Note: Descriptions are shown in the official language in which they were submitted.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
METHOD FOR PR-39 PEPTIDE REGULATED STIMULATION
OF ANGIOGENESIS
RESEARCH SUPPORT
The research effort for the invention was supported in part by grants from
the National Institutes of Health, grants ROl HL 53793 and P50 HL 56993 (MS),
F32 HL 10013 (RV); and by a grant from Chiron Corporation. The government
has certain rights in the invention.
CROSS REFERENCE
The present application is a Continuation-In-Part of prior pending United
States Patent Application Serial No. 09/276,868 filed March 26, 1999
FIELD OF THE INVENTION
The present invention is concerned generally with the induction of
angiogenesis within viable cells comprising living tissues and organs; and is
particularly directed to mechanisms regulated by PR-39 peptides which result
in a
stimulation of angiogenesis on-demand and may be used as a controlled
therapeutic
treatment.
BACKGROUND OF THE INVENTION
Angiogenesis, by definition, is the formation of new capillaries and blood
vessels within living tissues; and is a complex process first recognized in
studies of
wound healing and then within investigations of experimental tumors.
Angiogenesis is thus a dynamic process which involves extracellular matrix
remodeling, endothelial cell migration and proliferation, and functional
maturation
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-2-
of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo,
Trends
Cell Bio~ 6: 454-456 (1996)]. Clearly, in normal living subjects, the process
of
angiogenesis is a normal host response to injury; and as such, is an integral
part of
the host body's homeostatic mechanisms.
It will be noted and appreciated, however, that whereas angiogenesis
represents an important component part of tissue response to ischemia, or
tissue
wounding, or tumor-initiated neovascularization, relatively little new blood
vessel
formation or growth takes place in most living tissues and organs of mature
adults
(such as the myocardium of the living heart) [Folkman, J.and Y. Shing, J.
Biol.
Chem. 267: 10931-10934 (1992); Folkman, J., Nat. Med. 1: 27-31 (1995); Ware,
J.A. and M. Simons, Nature Med. 3: 158-164 (1997)]. Moreover, although
regulation of an angiogenic response in-vivo is a critical part of normal and
pathological homeostasis, relatively little is presently known about the
control
mechanisms for this process.
Overall, a number of different proteins, growth factors and growth factor
receptors have been found to be involved in the process of stimulation and
maintenance of angiogenic responses. For example, a number of cell membrane-
associated proteins are thought to be involved in the processes of
angiogenesis.
Such proteins include SPARC [Sage et al., J. Cell Biol. 109: 341-356 (1989);
Motamed K. and E.H. Sage, Ki. dnev Int. 51: 1383-1387 (1997)]; thrombospondin
1 and 2 respectively [Folkman, J., Nat. Med. 1: 27-31 (1995); Kyriakides et
al.,
J. Cell Biol. 140: 419-430 (1998)]; and integrins av~35 and av(33 [Brooks et-
al.,
Science 264: 569-571 (1994); Friedlander et al., Science 270: 1500-1502
(1995)].
In addition, a major role is played by heparin-binding growth factors such as
basic
fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF);
and thus the regulation of angiogenesis is believed today to involve matrix
components such as extracellular heparin sulfate and core proteins such as
syndecans which are found at the surface of endothelial cells.
However, while a number of heparin binding growth factors (including
VEGF, FGFl and FGF2) have been shown to promote angiogenesis in-vitro and
in-vivo, their process involvement appears limited to tissues demonstrating
some
form of inflammatory response to trauma (as defined by the presence of blood-
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-3-
derived macrophages), be it a direct tissue injury (such as wounding) or
ischemia.
Moreover, the presence of blood-derived macrophages is also routinely
associated
with localized secretion of a number of proteins including cytokines such as
IL-2
and TNF-a, growth factors such as VEGF and FGF-2, matrix metalloproteinases
as well as many other biologically active molecules. Accordingly, although
there
have been many investigations, publications, and developments of these
entities,
there remains a general ignorance and failure of understanding by research
investigators and clinicians alike regarding useful and effective specific
means and
methods for inducing angiogenesis on-demand within living cells, tissues, and
organs. Thus, while the value and desirability of initiating new
vascularization -
especially using cells in localized areas on an as needed basis as well as a
therapeutic treatment for individual patients - is well recognized, these aims
remain
a long sought goal yet to be achieved in a practical manner.
SUMMARY OF THE INVENTION
The present invention has multiple aspects and uses. A first aspect provides
a method for stimulating angiogenesis within a targeted collection of viable
cells in-
situ, said method comprising the steps of:
identifying a collection of cells comprising viable cells in-situ as a target
for
stimulation of angiogenesis;
providing means for effecting an introduction of at least one member
selected from the group consisting of the PR-39 oligopeptide collective to the
cytoplasm of said targeted collection of cells;
introducing at least one member of the PR-39 oligopeptide collective to the
cytoplasm of said targeted collection of cells using said effecting means;
allowing said introduced PR-39 oligopeptide collective member to interact
with such proteasomes as are present within the cytoplasm of said targeted
collection of cells whereby
(a) at least some of the proteasomes interact with said PR-39
oligopeptide collective member, and
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-4-
(b) at least a part of the proteolytic activity mediated by said
interacting proteasomes becomes selectively altered, and
(c) the selectively altered proteolytic activity of said interacting
proteasomes results in a stimulation of angiogenesis in-situ within the
targeted
S collection of viable cells.
A second aspect of the invention provides a method for selective inhibition
of proteasome-mediated degradation of peptides in-situ within a collection of
viable
cells, said method comprising the steps of:
identifying a collection of cells comprising viable cells in-situ as a target;
providing means for effecting an introduction of at least one member
selected from the group consisting of the PR-39 oligopeptide collective to the
cytoplasm of said targeted collection of cells;
introducing at least one member of the PR-39 oligopeptide collective to the
cytoplasm of said targeted collection of cells using said effecting means;
allowing said introduced PR-39 oligopeptide collective member to interact
with such proteasomes as are present within the cytoplasm of said targeted
collection of cells whereby
(a) at least some of the proteasome interacts with the PR-39
oligopeptide collective member, and
(b) at least a part of the proteolytic activity mediated by said
interacting proteasomes becomes markedly altered, and
(c) the markedly altered proteolytic activity of said interacting
proteasomes results in a selective inhibition of proteasome-mediated
degradation of
peptides in-situ within the targeted collection of cells.
BRIEF DESCRIPTION OF THE FIGURES
The present invention may be more fully understood and better appreciated
when taken in conjunction with the accompanying drawing, in which
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-5-
Figs. lA-1D are presentations of empirical data showing the direct
interaction between PR-39 peptide and the a,7 subunit of proteasomes
intracellularly;
Figs. 2A-2D are presentations of empirical data showing the effect of PR-
39 peptide upon proteasome activity in-vivo;
Figs. 3A-3D are graphs demonstrating the results of in-vitro proteasome
activity assays;
Figs. 4A-4C are presentations of empirical data showing the in-vivo effects
of PR-39 peptide expression;
Figs. SA-SC are photographs of representative sections showing differences
in vascularity among control, PR-39 peptide and FGF2 impregnated Matrigel
pellets;
Fig. 6 is a graph providing a quantitative analysis of vascularity for the
representative sections of Fig. 5; and
Fig. 7 is a graph showing the induction of angiogenesis in-vivo using PR-39
peptide and short-length PR11 peptide impregnated Matrigel pellets.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for stimulating angiogenesis via the
purposeful introduction of native PR-39 peptide or a member of the PR-39
derived
oligopeptide family to the cytoplasm of viable cells in-situ. The PR-39
peptide or
the derived member of the family will interact with such proteasomes as are
present intracellularly; and the consequence of PR-39 peptide/proteasome
interaction is the selective inactivation of proteasomes such that
intracellular
degradation of proteins such as HIF-la and IKBa is diminished and a marked
stimulation of angiogenesis in-situ consequently results.
A number of major benefits and advantages are therefore provided by the
means and methods comprising the present invention. These include the
following:
1. The present invention provides an in-situ stimulation of angiogenesis. By
definition, therefore, both in-vivo and in-vitro circumstances of use and
application
are envisioned and expected. Moreover, the viable cells which are the location
of
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-6-
PR-39 peptide and proteasome interaction, alternatively may be isolated cells;
be
part of living tissues comprising a variety of different cells such as
endothelial
cells, fibrocytes and muscle cells; and may also comprise part of specific
organs in
the body of a living human or animal subject. While the user shall choose the
specific conditions and circumstances for practicing the present invention,
the
intended scope of application and the envisioned utility of the means and
methods
described herein apply broadly to living cells, living tissues, functional
organs and
systems, as well as the complete living body unit as a viable whole.
2. The present invention has a variety of different applications and uses. Of
clinical and medical interest and value, the present invention provides the
opportunity to stimulate angiogenesis in tissues and organs in a living
subject which
has suffered defects or has undergone anoxia or infarction. A common clinical
instance is the myocardial infarction or chronic myocardial ischemia of heart
tissue
in various zones or areas of a living human subject. The present invention
thus
provides opportunity and means for specific site stimulation and inducement of
angiogenesis under controlled conditions. The present invention also has major
research value for research investigators in furthering the quality and
quantity of
knowledge regarding the mechanisms controlling angiogenesis under a variety of
different conditions and circumstances.
3. The present invention envisions and permits a diverse range of means for
introducing native PR-39 peptide or a shorter-length peptide of the
oligopeptide
family to a specific location, site, tissue, organ, or system in the living
body. A
variety of different routes of administration are available to the
practitioner; and a
wide and useful choice of delivery systems are conventionally available, and
in
accordance with good medical practice are adaptable directly for use. In this
manner, not only are the means for PR-39 peptide introduction under the
control of
the user, but also the manner of localized application and the mode of
limiting the
area of peptide introduction can be chosen and controlled.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
I. Underlying Direct And Indirect Mechanisms For Initiating A
Stimulation Of Angiogenesis
The present invention utilizes and relies upon novel and previously
unknown direct and indirect mechanisms of interaction between PR-39 peptide
(or
its shorter-length homologs) and proteasomes in-situ as the basis for
stimulation of
angiogenesis in cells, living tissues, and organs. Evidence of such multiple
intracellular interactions is provided by the experiments and empirical data
described hereinafter. Such interactions between proteasomes (and its a7
subunit
in particular) and the PR-39 peptides collectively (of any size) are
previously
unknown; in fact, no meaningful relationship or interaction between any
peptide
whatsoever and intracellular proteasome function has ever been proposed or
envisioned before the present invention was conceived or demonstrated
empirically.
As shown experimentally hereinafter, the PR-39 peptide (and the shorter-
length PR-39 derived oligopeptide family members) when introduced into the
cytoplasm of viable cells will interact, directly and indirectly, with
proteasomes.
In some instances, the interaction between the collective of PR-39
oligopeptides
and the proteasome is direct and a direct binding with the a7 subunit of the
proteasome occurs. In these instances, no intermediaries or cofactors are
involved
in the binding reaction; and such direct a7 subunit binding interactions
result in a
selective inactivation and inhibition of proteasome function intracellularly
such that
expression of certain proteins such as HIF-la is increased and stimulation of
angiogenesis subsequently occurs.
In addition, however, alternative mechanisms of interaction in addition to
and other than direct inactivation of a proteasome subunit are deemed to exist
and
be in effect in-situ for members of the PR-39 peptide collective (including
native
PR-39 and its substituted or homologous forms). As merely an examplary and
representative instance illustrating an alternative mechanism of action, the
experiments and empirical data presented hereinafter demonstrate that: the PR-
39
peptide acts as a selective inhibitor of tumor necrosis factor-a induced IxBa
degradation by proteasomes; and that PR-39 peptide inhibition of IxBa
degradation
is rapidly reversible (unlike the action of known inhibitory compounds such as
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
_g_
lactacysin); and that even in the presence of PR-39 peptide, the intracellular
expression of other proteasome-regulated proteins such as p105 and p50 NFKB
was
unchanged. These empirical findings are meaningful evidence of, harmonious
with, and more consistent as demonstrating indirect mechanisms of interaction
(rather than any direct action effect). Accordingly, the selective and
reversible
nature of PR-39 peptide activity with regard to inhibiting IxBa degradation is
perceived to be due to a prevention of recognition of ubiquitinated IKBa by
the
proteasome and/or by a physical blockage of its entry into the interior of the
proteasome in-situ. These indirect mechanisms of action are functional
alternatives
available on-demand and effective concurrently in addition to any direct
action
inactivation mechanisms.
To obtain a demonstrable proteasome interaction and effect in-situ, the
introduction of native PR-39 peptide (or one of its substituted forms or its
shorter-
length homologs) is a necessary prerequisite; and the presence of sufficient
PR-39
peptide (or its substituted or homologic equivalent) quantitatively to
interact
selectively with proteasomes intracellularly within viable cells can be
achieved
under both in-vivo conditions and in-vitro experimental circumstances.
The methodology and means provided by the present invention for
selectively inhibiting proteolysis and stimulating angiogenesis within viable
cells is
therefore directed at and focused upon the intracellular degradation
capability and
functional activity of proteasomes. Such selective inhibition and/or
disruption of
proteasome-mediated degradation is achieved via the introduction of native PR-
39
peptide or a member of the shorter-length PR-39 derived oligopeptide family in
a
therapeutic regimen of treatment.
II. Proteasomes
The proteasome is a component of the ubiquitin-proteasome-dependent
proteolysis system. This system plays a major role in the turnover of
intracellular
proteins, of misfolded proteins, and in the selective degradation of key
proteins.
Controlled protein degradation is an important and efficient way to remove
nonfunctional proteins and/or to regulate the activity of key proteins. Target
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-9-
proteins are selectively recognized by the ubiquitin system and subsequently
marked by covalent linkage of multiple molecules of ubiquitin, a small
conserved
protein. The polyubiquitinated proteins are degraded by 26S proteasome. This
complex, however, is composed of two large subcomplexes: the 20S proteasome
constituting the proteolytic core and the 19S regulatory complex which confers
polyubiquitin binding and energy dependence. A simplified scheme of the
ubiquitin pathway is depicted by Flow Scheme A below.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
- 10-
Flow Scheme A
Ubiquitin
E1
E1-S-Ubiquitin Protein
E~ E2
modification
E2-S-Ubiquitln anallary proteins
Ez E3 Substrate
E3-S-Ubiquitin
E3
Substrate-(Ubiquitin)"
I 19S complex
26S proteasome ( 20S proteasome
19S complex
Ubiquitin ~ + ~ Peptides
Schematic representation of the proteusome-ubiquitin
p:nhuav. l,'biquitin is first activated by a ubiquitin-activating
enyme (UBA or EII and passed on to a ubiquion-con~ueatine
protein (UBC or E3). Ubiquitin is then linked directly, or with the
help of ubiquitin ligases (E3). via an isopeptide bond to a lysine
residue of the substrate protein. Polyubiquitinated proteins are
recognized and selectively degraded by the ?6S proteasome. yield-
in~_ reusable ubiquitin molecules and peptides' of > to I> amino
acids. Conversion of a protein into a substrate for ubiquitination
can in certain cases occur after posttranslational modification or
associutiun with ancillary factors. Proteins can also be recoentzed
by an E3 ubiquitin liease without prior modification or associa-
tion
* Reproduced from Gerards et al., CMLS 54: 253-262 (1998)
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-11-
A substantial quantum of research has been conducted to understand the
architecture, assembly, and molecular biology of the proteasome. Merely
representative of scientific publications in this field are the following, the
individual texts of which are expressly incorporated by reference herein:
Goldberg
et al., Biol. Chem. 378: 131-140 (1997); Tanaka, K., Biochem. Bionhys. Res.
Commun. 247: 537-541 (1998); Baumeister et al., Cell 92: 367-380 (1998);
Gerards et al., CMLS 54: 253-262 (1998); Maurizi, M.R., Curr. Biol. 8: R453-
R456 (1998); Rechsteiner et al., J_. Biol. Chem. 268: 6065-6068 (1993);
Gerards
et al., J. Mol. Biol. 275: 113-121 (1998); Fenteany, G. and S. Schreiber, J.
Biol.
Chem. 273: 8545-8548 (1998); and Oikawa et al., Biochem. Bioph ~~s. Res.
Commun. 246: 243-248 (1998).
The 205 proteasome
The degrading component in ubiquitin-dependent protealysis is the 265
proteasome. The catalytic core of this complex is the 205 proteasome, which is
highly conserved and can be found in eukaryotes, archaebacteria, and some
eubacteria. In eukaryotes, the amount of proteasomes can constitute up to 1 %
of
the cell content, depending on the average protein breakdown rates of the
organ.
Proteasomes are localized in the nucleus and the cytosol, sometimes
colocalizing or
associating with the cytosketon. [See for example: Hilt, W. and D.H. Wolf,
Trends Biochem. Sci. 21: 96-102 (1996); Ciechanover, A., Cell 79: 13-21
(1994);
Jentseh, S. and S. Schlenker, Cell 82: 881-884 (1995); Coux et al., Annu. Rev.
Biochem. 65: 807-847 (1996); Dahlmann et al., FEBS Lett. 251: 125-131 (1989);
Tamura et al_, Curr. Biol. 5: 766-774 (1995); Machiels et al., Eur. J. Cell
Biol.
66: 282-292 (1995); Scherrer, K. and F. Bey, P_ roe. Nucleic Acid Res. Mol.
Biol.
49: 1-64 (1994); and Gerards et al., CMLS 54: 253-262 (1998)J.
The first description of a "cylinder-shaped" complex with proteasome-like
features dates back to the late 1960s. The plethora of names given to it
subsequently is a reflection of the problems that were encountered over a
period of
two decades in trying to define its biochemical properties and cellular
functions.
Enzymological studies revealed an array of distinct proteolytic activities and
led to
a consensus name, ' multicatalytic proteinase' . This name, however, was soon
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-12-
replaced by a new one, the 'proteasome' emphasizing its character as a
molecular
machine.
At about the same time, it was found that the occurrence of proteasomes
was not restricted to eukaryotic cells. A compositionally simpler, but
structurally
strikingly similar proteolytic complex was found in the archaeon ThernioplaSma
acidophilum, which later took a pivotal role in elucidating the structure and
enzymatic mechanism of the proteasome.
Nomenclature
The 20S proteasome was independently discovered by groups working in
different fields, and hence was given a variety of different names. In 1970,
Schemer and colleagues observed ring-shaped particles in ribosome-free
messenger
RNA (mRNA) preparations [Sporh et al., Eur. J. Biochem. 17: 296-318 (1970)].
Subsequently, in 1979, DeMartino and Goldberg isolated a 700-kDa 'neutral
protease' from rat liver [DeMartino, G.N. and A.L. Goldberg, J. Biol. Chem.
254: 3712-3715 (1997)]. Then, in 1980 Wilk and Orlowski isolated a large
protease complex from the pituitary that possessed three different catalytic
activities. They called it multicatalytic protease [Wilk, S. and M. Orlowski,
J. Neurochem. 35: 1172-1182 (1980); Wilk, S. and M. Orlowski, J. Neurochem.
40: 842-849 (1983)]. Later, Monaco and McDevitt immunoprecipitated
complexes consisting of low molecular weight proteins (LMPs) with a possible
role
in antigen presentation [Monaco, J.J. and H.O. McDevitt, Nature 309: 797-799
(1984)]. Also, in 1984 this particle was called prosome, referring to its
presumed
role in programming mRNA translation [Schmid et al., EMBO 3: 29-34 (1984)].
Altogether, this complex has been given 21 different names in the literature.
Since
all particles were shown to be identical the name 'proteasome' (which is now
generally accepted) was proposed first, referring to its proteolytic and
particulate
nature [Arrigo et al., Nature 331: 192-194 (1988); Faulkenburg et al., Nature
331:
190-192 (1988); Brown et al., Nature 353: 355-357 (1991)].
CA 02368638 2001-09-25
WO Ob/57895 PCT/US00/07050
-13-
Overall characteristics and properties
The 20S proteasome is the major cytosolic protease in eukaryotic cells and
is the proteolytic component of the ubiquitin-dependent degradative pathway.
Proteasomes are also found in some, but not all, archaebacteria and
eubacteria, and
in eukaryotes. True proteasomes are composed of 28 subunits, 14 each of two
different classes - non-catalytic alpha (a) and catalytically-active beta ([3)
subunits.
The subunits are arranged in rings of seven subunits, all of a single type.
The 20S
proteasome is a stack of four rings, two inner beta rings flanked by the alpha
rings. The junction between the beta rings produces a remarkable structural
feature of proteasomes - an interior aqueous cavity large enough to
accommodate
about 70 kDa of protein and accessible only through narrow axial channels in
the
rings. The catalytic sites are located on the beta subunits within the aqueous
cavity. Isolation of the catalytic sites in this way, and the limited access
via
narrow channels, serves to compartmentalize proteolysis, allowing degradation
of
only those proteins that can be actively translocated into the interior of the
proteasome.
Structure and subunit components
The 20S proteasome has a cylindrical or barrel-like structure, typically
14.8 nm in length and 11.3 nm in diameter. It is composed of 28 subunits and
arranged in four stacked rings, resulting in a molecular mass of about 700
kDa.
This overall structural architecture is conserved from bacteria to man.
In eukaryotes, including humans, 14 different subunits, ranging from
21 kDa to 32 kDa, are present in the complex. Based on the sequence homology
with the T. acidophilum a- or ~3-subunit, the eukaryotic subunits are divided
into
a-type and (3-type, respectively [Zwicki et al., Biochemistry 31: 964-972
(1992);
Heinemeyer et al., Biochemistry 33: 12229-12237 (1994); Coux et al., Mol. Gen.
Genet. 245: 769-780 (1994)). Table 2 shows some characteristics and
alternative
names of the subunits of the human and yeast 20S proteasome using the older
and
the new nomenclature proposed by Groll and coworkers [troll et al., Nature
386:
463-471 (1997)]. Immuno-electron microscopy (EM) studies also revealed that
the
eukaryotic a-type subunits reside in the outer rings and the [i-type subunits
in the
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-14-
inner rings. Furthermore, these studies indicated that in the eukaryotic 20S
proteasome seven different subunit constitute a ring, each subunit located at
a
defined position [Kopp et al. , J. Mol. Biol. 229: 14-19 ( 1993); Kopp et al.
,
J. Mol. Biol. 248: 264-272 (1995); Schauer et al., J. Struct. Biol. 111: 135-
147
(1993); Kopp et al., Proc Natl Acad Sci USA 94: 2939-2944 (1997)]. Therefore,
the eukaryotic proteasome assembles as an a,_~~,-~a,-~a,,-~ particle. The
typical
human structure and assembly is illustrated by Table 3.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-15-
Table 2: Nomenclature and molecular masses of proteasomal subunits
Systematic Molecular mass
of
name Human gene Yeast g-ene human subunit
al HsPROS27 HsIota C7 PRS2 27.4
a2 HsC3 Y7 25.9
a3 HsC9 Y 13 29.5
a4 XAPC7 HsC6 PRE6 27.9
a5 HsZeta PUP2 26.4
a6 HsPROS30 HsC2 PRES 30.2
a7 HsC8 C 1 PRS 1 28.4
~ 1 HsDelta Y PRE3 25.3 (21.9)
X31 i LMP2 23.2 (20.9)
X32 Z PUP 1 30.0 (24.5)
(32i MECLI 28.9 (23.8)
(33 HsClO-11 PUPS 22.9
/34 HsC7-1 PRE1 C11 22.8
(35 MB1 X PRE2 nd (22.4)
~i5i LMP7 30.4 (21.2)
(36 HsCS CS PRS3 26.5 (23.3)
(37 HsBPROS26 HsN3 Pre4 29.2 (24.4)
* Reproduced from Gerards et al., CMLS 54: 253-262 (1998)
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
- 16-
Table 3: Schematic representation of the human 20S proteasome*
cr.
J ~j
( i: (Y.
* Reproduced from Gerards et al., CMLS 54: 253-262 (1998)
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
- 17-
Proteolvtic activity
The first report on the multicatalytic properties of the proteasome stems
from 1983, when three different proteolytic activities were distinguished:
'trypsin-
like', 'chymotrypsin-like' and 'peptidylglutamyl-peptide hydrolase' activity
[Wilk,
S. and M. Orlowski, J. Neurochem. 40: 842-849 (1983)]. These three
proteasomal activities refer to peptide bond cleavage at the carboxyl side of
basic,
hydrophobic and acidic amino acid residues, respectively. They were identified
using short synthetic peptide substrates and are believed to be catalyzed at
independent sites - in part because the different proteolytic activities
respond
differentially to various activators and inhibitors. With similar approaches,
at least
two additional proteolytic activities have been recently described [Orlowski
et al. ,
Biochemistry 32: 1563-1572 (1993); Orlowski, M., Biochemistry 29: 10289-10297
(1990); Rivett, A.J., Biochem. J. 291: 1-10 (1993)].
The Progressive De;~radation Of Protein Substrates
Recent studies have also revealed a fundamental new property of the
proteasome that clearly distinguishes it from conventional proteases: i.e.,
this
particle degrades a protein substrate all the way to small peptides, before
attacking
another protein substrate [Akopian et al. , J. Biol. Chem. 272: 1791-1798 (
1997)] .
Because the proteasome's multiple active sites are located in its central
chamber
and because diffusion of a peptide substrate into this compartment must be a
slow
process, these particles function in a highly processive fashion; i.e., they
have
mechanisms of action to bind tightly protein substrates and to make multiple
cleavages in the polypeptide before releasing the peptide products. Moreover,
the
ratio of new peptides generated to the number of substrate molecules consumed
is
constant during the reaction. In other words, as peptides accumulated, they
were
not hydrolyzed further, even during prolonged incubations, where up to half of
the
substrate molecules were consumed. Equally important, the disappearance of
these
substrate molecules coincided exactly with the appearance of small peptide
products
[Goldberg et al., Biol. Chem. 37$: 131-140 (1997)). These observations,
together
with the finding that the pattern of the products is independent of time,
established
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-18-
that processive degradation is a general feature of the 20S proteasome [Gerard
et
al., CMLS 54: 253-262 (1998)].
The contribution of each individual active center and proteolytic activity to
the degradation of longer peptides and complete proteins is presently unknown.
Nevertheless, proteasomes are able to cleave behind most amino acids in a
protein.
Thus, the 20S proteasome is in fact a nonspecific endopeptidase. In addition,
however, the generated (degraded) peptides fall into a rather narrow size
range of
6 to 10 amino acids in length, demonstrating the existence of a kind of '
molecular
ruler' . The average length of the degradation products is typically 7 to 8
amino
acids; this finding is in agreement with the distance between the active sites
in the
proteasome. Similar nonspecific endopeptidase activity and size distribution
of
degration products from whole proteins was observed for proteasomes generally
and by proteasomes of human origin in particular.
Other features of the 20S proteasome degradation are also unique. While
unfolded peptides are usually digested, most native proteins are resistant to
proteolytic degradation by the 20S proteasome in vitro. However, denaturation
of
the substrate protein by oxidation or reduction of disulphide bridges can
render it
accessible to degradation by proteasomes. Also, small gold particles with a
diameter of 2 nm containing unfolded substrate cannot enter the proteasome.
These characteristics show that a relatively narrow opening controls access to
the
inner proteolytic compartment of the proteasome.
III. The PR-39 Oligopeptide Collective
Native PR-39 peptide is a substance belonging to the cathelin family of
proteins; the mature peptide is 39 amino acids in length in the naturally
occurring
state; and the peptide is able to exert a variety of activities and cause
different
cellular outcomes. Although first identified as a membrane permeating
antibacterial peptide found in the intestine of pigs [Agerberth et al.; Eur.
J.
Biochem. 202: 849-854 (1991)], this peptide was subsequently isolated from
wounds where it could simultaneously reduce infection and influence the action
of
growth factors, matrix components, and other cellular effectors involved in
wound
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-19-
repair [Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994); Gallo
et al., J. Invest. Dermatel. 104: 555 (1995)). The structure and membrane
interactions of native PR-39 peptide have also been elucidated [Cariaux et
al., Eur.
J. Biochem. 224: 1019-1027 (1994)) and the complete amino acid sequences of
native PR-39 peptide and its various substituted forms have been reported [PCT
Publication No. WO 92/22578 published 23 December 1992).
More recently, the native PR-39 peptide was shown to possess a syndecan-
inducing activity in furtherance of its wound healing capabilities; and while
renamed a "synducin", was shown to induce cellular production of two specific
proteoglycans, syndecan-1 and syndecan-4, within living mesenchymal cells
[U.S.
Patent No. 5,654,273). Overall, native PR-39 peptide has been shown to play a
role in several inflammatory events including wound healing and myocardial
infarction [Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994);
Li et al., Circ. Res. 81: 785-796 (1997)); and the native peptide has been
shown to
be taken up rapidly by a number of different cell types including meschymal
cells
and endothelial cells [Chan, Y.R. and R.L. Gallo, J. Biol. Chem. 273: 28978-
28985 (1998)).
The PR-39 peptide grouping
Native PR-39 peptide is composed of the 39 amino acid sequence shown
below (and also by Table 4).
PR-39: Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-
Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-Pro-Pro-Gly-Phe-
Pro-Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro
As conventionally known and reported [see for example, U.S. Patent No.
5,654,273), the specific peptide can be substituted using conservative
substitutions
of amino acids having the same or functionally equivalent charge and
structure,
except for the required amino acid sequence "Arg-Arg-Arg" at the N-terminus
and
the intermediate amino acid sequences "Pro-Pro-X-X-Pro-Pro-X-X-Pro" and "Pro-
Pro-X-X-X-Pro-Pro-X-X-Pro" where X can be substituted freely using any amino
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-20-
acid. Thus, all of the preferred substituted amino acid sequences are of about
the
same size and each differ from the native PR-39 peptide sequence only by
substitutions in the intermediate portions of the structure.
The PR-39 derived oligopeptide family
In addition to the conventionally known native PR-39 peptide amino acid
residue sequence and its readily recognizable substituted forms as described
above,
an entirely novel and unforeseen family of PR-39 derived oligopeptide
structures is
provided by the present invention for use. This previously unknown family of
PR-
39 derived oligopeptides is constituted of members which individually will
cause a
selective inhibition of proteasome-mediated degradation of peptides in-situ
after
introduction intracellularly to a viable cell.
Each member of this PR-39 derived oligopeptide family presents
characteristics and properties which are commonly shared among the entire
membership. These include the following:
(i) each peptide sequence is less than 39 amino acid residues in length
in every embodiment, and preferably is less than 20 residues in size in the
best
mode;
(ii) each short-length peptide sequence is at least partially homologous
(or analogous) with the N-terminal amino acid residues of the native PR-39
peptide, and preferably is completely identical or markedly similar to the
N-terminal end residues of the native PR-39 peptide;
(iii) each short-length peptide is able to interact in-situ with at least the
a7 subunit of such proteasomes as are present within the cytoplasm of the
cell; and
(iv) each short-length peptide sequence is able to alter markedly the
proteolytic activity of proteasomes with an interacting a7 subunit such that a
selective increased expression of specific proteins (such as IKBa and HIF-la)
occurs in-situ.
Merely as illustrative examples and preferred embodiments of the broad
membership constituting this PR-39 derived oligopeptide family, the members
comprising 15, 11 and 8 amino acid residues respectively in length are
presented
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-21 -
below as the PR1$, PR11, and PR8 entities respectively. For comparison
purposes
only, the complete amino acid sequence of the native PR-39 peptide is
presented as
well.
$ 1 2 3 4 5 6 7 8 9 10 11 12 13
PR-39: Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-
14 15 16 17 18 19 20 21 22 23 24 25 26
Pro-Pro-Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-
27 28 29 30 31 32 33 34 35 36 37 38 39
Pro-Pro-Gly-Phe-Pro-Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro
1$
PR-15: 1 2 3 4 5 6 7 8 9 10 11 12 13
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-
14 15
Pro-Pro
PR-11: 1 2 3 4 5 6 7 8 9 10 11
2$ Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg
PR-8: 1 2 3 4 5 6 7 8
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr
The PR-39 Oligopeptide Collective
Terminology and nomenclature often pose problems for the reader as to
3$ what precisely is meant. Accordingly, for definitional purposes, avoidance
of
ambiguities, and clarity of understanding, the following terms and titles will
be
employed herein. The term "PR-39 peptides grouping" includes by definition the
native PR-39 structure and all substituted forms conventionally known of the
naturally occurring 39 length amino acid sequence. In distinction, the term
"PR-39
derived oligopeptide family" and its members includes by definition all the
previously unknown shorter-length homologs and analogs of the native PR-39
structure as described above. Finally, the umbrella term and category title
"PR-39
oligopeptide collective" includes by definition both the ' PR-39 peptide
grouping' as
well as the 'PR-39 derived oligopeptide family' members, and identifies any
and
4$ all individual structures falling into either of the two subset categories.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-22-
Table 4:
(1) GfiNSRAL INFORMATION:
(i) APBLICANT~ Children's Medical Center Corporaton
(ii) TITLE OF INVBrJTION: Synducin Mediated Modulation of
Tissue Repair
(iii) NUMBER OF SEQUSNCHS: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Patrea L. PabsC
(B) STREET: 2800 One Atlantic Center
1201 West Peachtree
(C) CITYs Atlanta
(D) STATE: Georgia
(E ) COUNTRY : USA
(F) ZIPS 30309-3450
(v) COMPUTER RP.ADA9LE FORM:
(A) MEDIUM TYPE: Floppy disk
(H) COMP'OTER: IHM PC Compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) 80PTf~IA&E: PatentIn Release #1.0, Version #1.25
( ix ) TELECOIR~fLTNICATION INFORMATION
(A) TELEPHONE: (404)-873-8794
(B) TELEFAX: (409)-815-8795
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(H) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(x) PUBLICATION INFORMATION:
(A) AiTTHORS: Lee, Jong-Youn
Boman, Hans G.
Mutt, Viktor
Jornvall, Haas
(B) TITLE: Novel Polypeptides And Their Use
(C) JOURNAL: PCT WO 92/22578
(G) DATE: 12/23/92
(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Pro
1 5 10
Phe Phe Pro Fro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe
Pro Pro
25 30
Arg Phe Pro Pro Arg Phe Pro
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
- 23 -
Synthesis
The PR-39 peptide can be synthesized using standard amino acid synthetic
techniques. An example is the conventionally used solid phase synthesis
[Merrifield, J., J. Am. Chem. Soc. 85: 2149 (1964)] described in U.S. Patent
No.
4,244,946, wherein a protected alpha-amino acid is coupled to a suitable
resin, to
initiate synthesis of a peptide starting from the C-terminus of the peptide.
Other
methods of peptide synthesis are described in U.S. Patent Nos. 4,305,872 and
4,316,891, the teachings of which are incorporated herein. These methods can
be
used to synthesize peptides having identity with the native PR-39 peptide
amino
acid sequence described herein, or to construct desired substitutions or
additions of
specific amino acids, which can be screened for content and evaluated for
activity.
PR-39 can also be commercially obtained from Magainin, Inc. (Plymouth Meeting,
PA).
Pharmaceutical Formats
After synthesis or purchase, the PR-39 peptides (as a family of homologs
and analogs with substituted amino acid residues) can be introduced as a
peptide-
containing preparation in a pharmaceutically acceptable format.
The PR-39 can be administered and introduced in-vivo systemically,
topically, or locally. The peptide can be administered as the peptide or as a
pharmaceutically acceptable acid- or base-addition salt, formed by reaction
with an
inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid,
nitric
acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic
acid
(such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid,
pyruvic
acid, oxalic acid, malonic acid, succinic acid, malefic acid, and fumaric
acid); or
by reaction with an inorganic base (such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide); or with an organic base (such as mono-,
di-, trialkyl and aryl amines and substituted ethanolamines).
PR-39 peptide and any of the PR-39 derived oligopeptide family members
may also be conjugated to sugars, lipids, other polypeptides, nucleic acids
and
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-24-
PNA; and function in-situ as a conjugate or be released locally after reaching
a
targeted tissue or organ. The PR-39 family of peptides may also be linked to
targeting compounds for attachment in-situ to a specific cell type, tissue or
organ.
IV. Means For Introduction Of PR-39 Peptide
And/Or Its Shorter-Length Derived Homologs
DNA Fragments and Expression Vectors
A variety of means and methods are conventionally known and presently
available to the user or practitioner of the present invention in order to
introduce
PR-39 peptide (or a derived oligopeptide family member) to living cells and
tissues. One desirable means uses a prepared DNA sequence fragment encoding
the PR-39 peptide (or a shorter-length homology in a suitable vector as the
means
of introduction to the intended target in-situ. These means for delivery
envision
and include in-vivo use circumstances; ex-vivo specimens and conditions; and
in-
vitro cultures. In addition, the present invention intends and expects that
the
prepared DNA sequence fragment coding for PR-39 peptide (or shorter-length
homologs) has been inserted in a suitable expression vector and will be used
in a
route of administration for delivery to living tissues comprising endothelial
cells,
and typically vascular endothelial cells which constitute the basal layer of
cells
within capillaries and blood vessels generally. Clearly, the cell recipients
themselves are thus eukarytoic in origin, typically mammalian cells from human
and animal sources; and most typically would include the higher orders of
mammals such as humans and domesticated mammalian animals kept as pets or
sources of food intended for future consumption. Accordingly, the range of
animals includes all domesticated varieties involved in nutrition including
cattle,
sheep, pigs and the like; as well as those animals typically used as pets or
raised
for commercial purposes including horses, dogs, cats, and other living mammals
typically living with and around humans.
Clearly, the expression vectors must be suitable for transfection of
endothelial cells in living tissues of mammalian origin and thus be compatible
with
that type and condition of cells under both in-vivo and/or in-vitro
conditions. The
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
- 25 -
expression vectors thus typically include plasmids and viruses as expression
vectors.
Also, both the plasmid based vectors and the viral expression vectors
constitute conventionally known means and methods of introduction which are
conventionally recognized today as "gene therapy" modes of delivery. However,
this overall approach is not the only means and method of delivery available
for the
present invention.
Direct Introduction of Previousl~S~nthesized PR-39 Peptides or a PR-39 Derived
Ol~o~peptide Famil~Member
PR-39 peptide or an oligopeptide family member can be introduced directly
as a synthesized compound to living cells and tissues via a range of different
delivery means. These include the following.
1. Intracoronary delivery is accomplished using catheter-based deliveries of
synthesized PR-39 peptide (or homolog member) suspended in a suitable buffer
(such as saline) which can be injected locally (i.e., by injecting into the
myocardium through the vessel wall) in the coronary artery using a suitable
local
delivery catheter such as a lOmm InfusaSleeve catheter (Local Med, Palo Alto,
CA) loaded over a 3.Omm x 20mm angioplasty balloon, delivered over a 0.014
inch angioplasty guidewire. Delivery is typically accomplished by first
inflating
the angioplasty balloon to 30 psi, and then delivering the protein through the
local
delivery catheter at 80 psi over 30 seconds (this can be modified to suit the
delivery catheter).
2. Intracoronary bolus infusion of PR-39 peptide (or a short-length homology
synthesized previously can be accomplished by a manual injection of the
substance
through an Ultrafuse-X dual lumen catheter (SciMed, Minneapolis, MN) or
another
suitable device into proximal orifices of coronary arteries over 10 minutes.
3. Pericardial delivery of synthesized PR-39 peptide (or a shorter-length
homology is typically accomplished by instillation of the peptide-containing
solution
into the pericardial sac. The pericardium is accessed via a right atrial
puncture,
transthoracic puncture or via a direct surgical approach. Once the access is
established, the peptide material is infused into the pericardial cavity and
the
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-26-
catheter is withdrawn. Alternatively, the delivery is accomplished via the aid
of
slow-release polymers such as heparin-alginate or ethylene vinyl acetate
(EVAc).
In both cases, once the PR-39 peptide (or homology is integrated into the
polymer,
the desired amount of PR-39/polymer is inserted under the epicardial fat or
secured
to the myocardial surface using, for example, sutures. In addition, the PR-39/
polymer can be positioned along the adventitial surface of coronary vessels.
4. Intramyocardial delivery of synthesized PR-39 peptide (or a shorter-length
homology can be accomplished either under direct vision following thoracotomy
or
using thoracoscope or via a catheter. In either case, the peptide containing
solution
is injected using a syringe or other suitable device directly into the
myocardium.
Up to 2 cc of volume can be injected into any given spot and multiple
locations (up
to 30 injections) can be done in each patient. Catheter-based injections are
carried
out under fluoroscopic, ultrasound or Biosense NOGA guidance. In all cases
after
catheter introduction into the left ventricle the desired area of the
myocardium is
injected using a catheter that allows for controlled local delivery of the
material.
Pharmaceutical Carriers Of PR-39 Peptides or a PR-39 Derived Olig-onentide
Family Member
A range of suitable pharmaceutical carriers and vehicles are known
conventionally to those skilled in the art. Thus, for parenteral
administration, the
compound will typically be dissolved or suspended in sterile water or saline.
For enteral administration, the PR-39 peptide or homologous oligopeptide of
choice will be typically incorporated into an inert carrier in tablet, liquid,
or
capsular form. Some suitable carriers are starches and sugars; and often
include
lubricants, flavorings, binders, and other materials desirable in tablet
making
procedures.
The PR-39 peptide and oligopeptide family of compounds can also be
administered topically by application of a solution, cream, gel, or polymeric
material (for example, a PluronicTM, BASF).
As an alternative, the chosen peptide can be administered in liposomes or
microspheres (or microparticles), which can be injected for local or systemic
delivery. Methods for preparing liposomes and microspheres for administration
to
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-27-
a patient are conventionally known to those skilled in the art. For example,
U.S.
Patent No. 4,789,734 describes methods for encapsulating biological materials
in
liposomes. Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants if
required, and
the material dialyzed or sonicated, as necessary. See also, G. Gregoriadis,
Chapter 14, "Liposomes", Drug Carriers in Biolog~and Medicine, chap. 14, pp.
287-341 (1979). Microspheres formed of polymers or proteins are well known to
those skilled in the art, and can be tailored for passage through the
gastrointestinal
tract directly into the bloodstream. Alternatively, the compound can be
incorporated and the microspheres, or composite of microspheres, implanted
from
days to months. See, for example, U.S. Patent Nos. 4,906,474; 4,925,673; and
3,625,214.
Examplary Introductions And Preferred Routes of Administration
A variety of approaches, routes of administration, and delivery methods
have been identified herein and are available for introduction of PR-39
peptide and
the derived family of oligopeptides. It is envisioned, however, that a
majority of
the approaches and routes of administration described herein will be medical
applications and specific clinical approaches intended for use with individual
human
patients having specified medical problems and diagnosed pathologies. It is
expected, accordingly, that the reader is familiar generally with the typical
clinical
human problem, pathology, and medical conditions described herein; and
therefore
will be able to follow and easily understand the nature of the intervention
clinically
using the present invention and the intended outcome and result of the
clinical
treatment - particularly as pertains to the stimulation of angiogenesis under
in-vivo
treatment conditions. A representative listing of preferred clinical
approaches is
given by Table 5 below.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-28-
Table S
Preferred Routes of Administration
Catheter-based (intracoronary) injections and infusions;
Direct myocardial injection
(intramyocardial guided);
Direct myocardial injection
(direct vision-epicardial-open chest or under thorascope guidance);
Local intravascular delivery;
Liposome-based delivery;
Delivery in association with receptor-specific peptides;
Oral delivery;
In instances of peripheral vascular disease:
- intramuscular injection
- intraarterial injection and/or infusion.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-29-
V. Experiments and Empirical Data
To demonstrate the merits and value of the present invention, a series of
planned experiments and empirical data are presented below. It will be
expressly
understood, however, that the experiments described and the results provided
are
merely the best evidence of the subject matter as a whole which is the
invention;
and that the empirical data, while limited in content, is only illustrative of
the
scope of the invention envisioned and claimed.
Introduction
Proteolytic degradation in mammalian cells is known to proceed via two
distinct pathways: lysosome-dependent degradation and proteasome-dependent.
The proteasome in its pathway plays a key role in proteolysis of intracellular
proteins which are marked for degradation by the ubiquitin system. The
multienzyme complex involved in these events, the 26S proteasome, consists of
a
20S catalytic proteasome "core" and two 19S caps that bind uniquitylated
proteins,
as has been described in detail previously herein. Proteasome-mediated
proteolysis
is a principal event quantitatively controlling intracellular levels of a
number of
different proteins including hypoxia-inducing factor (HIF)-la, heat shock
protein
HSP70, protooncogenes c-Fos, c-Jun and c-Mos, NFKB inhibitor IKBa, and
various cyclins. In addition, the proteasome also is known to play a critical
role in
the specific processing and presentation of major histocompatibility complex
(MHC) class I-restricted antigens as well as provide partial proteolytic
cleavage of
p 105 NFKB to the active p50 subunit.
The PR-39 peptide, belonging to the cathelin family of proteins, plays an
important role in several inflammatory events including wound healing and
myocardial infarction. The PR-39 peptide typically is rapidly taken up by a
number of different cell types including endothelial cells; and prolonged
treatment
with PR-39 peptide leads to increased cell growth and angiogenesis. However,
the
mechanism of action for this peptide activity has yet to be understood or
defined.
The experiments and data presented below reveal for the first time the nature
and
detailed intracellular actions exerted by the PR-39 peptide.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-30-
Methods and Materials:
Yeast two-hybrid screening
Two-hybrid screening was carried out using MATCHMAKER GAL4
System 2 (Clontech) with exon 4 of the porcine PR-39 gene as a bait to screen
the
mouse embryo 3T3 cDNA library in yeast CG 1945.
Cell culture studies
U937 cells (ATCC) grown in RPMI medium 1640 with 10% FBS (Gibco-
BRL) and ECV cells were treated with synthetic PR-39, lactacystin (CalBiochem,
426100) or MG 132 (CalBiochem, 474790) at concentration indicated in the
presence of 100 mM cyclohexamide 20 mM chloroquine [Merin et al., J. Biol.
Chem. 273: 6373-6379 (1995)]. After 45 min. of incubation, TNFa (1 ng/ml) was
added. After 5 min of 37°C incubation, the cells were lysed in SDS-PAGE
loading
buffer. Following SDS-PAGE of the total protein extract, IKB-a and NF-KB p105
p50 expressions were determined by Western blotting with anti-human antibodies
(Santa Cruz, sc-203, sc114G). For studies of HIF-la and VEGF expression, ECV
cells were cultured in a hypoxia chamber (5 % COZ/95 % Nz) at 37°C for
16 hr.
HIF-la was immunoprecipitated with anti-HIF-la mAb (OZ12 1:5) in RIPA buffer
and Western blotting with anti-HIF-la mAb (OZ15 1:10) (courtesy of
Dr. A. King, DFCI, Boston). VEGF expression was shown by Western blotting
of hypoxia treated ECV cell lysate with anti-human VEGF antibody (Santa Cruz,
sc-152). For HSP70 expression, U937 was treated for 3 hr, harvest with SDS-
PAGE loading buffer, Western blotting with anti-human HSP70 polyclonal
antibody (Santa Cruz, sc-1060).
In-vitro proteasome activity assays
Rabbit muscle 20S proteasome preparation (courtesy of Dr. M. Sherman,
BBRI, Boston) was used for all studies. For determination of proteasome
activity,
5 ~,1 of 1:10 diluted proteasome preparation was incubated at room temperature
in
eukaryotic proteasome assay buffer (20 mM Tris-HCl pH 8.0, 0.5 mM EDTA and
0.01 % SDS) with 20 ~cM proteasome substrates (CalBiochem, 539140-3) and
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-31-
PR-39 or other proteasome inhibitor at indicated concentration [Rock et al. ,
Cell
78: 761-771 (1994)]. The extent of substrate degradation was monitored
continuously by fluorescence spectrophotometry (380 nm excitation, 460nm
emission Hitachi F-2000) for 10 min.
Experiment l:
This experiment was designed to reveal the ability of PR-39 peptide to
affect proteasome function. To test this capability, the effect of PR-39
administration upon a7 subunit processing was empirically determined. The
results
are illustrated by Figs. lA-1D respectively.
Experimentally, a peptide corresponding to the 4th exon porcine PR-39
gene sequence was used to generate a rabbit polyclonal antibody RPE4. Full
length porcine cDNA (containing leader sequence) and a sequence corresponding
to
the 4th exon of porcine PR-39 gene were cloned into eukaryotic expression
vector
pGRES-2 (USB). These expression constructs were then used to stably transfect
an
immortalized human endothelial cell line (ECV304, ATCC). For co-
immunoprecipitation, wild type ECV, full length PR-39 (ECV-PR39) and exon 4
PR39 and exon 4 PR39 (ECV-F~l) transfected cells were cultured in Medium 199
with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were
lysed
with RIPA buffer; immunoprecipitated with 10 ~cg affinity purified rabbit anti-
PR39
antibody; and following Protein A-Sepharose purification and SDS-PAGE,
subjected to immunoblotting with 1:1000 mouse anti-HC8 mAb (Affiniti Research
Products Limited UK, PW8110).
Figs. 1 A-1 D show the interactions of PR-39 peptide and the a7 subunit of
proteasomes. Fig. lA recites the cDNA sequence of cloned mouse a7 subunit
(top; GeneBank accession number AF055983) and corresponding human HC8
subunit of 205 proteasome. Fig. 1B shows the sequence alignment of C-terminal
tails mouse a subunits of 20S proteasome. Fig. 1C shows a deletion analysis of
a7-PR39 binding. Deletion mutants of the mouse a7 subunit were cloned into an
yeast-two hybrid vector and the extent of growth of IacZ+ colonies on
selective
medium following co-transformation with PR-39 construct in the yeast CG 1945
was determined. It is noted that only full length a7 construct was able to
bind to
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-32-
PR-39. Finally, Fig. 1D shows the co-immunoprecipitation of PR-39 and a7
subunit in ECV cells.
It will be noted also that Fig. 1 represents the evidence of four clones
growing on selective media and demonstrating IacZ staining. All four clones
encoded overlapping identical cDNA sequences highly homologous to the human
sequence of a7 (HC8) subunit of proteasome (Fig. lA). Similar to all a
subunits
of the 20S proteasome, the cloned mouse protein possesses a highly conserved
N-terminal region; in addition it demonstrated the presence of 16 amino acid
long
C-terminal sequence found in some but not all a subunits (Fig. 1B). Deletion
analysis showed that the presence of both C-terminal as well as N-terminal
amino
acids sequences was required for PR-39 binding (Fig. 1C). In order to confirm
the
PR39-a7 subunit interaction in-vivo, anti-PR39 antibody was used to
immunoprecipitate PR39 protein from ECV-PR39, ECV-E4 and mock-transfected
ECV cells. Western blotting of the immunoprecipitate from ECV-PR39 and ECV-
E4 but not wild type ECV cells with anti-a7 subunit antibody demonstrated the
presence of a 29kDa band corresponding to the known size of a7 subunit protein
(Fig. 1D). The evidence therefore reveals that PR39 peptide interacts with a7
subunit of proteasome in ECV cells.
Experiment 2:
To test the ability of PR-39 peptide to affect proteasome function in-vivo,
the effect of PR-39 peptide administration on IKBa processing was assessed.
The
results are illustrated by Figs. 2A-2D respectively.
Fig. 2A shows a Western analysis of IKBa expression in ECV cells. The
results show that pretreatment of cultured ECV cells with lactacystin (10 ~,M,
4th
lane) or stable expression of full length (ECV-PR39) or PR39 exon 4 (ECV-E4)
constructs inhibited TNF-a-induced degradation of IxB.
Thus, tumor necrosis factor (TNF)-a induces rapid degradation of IKBa - a
function that is blocked by the proteasome inhibitor lactacystin. However,
Western
analysis of IKBa levels after TNF-a treatment demonstrated comparable levels
of
IKBa expression in both ECV-PR39 and ECV-E4 cells to that seen in ECV cells
pre-treated with lactacystin.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-33-
Fig. 2B shows the effect of PR39, MG132 and lactacystin pretreatment on
IKBa expression in U937 cells following TNF-a treatment. Note similar extent
of
inhibition of IKBa degradation by TNF-a following pretreatment with PR39,
MG132 or lactacystin. Thus, it is clear that pretreatment of U937 cells with
PR39
blocked TNF-a induced IxBa degradation in a manner that was similar to the
degree of inhibition seen with MG132 and lactacystin.
Fig. 2C demonstrates the reversibility of PR39 inhibition of proteasome
activity. U937 cells were pretreated with PR39, MG132 or lactacystin for 45
min.
After that time, the cells were extensively washed with fresh medium. 45 min
later TNF-a (1 ng/ml) was added to the medium and the extent of IKBa
degradation was determined 10 min later by Western blotting. Note preservation
of IKBa in lactacystin-treated cells but not PR39-treated cells. Thus, unlike
lactacystin but similar to MG 132, PR-39 peptide mediated inhibition of IKBa
degradation was rapidly reversible.
Finally, to show that PR-39 inhibition of IKBa degradation affected NFKB-
dependent transcription, ECV cells were transiently transfected with a NFKB-
Luc
reporter construct containing a tandem of four NFxB binding sites in front of
luciferase cDNA. The results of Fig. 2D show that stimulation with TNF-a
induced a significant increase in luciferase activity that was completely
inhibited by
pretreatment with PR39.
Accordingly, the true functional significance of PR39-mediated inhibition of
ItcBa degradation in ECV cells transiently transfected with pNFxB-Luc reporter
vector (Clontech) is clearly shown by Fig. 2D. Pre-treatment with PR39
completely inhibited TNF-a-induced increase in luciferase activity. *p < 0.01
vs.
control (Luc activity in the absence of TNF-a).
Experiment 3:
To demonstrate directly the ability of PR-39 peptide to inhibit proteasome-
mediated protein degradation, preparations of eukaryotic 20S proteasomes were
tested for their ability to induce proteolysis of various synthetic peptides
in-vitro.
The results are graphically illustrated by Figs. 3A-3D respectively.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-34-
For determination of proteasome activity, 5 ~,1 of 1:10 diluted rabbit muscle
20S proteasome preparation (courtesy of Dr. M. Sherman, BBRI, Boston) was
incubated at room temperature in an assay buffer (20 mM Tris-HCl pH 8.0,
0.5 mM EDTA and 0.01 % SDS) with 20 ~cM of four different proteasome
substrates (CalBiochem, 539140-3) and PR39 or other proteasome inhibitor at
indicated concentration. The extent of substrate degradation was monitored
continuously by fluorescence spectrophotometry (380 nm excitation, 460 nm
emission Hitachi F-2000) for 10 min.
Figs. 3A-3D reveal that the PR-39 peptide inhibited, in a dose-dependent
manner, degradation of all 4 peptides tested. PR-39 peptide was as potent as
lactacystin or MG 132 in inhibiting degradation in three of the four peptides
tested
and was considerably more potent in inhibiting degradation of the Z-Leu-Leu-
Glu-
AMC peptide.
Experiment 4:
To test the effect of PR39 treatment on cellular levels of other proteasome-
dependent proteins, the in-vivo expression of p105 and p50 NFKB, HSP70 and
HIF-la within transfected and wild type ECV cells was determined. The results
are illustrated by Figs. 4A-4C respectively.
Fig. 4A shows the results of a Western blot analysis of HIF-la, p50 and
p105 NFxB expression in wild type ECV cells and ECV-E4 and ECV-PR39
clones. It is noted that an increase in HIF-la expression occurs (but not p105
or
p50 NFxB expression) in ECV-E4 and ECV-PR 39 clones. Thus, while there was
a significant increase in expression of HIF-la and IxB in PR39 transfected or
treated compared to wild type cells, there was no significant change in
expression
of either HSP70 or NFKB - demonstrating that effects of PR39-proteasome
interaction are selective.
Since increased expression of HIF-la is known to result in increased
transcription of a number of angiogenesis-related molecules including VEGF,
Northern analysis of VEGF mRNA levels in wild type and PR39 transfected ECV
cells was performed. The results are shown by Fig. 4B. As expected, there was
a
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-35-
significant increase in expression of both of these genes in ECV-PR39 and ECV-
E4
cells compared to ECV controls.
Finally, exposure to proteasome inhibitor lactacystin is known to induce
rapid cell death. To test the effect of PR39 on cell survival, growth rates of
ECV
cells treated with PR39 peptide were assessed. The results are shown by Fig.
4C
50,000 ECV cells were cultured in 10% FBS-M199 in the absence (control) or
presence of 10 ~cM of PR39, lactacystin or MG 132. Note that while exposure to
PR39 did not affect cell growth, exposure to lactacystin or MG 132
substantially
inhibited cell growth. Thus, following 3 days of PR39 exposure, treated cells
demonstrated normal growth compared to controls while those cells exposed to
lactacystin demonstrated markedly reduced survival.
Experiment 5:
To demonstrate the stimulation of angiogenesis directly in living cells and
tissues via the introduction of PR-39 peptide, a mice matrigel assay system
was
employed. Growth factor-depleted Matrigel pellets containing 5 ~.g of PR39,
50 ng of FGF2 or saline (control) were inserted intraperitoneally into C57BL/6
mice. Ten days later the pellets were removed, sectioned and stained with anti-
CD31 antibody. The number of vessels was determined in multiple sections using
a digital camera and Optimas 5.0 software. The results are shown by Figs. SA-
SC
and Fig. 6 respectively.
Figs. SA-SC are representative sections from control, PR39 and FGF2
impregnated Matrigel pellets and Fig. 6 provides a quantitative analysis of
vascularity. Clearly, the results of the representative sections and the
graphic
quantitative evaluations demonstrate that insertion of growth-factor depleted
Matrigel pellet containing PR-39 peptide induced intense vessel growth that
exceeded that seen with implantation of pellets containing 50 ng/ml of bFGF.
Experiment 6:
To demonstrate the efficiency of shorter-length peptides which collectively
are members of the PR-39 derived oligopeptide family in stimulating
angiogenesis
in-vivo, a novel peptide, PRl l, composed of the first 11 amino acid residues
CA 02368638 2001-09-25
WO OD/57895 PCT/US00/07050
-36-
[N-terminal end] of the native PR-39 sequence was purposely synthesized. The
amino acid sequence of PR11 is as follows:
1 2 3 4 5 6 7 8 9 10 11
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg
To introduce the short-length PR 11 peptide in-vivo, a mouse Matrigel assay
system was utilized. In sum, either 5 ~g/ml of PR11 peptide or 5 ~.g/ml of
native
PR-39 peptide were individually placed into a growth factor-depleted Matrigel
pellet; and then each prepared Matrigel pellet was inserted into the
peritoneal
cavity of a mouse. After 14 days intraperitoneal placement, each pellet was
removed from its living host; and each pellet was examined for evidence of new
vascularity. The results are graphically presented by Fig. 7. Note that the
bar
graph of Fig. 7 shows the number of blood vessels [mean~SD] per 10 high power
fields (HPF).
As evidenced by Fig. 7, the analysis of Matrigel pellet vascularity after 14
days incubation in-vivo demonstrated significant induction of angiogenesis in
both
the PRl l and the native PR-39 pellets. The control Matrigel pellets, however,
showed no evidence of angiogenesis as such. Clearly therefore, the short-
length
PR11 peptide is fully efficacious and effective in stimulating angiogenesis in-
vivo.
Conclusions:
(1) The described experiments and empirical data have demonstrated that PR-39
peptide has the ability to selectively alter activity of 20S proteasome in
human
endothelial cells by interacting with proteasomes in a reversible manner. This
interaction leads to suppression of IKB and HIF-la degradation while not
affecting
expression of other proteasome-dependent proteins such as p105 NFKB or HSP70.
Unlike other proteasome inhibitors, treatment with PR39 is not associated with
any
cellular cytotoxicity. Thus, PR39 and its related peptides provide a unique
and
unforeseen means of regulating cellular function and stimulating angiogenesis.
CA 02368638 2001-09-25
WO 00/57895 PCT/US00/07050
-37-
(2) Several observations also set PR39 apart from the conventionally known
proteasome inhibitors. First, PR39-mediated inhibition of IKBa degradation is
demonstrably reversible, unlike that of lactacystin. Second, long-term
exposure of
several cell types to PR39 did not result in any cytotoxicity, in contrast to
the rapid
cell death typically observed following cell treatment with lactacystin or MG
132.
This observation shows that PR39 peptide differentially affects processing of
various and different intracellular proteins. Also supporting this view is the
observation that while increasing HIF-la expression, PR39 administration had
no
meaningful effect on the expression of either NFKB or HSP70. Third, PR-39
peptide modulation of proteasome activity plays a functional role since the
observed increased expression of HIF-la was directly associated with an
increased
expression of its target genes, VEGF and flt-1.
The present invention is not to be limited in form nor restricted in scope
except by the claims appended hereto.
