation.
It is known that fibril formation is pH dependent.
To study this e~fect using fluorescence energy transfer,
spectra were collected at varying pH. AEDANS intensities
~rom 425-525 nm were integrated and this value was
expressed as a ratio versus the integrated intensities o~
Trp from 340-375 nm. The degree of fibril formation
would thus be directly proportional to this
acceptor/donor ratio. The advantages of this
manipulation are two-fold: 1) taking the ratios
~; m; n; shes the within experiment errors such as noise in
the intensity measurements, and 2) results can now be
compared between experiments because the acceptor/donor
ratio is concentration-independent and it depends only on
the fraction of A~t9-25) molecules in multimeric states.
Fibril formation reached a m,i~;mllm at pH 5 and
decreased both towards more acidic and more basic pH
(Figure 2). Basal acceptor/donor ratios occurred below
pH 2 in the acidic limb and above pH 8 in the basic limb
of the titrations suggesting very few fibrils were
present beyond these values. The pH dependence of fibril
formation was modelled as a sequential equilibrium
between 4 ionic species, and the data were fit to the
following equation (I) which relates the acceptor/donor
ratio to the concentrations of each ionic species and 3
pKa constants:
(I)
¦'10 ~ pH-pK i)i~lt~eii~e2 + 10(-pH-p~l-pK2)il~ eli e3 + 1o~ -pl~ l-pK 2-pEC3~2elirle4 + 1o(-3pH)b~eli el
~cccptor
~ ~io 10 + 10( pH p~l-p}C2~ + 1o(-plCl-plC2-p~3~ + 10(-3pH)
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-13-
The fitted equation gave apparent pKa's of 3.98, 5.86 and
7.62.
The pH titration in the presence of 500 mM NaCl
showed a dramatic decrease in the level of m~; mllm
fibrillogenesis (Figure 2). The salt titration curve at
pH 5.25 ~Pm~n~trated the inverse relationship between
NaCl concentration and fibril formation (Figure 2 inset).
The effect of pH on fibrillogenesis was also studied
using electron microscopy. The analysis indicates that
equimolar mixtures of Trp/AEDANS-A~(9-25) give rise to
fibrils that are several mm long and approximately 60 A
in diameter. The morphology of these fibrils are very
similar to fibrils formed by A~(11-25) (Fraser et al.,
1994) and by full-length A~ (Fraser et al., 1991). The
amount of fibrils in the micrograph~ at three dif~erent
pH values 2, 5 and 8 paralleled the observations made
using fluorescence, with a great number of fibrils
present at pH 5 and few at pH 2 and pH 8. Furthermore,
continued aging o~ the fibrils for 1 week showed a
time-dependent change in morphology from loose short
aggregates to well-structured, long fibrils with a
"braided" appearance. These braided fibrils were most
evident in the pH 5 samples.
Kinetics of exchange between mo~nm~s and fibrils.
Fibrils composed of Trp-A~(9-25) alone and
AEDANS-A~(9-25) alone were formed separately, aged for 24
hrs, mixed, and then fluorescence emission spectra of the
mixed fibril preparation were acquired at timed intervals
for the next 8 hrs. The acceptor/donor ratio as a
function of time after mixing is shown in Figure 3.
Fluorescent fibrillogenesis assay.
The successful development of a fluorescent
fibrillogenesis assay depends on two prerequisites: a)
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W O 97/07402 PCT/CA96,'~C~'5
the extrinsic fluorescent groups must not interfere with
the process of fibril formation, and b) significant
energy transfer must occur between donor and acceptor
fluorophores in the fibril state. The data presented
show that both prerequisites have been met. The
fluorescent-labelled A~(9-25) peptides formed fibrils, as
observed by electron microscopy, that had very similar
morphology to fibrils formed by native A~ peptides and A~
fragments (Fraser et al., 1991; Fraser et al., 1994).
Significant donor qu~nrhing of Trp and sensitized
emission of AEDANS were observed with fibrils composed of
equimolar mixtures of Trp- and AEDANS-A~(9-25), compared
to ~ibrils composed of Trp-A~(9-25) alone and
AEDANS-A~(9-25) alone (Figure 1). These features are
typical of energy transfer processes; therefore,
supporting the conclusion that energy transfer was
induced by fibril formation in the mixed fibril
preparation. The dependence of the acceptor/donor ratio
on pH (Figure 2) correlated with the electron microscopic
observations of the number of fibrils present in
solutions of Trp- and AEDANS-A~(9-25) at different values
of pH. This correlation cross-validates the fluorescent
fibrillogenesis assay with electron microscopy, and it
indicates that the acceptor/donor ratio is a ~uantitative
measure of the fraction of A~(9-25) molecules present in
the fibril state.
pH dependence of f~ibrillogenesis
FTIR and electron microscopy studies have
established that A~ fibrillogenesis is very pX dependent;
thus, it was concluded that fibrillogenesis is
stabilized, at least in part, by electrostatic
interactions (Fraser et al., 1991; Fraser et al., 1994).
However, the number and type of ionizable groups involved
in the electrostatic interaction has not been determined.
The sensitive and quantitative nature of the fluorescent
fibrillogenesis assay allows such quantitation of the pH
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-15-
dependence. The simplest model (equation I) that
accurately describes the pH effects shown in Figure 2 is
a sequential equilibrium between 4 ionic species, where
~ each species differ in the ionization state of three
chemical groups. The ~itted equation gave apparent pKa's
- of 3.98, 5.86 and 7.62. The apparent pKa of 3.98 is
suggestive of carboxyl groups on Asp and Glu. The
apparent pKa's of 5. 86 and 7.62 is suggestive of
imidazole groups on His. In light of this information
one can conclude that fibrillogenesis in A~ (9-25) is
m~;m~l when His13, His 14, Glull, Glu22 and Asp23 are in
their ionized states. The involvement of Lysl6 cannot be
assessed because fibril formation does not occur around
pH 10 where titration of Lys sidechain occurs.
As regards the mechanism of electrostatic
stabilization of fibrillogenesis, two different scenarios
could apply: the pH dependence could be caused by either
isoelectric precipitation or formation of salt bridges.
In the case of isoelectric precipitation, the ~im~l
fibrillogenesis at pH 5.00 (Figure 2) is a result of the
peptide showing a greater tendency to associate at its
isoelectric point. Alternatively, the pH dependence
could be caused by formation of imidazole-carboxylate
salt bridges between His side~h~; n~ and sidech~i ns of Asp
and/or Glu. To differentiate between these two
possibilities the effect of counter ion screening by NaCl
was ~m; ned~ If the pH dependence of fibrillogenesis is
an isoelectric effect, then addition of NaCl to the
solution should screen the repulsive interactions between
peptides at pH values that are away from the isoelectric
point causing a bro~n;ng of the pH titration curve.
If, on the other hand, the pH dependence is caused by
formation of imidazole-carboxylate salt bridges, then
~ addition of NaCl will screen these attractive
interactions and the pH titration curve will be
attenuated. Addition of 0.5 M NaCl resulted in
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WO 97/07402 PCT/CA96/00555
-16-
attenuation of the pH titration curve, supporting the
conclusion that imidazole-carboxylate salt bridges
contribute to the stability of the fibril formed by
A~(9-25). This conclusion was further substantiated by
~m~n~trating that NaCl titration causes a reduction in
fibrillogenesis (Figure 2 inset). In addition, since two
imidazole titrations were detected, it can also be
concluded that there are two stabilizing
imidazole-carboxylate salt bridges per monQm~r
Kinetics of exchange between monom~rs and fibrils.
In the ~ch~nge experiments, fibrils of Trp-A~(9-25)
and AEDANS-A~(9-25) were formed separately and then
mixed. Upon mixing these fibrils, little if any energy
transfer was expected because ~ibrils would be composed
of either Trp-A~(9-25) or AEDANS-A~(9-25), but not both.
Interaction between fluorophores would be limited by
fibril structure. This indeed was the case; ;mm~ately
after mixing, the acceptor/donor ratio was only slightly
greater than the basal ratio (Figure 3). However, the
acceptor/donor ratio increased signi~icantly over the
next 8 hrs, which suggested that fibrils, though
insoluble, existed in a dynamic state in which monomers
were in constant exchange between fibrils. The kinetics
of exchange was ade~uately fit to two exponentials, which
had relaxation times of 21 seconds and 77 minutes (Figure
3).
The use of amyloid-enhancing factors to promote fibril
formation under physiological conditions.
Studies of the pH dependence of A~ fibrillogenesis
have revealed that fibrils do not readily form at the
physiological pH of 7.00. Amyloid-enhancing factors such
as heparin are known, however, to promote fibril
formation under physiological conditions (Fraser et al.,
1993). Thus, by using the fluorescent fibrillogenesis
assay in the presence of an amyloid-enhancing factor, A~
CA 022428~l l998-07-l3
W O 97/07402 PCT/CA9~ 5
-17-
~ibrillogenesis can be investigated at the physiological
pH of 7.00. As ~hown in Figure 4, increasing the amount
of heparin present results in an increased level o~
fluorescence energy transfer as evidenced by a decrease
in donor fluorescence. The data shown in Figure 4 were
- obtained using a concentration of Trp/AEDANS-A~(9-25) of
100 nM, and the buf~er was phosphate-buffered saline, pH
7.00.
The invention is a new technique for monitoring
fibrillogenesis by A~. Using this technique, it has been
~Pmon~trated that fibril formation by Trp-A~(9-25) and
AEDANS-A~(9-25) is stabilized by 2 imidazole-carboxylate
salt bridges. It has also been ~Pmon~trated that
monom~rs that compose the fibril are not kinetically
trapped in the fibril state, and that they exchange with
the soluble fraction. The exchange kinetics are
multiphasic. This information will be extremely valuable
when designing pharmacological agents that inhibit fibril
formation. By obt~in;ng a greater underst~n~;ng of the
fibril assembly process under physiological conditions, a
better idea of which features of the process are the most
susceptible to attack can be obtained and exploited.
The st~n~rdized fluorescence assay for
fibrillogenesis of the invention should also be of
immediate value for screening compounds that have
potential for being inhibitors of fibril formation.
MAT~212 T~T-.S AND I~sl~~Ol~S
Peptide synthesis and fluorescent labeling
Peptides were synthesized, by the solid phase method
on the Milligen 9050~ peptide synthesizer, as peptide-
amides using Rink-resin (Advanced Chemtech). An active
ester coupling procedure, employing pentafluorophenyl
esters of 9-fluorenylmethoxycarbonyl amino acids, was
used. The N-termini was acetylated with acetic anhydride.
-
CA 022428~l l998-07-l3
W O 97/07402 PCT/CA~ '5
-18-
The peptides were cleaved from the resin with 95:2.5: 2. 5
trifluoroacetic acid: thioanisole: eth~n~;thiol mixture.
The peptides were purified by C18 reverse phase
chromatography, and peptide identity was confirmed by FAB
mass spectrometry and amino acid analysis. Peptide
purity was assessed by analytical C18 reverse phase
chromatography using the Pharmacia FPLC system.
5-Acetylethyl~; ~m; nonaphthalene-1-sulfonic acid
(AEDANS) labelling was perfonmed by incubation of 1 mM
Cys-contA;n;ng peptide with 5 mM (1,5)-IAEDANS (Molecular
Probes) in 50 mM Tris-HCl, 6 M guanidine hydrochloride,
pH 8.0 for 18 hrs. AEDANS-labelled peptide was separated
from unreacted peptide and 1,5-IAEDANS by Cl8 reverse
phase chromatography.
Preparation of peptide stock solutions
The lyophilised Trp-A,B(9-25) was dissolved in a 2 ml
solution of 40~ (v/v) trifluoroethanol (TFE) cont~;n;ng
10 mM acetic acid. This stock solution was diluted 1:10
with 7.5 M guanidine hydrochloride (GdnHCl) and the
peptide concentration was determined via tryptophan
absorbance at 280 nm with an absorbance coefficient (~)
of 5690 M~1cm~1 (~lhochl 1967).
Lyophilised AEDANS-A,~ (9-25) was dissolved in a 2ml
solution of 40~ (v/v) TFE adjusted to pH 9 with ammonium
hydroxide. Hudson and Weber have shown that ~ for AEDANS
in 40~ ethanol is 6500 M~lcm~l and the absorbance A~ is
338.0 nm. It was found that the difference in absorbance
between AEDANS in ethanol and TFE is less than 1~, well
within experimental error; therefore, an ~ of 6500 M~1cm~
were used in all concentration determinations of
AEDANS-A~(9-25) in 40~ TFE.
Absorbance measurements were made on a Perkin Elmer
Lambda 3B~ spectrophotometer. The stock solutions were
CA 022428~1 1998-07-13
WO 97/07402 PCT/CA9C,'~ ~'=5
--19 -
~tored at -20 C.
Electron Microscopy
~ Negatively stained ~ibrils were prepared by floating
charged pioloform, carbon-coated grids on peptide
solutions (0.025 mg/mL Trp:A~(9-25) and 0.025 mg/mL
AEDANS:A~(9-25), pH2-pH8). These solutions were pre-aged
24 h and 1 week. To control pH, the peptide solutions
were made using a bu~er of 1 mM borate, 1 mM citrate and
1 mM phosphate. After the grids were blotted and
air-dried, the samples were stained with 1~ (w/v)
phosphotungstic acid which was prepared using the same
borate, citrate, phosphate buf~er and pH adjusted to
correspond to the respective samples. The Hitachi
H-7000~ instrument used ~or visualization was operated at
75 kV. The samples were inspected and images of
representative fibrils were recorded.
Fluorescence Spectroscopy
Steady-state ~luorescence was measured at room
temperature using a Photon Technologies International
QM-1~ fluorescence spectrophotometer equipped with
excitation intensity correction, a temperature controlled
cell holder and a magnetic stirrer. Emission spectra
from 300nm to 550 nm were collected with excitation set
at 281 nm. Both the excitation and emission
monochromator slit widths were 4 nm.
In the energy transfer experiments, four spectra
were taken: a blank of the bu~fer containiny 1 mM
borate, 1 mM citrate and 1 mM phosphate; 3 ~M
Trp:A~(9-25) alone; 3 ~M AEDANS:A~(9-25) alone; and a
mixture of 3~M of each labeled peptide. The samples were
pre-aged at pH 5 ~or 18 to ~4 hours prior to spectral
acquisition. The pH titrations were performed using
hydrochloric acid and sodium hydroxide. For the salt
studies, samples were pre-aged with the appropriate
-
CA 02242851 1998-07-13
WO 97/07402 PCT/CA96~'~~5~'
-20-
concentration o~ sodium chloride.
In the exchange experiments 6 ~M Trp:A~(9-25) and 6
~M AEDANS:A,B (9-25) were incubated separately overnight at
pH 5. These solutions were mixed giving a final
concentration of 3 ~M each labeled peptide at which time
~pectra were collected.
CA 022428~l l998-07-l3
WO 97/07402 PCTtCA96i'~ 5
-21-
K~ ~S
Barrow, C.J., and Zagor~ki, M.G. (1991) Solution
structures of ~-peptide and its constituent fragments:
relation to amyloid deposition. Science 253, 179-182.
Crowther, R.A. (1991) Structural aspects of pathology in
Alzheimer's disease. Biochim. Biophys. Acta 1096, 1-9.
~APlhoch~ H. (1967) Biochemistry 6, 1948 - 1954.
Fairclough, R.H., and Cantor, C.R. (1978) The use of
singlet-singlet energy transfer to study macromolecular
assemblies. Methods Enzymol. 48, 347-379.
Fraser, P.E., Nguyen, J.T., Surewicz, W.K., and
Kirschner, D.A. (1991) pH-dependent structural
transitions of Al~he~mPr amyloid ~/A4 peptides.
Biophysical J. 60, 1190-1201.
Fraser, P.E., Nguyen, J.T., Inouye, H., Surewicz, W.K.,
Selkoe, D.J., Podlisny, M.B., and Kirschner, D.A. (1992)
Fibril formation by primate, rodentl and
Dutch-hemorrahagic analogues of Alzheimer amyloid
~-protein. Biochemistry 31, 10716-10723.
Fraser, P.E., Leves~ue, L., and McLachlan, D.R. (1993)
Biochemistry of Alzheimer's Disease Amyloid Plaques.
Clin. Biochem. 26, 339-349.
Fraser, P.E., McLachlan, D.R., Surewicz, W.K., Mizzen,
C.A., Snow, A.D., Nguyen, J.T., and Kirschner, D.A.
(1994) Conformation and fibrillogenesis of Al~h~;mer A~
peptides with selected substitutions of charged residues.
J. Mol. Biol. (in the press).
CA 022428~1 1998-07-13
WO 97/07402 PCT/CA96/00555
-22-
Games, D., Adams, D., Alessandrini, R., Barbour, R.,
Berthelette, P., Blackwell, C., Carr, T., Clemens, J.,
Donaldson, T., Gillespie, F., et al. (1995)
Alzheimer-type neuropathology in transgenic mice
overexpressing V717F beta-amyloid precursor protein.
Nature 373, 523-527.
Goate A. Chartier-Harlin MC. Mullan M. Brown J. Crawford
F. Fidani L. Giu~fra L. Haynes A. Irving N. James L. et
al. (1991) Segregation of a missense mutation in the
amyloid precursor protein gene with familial Al~he;m~r's
disea~e. Nature 349, 704-706.
Haass, C., Schlossmacher, M.G., Hung, A.Y., Vigo-Pelfrey,
C., Mellon, A., Ostaszewski, B.L., Lieberburg, I., Koo,
E.H., Schenk, D., Teplow, D.B., and Selkoe, D.J. (1992)
Amyloid ~-protein is produced by cultured cells during
normal metabolism. Nature 359, 322-325.
Halverson, K., Fraser, P.E., Kirschner, D.A., and
Lansbury, P.T. (1990) Molecular determinants of amyloid
deposition in Alzheimer's disease: conformational
studies of synthetic beta-protein fragments.
Biochemistry 29, 2639-2644.
Hilbich, C., Kister-Wolke, B., Reed, J., Masters, C.L.,
and Beyreuther, K. (1992) Substitutions of hydrophobic
amino acids reduce the amyloidogenicity of Alzheimer's
disease ~A4 peptides. J. Mol. Biol. 228, 460-473.
Mattson, M.P., Barger, S.W., Cheng, B., Lieberburg, I.,
Smith-Swintosky, V.L., and Rydel, R.E. (1993) ~-Amyloid
precursor protein metabolites and loss of neuronal Ca
homeostasis in Alzheimer's disease. Trends Neurochem.
Sci. 16, 409-414.
CA 0224285l l998-07-l3
W 097~07402 PCT~CAgC~a'~
-23-
Pike C.J., Burdick, D., Walencewicz, A.J., Glabe, C.G.,
Cotman, C.W., (1993) Neurodegeneration induced by
beta-amyloid peptides in vitro: the role of peptide
assembly state. J. Neurosci. 13, 1676-1687.
Selkoe, D.J. (1993) Physiological production o~ the
~-amyloid protein and the mechanism of Alzheimer's
disease. Trends Neurochem. Sci. 16, 403-409.
Seubert, P., Vigo-Pel~rey, C., Esch, F., Lee, M., Dovey,
H., Sinha, S., Schlossm--acher~ M.G., Whaley, J.,
Swindlehurst, C., McCormack, R., Wol~ert, R., Selkoe,
D.J., Lieberburg, I., and Schenk, D. (1992) Isolation
and quantification of soluble Al~heimer's ~B-peptide ~rom
biological fluids. Nature 359, 325-327.
Yankner, B.~., Dawes, L.R., Fisher, S., et al. (1989)
Neurotoxicity of a ~ragment o~ the amyloid precursor
associated with Alzheimer's disea~e. Science 245,
417-420.
CA 022428~1 1998-07-13
WO 97/07402 PCT/CA9f/~CS''
-24-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
.
(1) APPLICANT: THE ONTARIO CANCER lNS'l'l'l'U'l'~;
(ii) TITLE OF INVENTION: PROTEIN FIBRIL ASSEMBLY
ASSAY
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Ridout & Maybee
(B) STREET: Suite 2400, One Queen Street East
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: C~ n A ~ ~
(F) ZIP CODE: M5C 3B1
(v) COM~uL~ READABLE FORM:
(A) MEDIUM TYPE: Diskette - 3.5 inch, 1.4 Mb
storage
(B) COM~u~L~: IBM PC Compatible
(C) OPERATING ~:iY~L~:M: MS-DOS 6.00
(D) SOFTWARE: Wordperfect 5.1
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lake, James R.
(B) REFERENCE/DOCKET NUMBER: ONTCA/46B
(ix) TELECOMMUNICATION INFORMATION:
(A) TEL~O~: (416) 868-1482
(B) TELEFAX: (416) 362-0823
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acid residues
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly Tyr Glu Val His His Glu Lys Leu Val Phe Phe Ala Glu
Asp Val Gly
CA 02242851 1998-07-13
W O 97/07402 PCT/CA9~ '5
-25-
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~N~ CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: The N-terminal amino
acid residue Xaa is
AcTRP, and the
C-terminal amino acid
residue Xaa is
Gly-CONH2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Xaa Gly Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe
Ala Glu Asp Val Xaa
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: The N-terminal amino
acid residue Xaa is
AcCYS (AEDANS), and
the C-terminal amino
acid residue Xaa is
Gly-CONH2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Gly Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe
Ala Glu Asp Val Xaa
