U.S. patent application number 12/815893 was filed with the patent office on 2010-11-18 for methods of regulating apoptosis.
This patent application is currently assigned to Sanford-Burnham Medical Research Institute. Invention is credited to Siva Kolluri, Arnold Satterthwait, Xiao-kun Zhang, Xiuwen Zhu.
Application Number | 20100292145 12/815893 |
Document ID | / |
Family ID | 36968601 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100292145 |
Kind Code |
A1 |
Satterthwait; Arnold ; et
al. |
November 18, 2010 |
METHODS OF REGULATING APOPTOSIS
Abstract
Compounds that modulate the function of anti-apoptotic proteins
such as Bcl-2 and related Bcl-2 family members are identified.
These compounds have the ability to convert the activity of
Bcl-2-family member proteins from anti-apoptotic to pro-apoptotic.
Methods for inducing or preventing apoptosis are described,
together with methods for identifying molecules that induce or
prevent apoptosis through interaction with Bcl-2-family members.
Methods for treatment of proliferative diseases and
neurodegenerative diseases using the modulators of Bcl-2 and
related family members are also disclosed.
Inventors: |
Satterthwait; Arnold; (San
Diego, CA) ; Zhang; Xiao-kun; (San Diego, CA)
; Zhu; Xiuwen; (San Deigo, CA) ; Kolluri;
Siva; (San Diego, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
Sanford-Burnham Medical Research
Institute
La Jolla
CA
|
Family ID: |
36968601 |
Appl. No.: |
12/815893 |
Filed: |
June 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11433783 |
May 11, 2006 |
7745574 |
|
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12815893 |
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60689206 |
Jun 9, 2005 |
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60680645 |
May 12, 2005 |
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Current U.S.
Class: |
514/8.4 ;
435/375 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 35/00 20180101; C07K 14/70567 20130101; G01N 33/6872 20130101;
C07K 14/4747 20130101 |
Class at
Publication: |
514/8.4 ;
435/375 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C12N 5/071 20100101 C12N005/071; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] This invention was made in part with United States
government support under grant numbers NIH CA87000, awarded by the
National Institutes of Health, DAMD 17-03-1-0427 awarded by the US
Army, and both BCRP 8WB-017 and TRDRP 11RT-0081 awarded by the
State of California. The U.S. Government has certain rights in this
invention.
Claims
1. A method of inducing apoptosis in a mammalian cell, comprising:
providing a compound comprising: Nur 77 or a fragment thereof; and
a cell-penetrating peptide; and contacting said mammalian cell with
an effective amount of said compound, wherein said contacting
results in apoptosis of said mammalian cell.
2. The method of claim 1, wherein contacting said mammalian cell is
performed in vitro.
3. The method of claim 1, wherein contacting said mammalian cell is
performed in vivo.
4. The method of claim 1, wherein said mammalian cell is a breast
cancer cell.
5. The method of claim 1, wherein said compound comprises D-amino
acids.
6. The method of claim 1, wherein said compound comprises at least
one non-naturally occurring amino acid.
7. The method of claim 1, wherein said compound is a peptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 60.
8. The method of claim 1, wherein said compound is a Nur77-derived
Bcl-2-converting peptide (NuBCP).
9. The method of claim 8, wherein said NuBCP peptide is
D-NuBCP-9-r8.
10. The method of claim 1, wherein said compound is a DC-3
peptide.
11. The method of claim 1, wherein said compound is 4-20 amino
acids in length.
12. A method of inducing apoptosis in a mammalian cell, comprising:
providing a compound consisting of: peptide having an amino acid
sequence of SEQ ID NO:60 or mutation or fragment thereof, wherein
the peptide is pro-apoptotic; and a cell-penetrating-peptide,
wherein said compound induces apoptosis of mammalian cells
expressing Bcl-2 or Bcl-2 related proteins; and contacting said
mammalian cell with an effective amount of said compound, wherein
said contacting results in apoptosis of said mammalian cell.
13. The method of claim 12, wherein said cell-penetrating peptide
consists of transportan10 or penetratin (SEQ ID NO: 65).
14. The method of claim 12, wherein said pro-apoptotic fragment
consists of the amino acid sequence Phe Gly Asp Trp Ile Asp Ser Ile
Leu (SEQ ID NO:16).
15. The method of claim 12, wherein said pro-apoptotic fragment
consists of the amino acid sequence Phe Ser Arg Ser Leu His Ser Leu
Leu (SEQ ID NO:9).
16. The method of claim 12, wherein said pro-apoptotic fragment
consists of the amino acid sequence Phe Ala Cys Leu Ser Ala Leu Val
Leu (amino acid residues 37-45 of SEQ ID NO:60).
17. The method of claim 12, wherein said pro-apoptotic fragment
consists of the amino acid sequence Phe Tyr Leu Lys Leu Glu Asp Leu
Val (SEQ ID NO:18).
18. The method of claim 12, wherein said mutation is selected from
the group consisting of an amino acid deletion, an amino acid
insertion, a conservative amino acid substitution, an amino acid
substitution with alanine, and an amino acid substitution with a
non-naturally occurring amino acid.
19. The method of claim 12, wherein said compound comprises one or
more D-amino acids.
20. A method of inducing apoptosis in a breast cancer cell,
comprising: providing a compound comprising: Nur 77 or a fragment
thereof; and a cell-penetrating peptide; and contacting said breast
cancer cell with an effective amount of said compound, wherein said
contacting results in apoptosis of said breast cancer cell.
21. The method of claim 20, wherein contacting said breast cancer
cell is performed in vitro.
22. The method of claim 20, wherein contacting said breast cancer
cell is performed in vivo.
23. The method of claim 20, wherein said compound is a NuBCP.
24. The method of claim 21, wherein said NuBCP is L-NuBCP-9-r8 or
D-NuBCP-9-r8.
25. The method of claim 20, wherein said cell-penetrating peptide
is Lyp-1 or F3.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/433,783, filed on May 11, 2006, which
claims the benefit of priority of U.S. Provisional Application No.
60/680,645, filed May 12, 2005, and U.S. Provisional Application
No. 60/689,206, filed Jun. 9, 2005, both of which are hereby
expressly incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] Compounds are provided herein that bind to Bcl-2-family
member proteins and alter their apoptosis regulatory function. More
specifically, the compounds are peptides, peptide analogs and small
molecules based on the Nur77 protein or functionally related
proteins that mimic the effects of Nur77 by binding Bcl-2 or
related anti-apoptotic Bcl-2 family members, converting them to
pro-apoptotic or neutral states resulting in apoptosis.
DESCRIPTION OF THE RELATED ART
[0004] Apoptosis, also known as programmed cell death, is a
physiological process through which the body disposes of unneeded
or undesirable native cells. The process of apoptosis is used
during development to remove cells from areas where they are no
longer required, such as the interior of blood vessels or the space
between digits. Apoptosis is also important in the body's response
to disease. Cells that are infected with some viruses can be
stimulated to undergo apoptosis, thus preventing further
replication of the virus in the host organism.
[0005] Impaired apoptosis due to blockade of the cell
death-signaling pathways is involved in tumor initiation and
progression, since apoptosis normally eliminates cells with
increased malignant potential such as those with damaged DNA or
aberrant cell cycling (White, 1996 Genes Dev 10:1-15). The majority
of solid tumors are protected by at least one of the two cell death
antagonists, Bcl-2 or Bcl-X.sub.L. Members of the Bcl-2-family are
known to modulate apoptosis in different cell types in response to
various stimuli. Some members of the family act to inhibit
apoptosis, such as Bcl-2 and Bcl-X.sub.L, while others, such as
BAX, BAK, Bid, and Bad, promote apoptosis. The ratio at which these
proteins are expressed can decide whether a cell undergoes
apoptosis or not. For instance, if the Bcl-2 level is higher than
the BAX level, apoptosis is suppressed. If the opposite is true,
apoptosis is promoted. Bcl-2 overexpression contributes to cancer
cell progression by preventing normal cell turnover caused by
physiological cell death mechanisms, and has been observed in a
majority of cancers (Reed, 1997 Sem Hematol 34:9-19; Buolamwini,
1999 Curr Opin Chem Biol 3:500-509). The expression levels of Bcl-2
proteins often correlate with resistance to a wide spectrum of
chemotherapeutic drugs and .gamma.-radiation therapy.
Paradoxically, high levels of Bcl-2 also associate with favorable
clinical outcomes for patients with some types of cancers.
[0006] Biological approaches targeted at reducing Bcl-2 levels
using antisense oligonucleotides have been shown to enhance tumor
cell chemosensitivity. Antisense oligonucleotides targeted to Bcl-2
in combination with chemotherapy are currently in phase II/III
clinical trials for the treatment of patients with lymphoma and
malignant melanoma, and further trials with patients with lung,
prostate, renal, or breast carcinoma are ongoing or planned (Reed,
1997 Sem Hematol 34:9-19; Piche et al. 1998 Cancer Res 2134-2140;
Webb et al. 1997 Lancet 349:1137-1141; Jansen et al. 1998 Nat Med
4:232-234; Waters et al. 2000 J Clin Oncol 18:1812-1823). Recently,
cell-permeable Bcl-2 binding peptides and chemical inhibitors that
target Bcl-2 have been developed, and some of them have been shown
to induce apoptosis in vitro and in vivo (Finnegan et al. 2001 Br J
Cancer 85:115-121; Enyedy et al. 2001 J Med Chem 44:4313-4324;
Tzung et al. 2001 Nat Cell Biol 3:183-191; Degterev et al. 2001 Nat
Cell Bio 3:173-182; Walensky et al. 2004 Science 305: 1466-1470;
Oltersdorf et al. 2005 Nature 435: 677-681).).
[0007] One well-established apoptotic pathway involves mitochondria
(Green and Reed, 1998 Science 281:1309-1312; Green and Kroemer,
1998 Trends Cell Biol 8:267-271). Cytochrome C is exclusively
present in mitochondria and is released from mitochondria in
response to a variety of apoptotic stimuli. Many Bcl-2-family
proteins reside on the mitochondrial outer membrane. Bcl-2 prevents
mitochondrial disruption and the release of cytochrome C from
mitochondria, while BAX and BAK create pores in mitochondrial
membranes and induce cytochrome C release. Recent evidence has
indicated, however, that Bcl-2 under certain conditions can
function as a pro-apoptotic molecule (Finnegan et al. 2001 Br J
Cancer 85:115-121; Fujita et al. 1998 Biochem Biophys Res Commun
246:484-488; Fadeel et al. 1999 Leukemia 13:719-728; Grandgirard et
al. 1998 EMBO J 17:1268-1278; Cheng et al. 1997 Science
278:1966-1968; Del Bello et al. 2001 Oncogene 20:4591-4595). Bcl-2
can be cleaved by caspase-3 and thus be converted to a
pro-apoptotic protein similar to BAX (Cheng et al. 1997 Science
278:1966-1968). Conversely, BAX has also been shown to inhibit
neuronal cell death when infected with Sinbis virus (Lewis et al.
1999 Nat Med 5:832-835). These observations suggest that members of
the Bcl-2-family have reversible roles in the regulation of
apoptosis and have the potential to function either as a
pro-apoptotic or anti-apoptotic molecule.
[0008] Members of the Bcl-2-family of proteins are highly related
in one or more specific regions, commonly referred to as Bcl-2
homology (BH) domains. BH domains contribute at multiple levels to
the function of these proteins in cell death and survival. The BH3
domain, an amphipathic .alpha.-helical domain, was first delineated
as a stretch of 16 amino acids in Bak that is required for this
protein to heterodimerize with anti-apoptotic members of the
Bcl-2-family and to promote cell death. All proteins in the
Bcl-2-family contain a BH3 domain, and this domain can have a
death-promoting activity that is functionally important. The BH3
domain acts as a potent "death domain" and there is a family of
pro-apoptotic proteins that contain BH3 domains which dimerize via
those BH3 domains with Bcl-2, Bcl-X.sub.L and other anti-apoptotic
members of the Bcl-2 family. Structural studies revealed the
presence of a hydrophobic pocket on the surface of Bcl-X.sub.L and
Bcl-2 that binds the BH3 peptide. Interestingly, the anti-apoptotic
proteins Bcl-X.sub.L and Bcl-2 also possess BH3 domains, but in
these anti-apoptotic proteins, the BH3 domain is buried in the core
of the protein and not exposed for dimerization (Kelekar and
Thompson 1998 Trends Cell Biol 8:324). NMR structural analysis of
the Bcl-X.sub.L/BAK BH3 peptide complex showed that the Bak BH3
domain binds to the hydrophobic cleft formed in part by the BH1,
BH2 and BH3 domains of Bcl-X.sub.L (Sattler 1997 Science 275:983;
Degterev 2001 Nature Cell Biol 3:173-182). BH3-domain-mediated
homodimerizations and heterodimerizations have a key role in
regulating apoptotic functions of the Bcl-2-family (Diaz et al.
1997 J Biol Chem 272:11350; Degterev 2001 Nature Cell Biol
3:173-182).
[0009] The orphan receptor Nur77 (also known as TR3 or nerve growth
factor-induced clone B NGFI-B, GenBank Accession No.: L13740, SEQ
ID NO: 55) (Chang and Kokontis 1988 Biochem Biophys Res Commun
155:971; Hazel et al. 1988 PNAS USA 85:8444) functions as a nuclear
transcription factor in the regulation of target gene expression
(Zhang and Pfahl 1993 Trends Endocrinol Metab 4:156-162; Tsai and
O'Malley 1994 Annu Rev Biochem 63:451; Kastner et al. 1995 Cell
83:859; Mageldorf and Evens 1995 Cell 83:841). Nur77 was originally
isolated as an immediate-early gene rapidly expressed in response
to serum or phorbol ester stimulation of quiescent fibroblasts
(Hazel et al. 1988 PNAS USA 85:8444; Ryseck, et al. 1989 EMBO J
8:3327; Nakai et al. 1990 Mol Endocrinol 4:1438; Herschman 1991
Annul Rev Biochem 60:281). Other diverse signals, such as membrane
depolarization and nerve growth factor, also increase Nur77
expression (Yoon and Lau 1993 J Biol Chem 268:9148). Nur77 is also
involved in the regulation of apoptosis in different cell types
(Woronicz et al. 1994 Nature 367:277; Liu et al. 1994 Nature
367:281; Weih et al. PNAS USA 93:5533; Chang et al, 1997 EMBO J
16:1865; Li et al. 1998 Mol Cell Biol 18:4719; Uemura and Chang
1998 Endocrinology 129:2329; Young et al. 1994 Oncol Res 6:203). It
is rapidly induced during apoptosis of immature thymocytes and
T-cell hybridomas (Woronicz et al. 1994 Nature 367:277; Liu et al.
1994 Nature 367:281), in lung cancer cells treated with the
synthetic retinoid
6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid
(AHPN) (Li et al. 1998 Mol Cell Biol 18:4719) (also called CD437),
and in prostate cancer cells treated with different apoptosis
inducers (Uemura and Chang 1998 Endocrinology 129:2329; Young et
al. 1994 Oncol Res 6:203). Inhibition of Nur77 activity by
overexpression of dominant-negative Nur77 or its antisense RNA
inhibits apoptosis, whereas constitutive expression of Nur77
results in massive apoptosis (Weih et al. PNAS USA 93:5533; Chang
et al, 1997 EMBO J 16:1865).
[0010] Further studies of Nur77 have yielded a better understanding
of its mechanism of action in apoptosis (Li et al. 2000 Science
289:1159). First, several apoptosis inducing agents which also
induced Nur77 expression in human prostate cancer cells were
identified. These included the AHPN analog
6-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chloro-2-naphthalenecarboxylic
acid (MM11453), the retinoid
(Z)-4-[2-bromo-3-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl-
)propenoyl]benzoic acid (MM11384), the phorbol ester
12-O-tetradecanoyl phorbol-13-acetate (TPA), the calcium ionophore
A23187, and the etoposide VP-16. Second, it was found that the
transactivation activity of Nur77 was not required for its role in
inducing apoptosis, as demonstrated by an experiment that showed
that apoptosis inducing agents blocked the expression of a Nur77
target reporter gene. This was further supported by the finding
that a Nur77 mutant deprived of its DNA binding domain (DBD) was
still competent for inducing apoptosis. Third, Nur77 was found to
relocalize to the outer surface of the mitochondria in response to
some apoptotic stimuli, and mitochondrial association of Nur77 is
essential for its apoptotic effects.
[0011] Nur77, visualized in vivo by tagging with Green Fluorescent
Protein (GFP), was shown to relocalize from the nucleus to the
mitochondria in response to apoptosis-inducing agents.
Fractionation studies showed that Nur77 was associating with the
mitochondria-enriched heavy membrane fraction, and proteolysis
accessibility studies on purified mitochondria confirmed that Nur77
was associating with the outer surface of the mitochondria, where
Bcl-2-family members are also found. Fourth, Nur77 was show to be
involved in the regulation of cytochrome c release from the
mitochondria. Inhibition of Nur77 activity by expression of Nur77
antisense RNA blocked the release of cytochrome c and mitochondrial
membrane depolarization in cells stimulated with TPA and MM11453.
Furthermore, incubating purified mitochondria with recombinant
Nur77 protein resulted in cytochrome c release.
[0012] Li et al. (2000 Science 289:1159) further explored the
function of Nur77 through mutation of the protein. A Nur77 mutant
which had the DNA binding domain (amino acid residues168-467)
removed (Nur77/.DELTA.DBD) no longer localized in the nucleus in
non-stimulated cells, but instead was consistently found in
mitochondria. This localization phenotype was accompanied by a
constant release of cytochrome c from the mitochondria. Three other
deletion mutants were also generated and assayed: an amino-terminal
deletion of 152 amino acids referred to as Nur77/.DELTA.1, a 26
amino acid carboxy-terminal deletion referred to as Nur77/.DELTA.2,
and a 120 amino acid carboxy-terminal deletion referred to as
Nur77/.DELTA.3. The Nur77/.DELTA.1 protein did not relocalize to
the mitochondria in response to TPA, but maintained a nuclear
localization. Nur77/.DELTA.1 had a dominant negative effect,
preventing the relocalization of full-length Nur77 to the
mitochondria and inhibiting apoptosis. Mitochondrial targeting was
still observed in Nur77/.DELTA.2 protein expressing cells, but not
in Nur77/.DELTA.3 protein cells in response to TPA treatment. These
results indicated that carboxy-terminal and amino-terminal
sequences are crucial for mitochondrial targeting of Nur77 and its
regulation.
[0013] Experiments designed to alter the localization of
Nur77/.DELTA.DBD by fusing it to various cellular localization
signals showed that Nur77 must have access to the mitochondria in
order to induce its pro-apoptotic effect. When Nur77/.DELTA.DBD was
fused to a nuclear localization sequence, a plasma membrane
targeting sequence, or an ER-targeting sequence, Nur77/.DELTA.DBD
was not targeted to the mitochondria and no induction of cytochrome
c release was observed.
SUMMARY OF THE INVENTION
[0014] One aspect of the invention pertains to the discovery of
molecules that modulate the activity of Bcl-2-family members in
their regulation of apoptosis. More specifically, this aspect
relates to regulators of apoptosis which inhibit proteins such as
Bcl-2 and related Bcl-2 family members and induce a conformational
change in these proteins resulting in pro-apoptotic properties. In
this embodiment, the pro-apoptotic modulators of Bcl-2 and related
Bcl-2 family members have the formula:
Phe-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu, or the formula
Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Phe, where n is between 0
and 3, Xaa can be any amino acid, the compound has 30 or fewer
amino acids and wherein the compound induces apoptosis in a
mammalian cell expressing Bcl-2 or a Bcl-2 related protein. Any
amino acid in the compound of the above formula can be an L- or
D-amino acid, or a non-naturally occurring amino acid.
[0015] Still another embodiment is a compound that has an amino
acid sequence Phe-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu, or
Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Phe, wherein n is between
0 and 3, Xaa is any amino acid, and the compound has one or more D
amino acids and wherein the compound induces apoptosis of mammalian
cells expressing Bcl-2 or Bcl-2 related proteins.
[0016] Another embodiment is a compound that has an amino acid
sequence Phe-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu, or
Leu-Xaa.sub.n-Leu-Xaa.sub.n-Leu-Xaa.sub.n-Phe, wherein n is between
0 and 3 and Xaa is any amino acid; and a cell-penetrating-peptide,
wherein the compound induces apoptosis of mammalian cells
expressing Bcl-2 or Bcl-2 related proteins.
[0017] Yet another embodiment of the invention is a method of
inducing apoptosis in a mammalian cell. The method includes
providing the non-naturally occurring compound described above, and
then contacting the mammalian cell with an effective amount of the
compound, wherein contacting results in apoptosis of said mammalian
cell.
[0018] Still another embodiment is a method of treating a
proliferative disease in a patient, that includes selecting a
patient suffering from a proliferative disease; and administering
to the patient a therapeutically effective amount of the compound
described above.
[0019] One other embodiment is the use of the compound described
above in the preparation of a medicament for the treatment of a
proliferative disease.
[0020] The scope of the compositions and methods described herein
includes the use of peptides, peptide analogs, and small molecules
to regulate the apoptotic effect of Bcl-2-family members.
[0021] The scope of the compositions and methods described herein
includes the use of antagonists of peptides, peptide analogs, and
small molecules to regulate the apoptotic effect of Bcl-2-family
members.
[0022] Yet another embodiment of the invention is a method of
inducing apoptosis in a mammalian cell, comprising contacting the
cell with an effective amount of a compound which binds to Bcl-2
and modulates the activity of Bcl-2 so as to be inductive of
apoptosis.
[0023] Yet another embodiment of the invention is a method of
preventing apoptosis in a mammalian cell, comprising contacting the
cell with an effective amount of a compound which binds to Bcl-2
and modulates the activity of Bcl-2 so as to be preventive of
apoptosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is a schematic representation of location of
Nur77-derived Bcl-2-converting peptides (NuBCP in Nur77) and
peptide sequences. NuBCP-20-r8 (SEQ ID NO: 1); NuBCP-9-r8 (SEQ ID
NO: 9); D-NuBCP-9-r8 (SEQ ID NO: 13); NuBCP-9-Penetratin (SEQ ID
NO: 48); NuBCP-9-Transportan (SEQ ID NO: 54); Smac-peptide-r8 (SEQ
ID NO: 56); t-Bid BH3 peptide-r8 (SEQ ID NO: 57); Bad BH3
peptide-CC-Ant (SEQ ID NO: 58); D-Bad BH3 peptide-CC-Ant (SEQ ID
NO: 59). Single letter code for amino acid with upper case for
L-amino acid and lower case for D-amino acid. X--6-Aminohexanoic
acid; CX--covalent linkage between cysteine thiol and acetyl group;
CC--disulfide link. Bad peptides were modified by replacing Ser
with Ala.
[0025] FIG. 1B is a bar graph showing that NuBCP-20-9-r8 causes
apoptosis in ZR-75-1 cancer cells. The bars represent means.+-.SD
from three independent experiments.
[0026] FIG. 1C is a bar graph showing that NuBCP-20-9-r8 does not
induce apoptosis in normal primary mammary epithelial cells.
[0027] FIG. 1D is a list of NuBCP peptides created by serial
deletion analysis of NuBCP-20 and the apoptotic effect of the
peptides conjugated with polyarginine (10 .mu.M) in H460 lung
cancer cells. NuBCP-20 (SEQ ID NO: 1); NuBCP-15 (SEQ ID NO: 2);
NuBCP-14 (SEQ ID NO: 3); NuBCP-13 (SEQ ID NO: 4); NuBCP-12 (SEQ ID
NO: 5); NuBCP-11 (SEQ ID NO: 6); NuBCP-10 (SEQ ID NO: 7); NuBCP-N10
(SEQ ID NO: 8); NuBCP-9 (SEQ ID NO:9); NuBCP-8 (SEQ ID NO: 10);
NuBCP-7 (SEQ ID NO: 11); NuBCP-9/AA (SEQ ID NO: 12).
[0028] FIG. 1E is a bar graph showing apoptotic effect of
NuBCP-9-r8 conjugated with CPPs (10 .mu.M) in H460 cells. The bars
represent means.+-.SD from three independent experiments.
[0029] FIG. 1F is a bar graph showing that apoptotic effect of
NuBCP-9-r8 is retained with D-analog in H460 cells. The bars
represent means.+-.SD from three independent experiments.
[0030] FIG. 2A is a photograph of a gel showing that
GFP-Nur77/489-497 (GFP-NuBCP-9) is precipitated by anti-Bcl-2
antibody only when Bcl-2 was coexpressed.
[0031] FIG. 2B is a photograph of gel showing that Nur77 lacking
its DNA-binding domain (DBD), Nur77/.DELTA.DBD bound strongly with
Bcl-2 and the binding was abrogated in the presence of NuBCP-9-r8
or D-NuBCP-9-r8. The bars represent means.+-.SD from 3-4
experiments.
[0032] FIGS. 2C-2E are graphs showing fluorescence polarization
(FP) assay of NuBCPs binding to Bcl-2.
[0033] FIG. 2F-H are graphs showing FP competition assay.
[0034] FIG. 3A is a bar graph showing that Bcl-2 siRNA suppressed
apoptosis induced by Nur77 peptides.
[0035] FIG. 3B is a bar graph showing that Bcl-2 antisense
oligonucleotides suppressed apoptosis induced by Nur77
peptides.
[0036] FIGS. 3C-E are graphs showing that stable expression of
Bcl-2 enhances apoptosis by NuBCP. The bars represent means.+-.SD
from 3-4 experiments
[0037] FIG. 3F is a bar graph showing that Bax and Bak are required
for apoptotic effect of NuBCP. The bars represent means.+-.SD from
3-4 experiments.
[0038] FIG. 4A is a series of graphs showing that NuBCPs induce
Bcl-2 conformational change in vitro. Bcl-2 fluorescence from
peptide-treated cells (white histogram) was compared to that from
the non-treated cells (shaded histogram). Numbers represent % of
treated cells showing increased Bcl-2 immunofluorescence compared
to the auto-fluorescence of the non-treated cells from the same
experiment.
[0039] FIG. 4B is a photograph of a gel showing that the
anti-Bcl-2/BH3 antibody precipitated endogenous Bcl-2 in cells
treated with L-NuBCP-9-r8 or D-NuBCP-9-r8.
[0040] FIG. 4C is a photograph of a gel showing that incubation
with NuBCP-9-r8 resulted in a strong precipitation of GST-Bcl-2
protein by the anti-Bcl-2/BH3 antibody.
[0041] FIG. 4D is a graph showing circular dichroism (CD) spectra
for the binding of the NuBCP peptides (30 .mu.M) to GST-Bcl-2 (2
.mu.M).
[0042] FIG. 4E is a non-linear regression analysis of the CD
spectra showing the binding of NuBCP-9 with Bcl-2 is saturating and
stoichiometric with a Kd=2.1.+-.0.2 .mu.M.
[0043] FIG. 4F is a graph showing Inhibition of tumor growth by
NuBCPs.
[0044] FIG. 5A is a series of photographs showing inhibitory effect
of various concentrations of Nur77-peptide on cell growth.
[0045] FIG. 5B is a series of graphs showing apoptotic effect of
various concentrations of Nur77-peptide.
[0046] FIG. 5C is a series of photographs showing a comparison of
growth inhibitory effects of NuBCP peptide and t-Bid peptide.
[0047] FIG. 5D is a series of graphs showing a comparison of
apoptotic effects of NuBCP-20-r8-peptide and t-Bid peptide.
[0048] FIG. 6 is a photograph of an immunoprecipitation gel showing
binding of NuBCP-20-r8-peptide with Bcl-2.
[0049] FIG. 7A is a photograph of an immunoblot gel showing
inhibition of Bcl-2 expression by Bcl-2 siRNA.
[0050] FIG. 7B is photograph of a gel showing stable expression of
Bcl-2 in Jurkat/Neo and Jurkat cells stably expressing Bcl-2.
[0051] FIG. 7C is a photograph of a gel showing expression of Bcl-2
in CEM and CEM cells stably expressing Bcl-2.
[0052] FIG. 8A is a bar graph showing that Bcl-2 potentiates the
apoptotic effect of NuBCP in Jurkat cells.
[0053] FIGS. 8B and C are graphs showing that NuBCP-20-r8 and t-Bid
BH3-r8 peptides induce apoptosis in Jurkat cells.
[0054] FIG. 8D is a series of graphs showing that various NuBCP
peptides induce Bcl-2 conformational change which is not the
consequence of apoptosis. Bcl-2 fluorescence from peptide-treated
cells (light histogram) was compared to that from the non-treated
cells (shaded histogram). Numbers represent % of treated cells
showing Bcl-2 immunofluorescence compared to the auto-fluorescence
of the non-treated cells from the same experiment.
[0055] FIGS. 9A-C are graphs showing CD spectra for binding NuBCPs
to GST-Bcl-2.
[0056] FIG. 10A-C are graphs showing CD spectra for binding NuBCPs
to GST-Bcl-xL.
[0057] FIG. 11A-C are graphs showing CD spectra for binding NuBCPs
to GST.
[0058] FIG. 12 is a bar graph showing induction of apoptosis of
MDA-MB435 breast cancer cells in vitro by NuBCP peptides.
[0059] FIG. 13 is a bar graph showing that replacement of Phe489,
Leu493, Leu497 and Leu498 amino acids in NuBCP-9-r8 peptide with
alanine largely impaired, while simultaneous substitutions of
Phe489 and Leu497 completely abolished, the apoptotic effect of
NuBCP-9-r8.
[0060] FIG. 14 is a bar graph showing induction of apoptosis by
reverse Nur77 peptides.
[0061] FIG. 15 shows a sequence of a Nur77 fragment, DC-3 (SEQ ID
NO: 60). Underlined sequences identify pro-apoptotic sequences that
share pro-apoptotic motif.
[0062] FIG. 16 is a bar graph showing induction of apoptosis by
Nur77-DC3 derived peptides (FGDWIDSIL, SEQ ID NO: 16, FSRSLHSLL,
SEQ ID NO: 9, FAALSALVL, SEQ ID NO: 17, and FYLKLEDLV, SEQ ID NO:
18).
[0063] FIG. 17 is a bar graph showing induction of apoptosis by
short Nur77 peptides.
[0064] FIG. 18 is a bar graph showing that Nur77 peptides linked to
various cell penetrating peptides are apoptotic.
[0065] FIG. 19 is a photograph of an immunoblot gel showing that
Nur77 peptide binds anti-apoptotic Bcl-2 family members.
DETAILED DESCRIPTION
[0066] Embodiments of the invention relate to the discovery that
Nur77 interaction with Bcl-2 converts Bcl-2 from an anti-apoptotic
to a pro-apoptotic molecule by inducing a Bcl-2 conformational
change. This finding provides novel and effective approaches to
induce cancer cell apoptosis by targeting Bcl-2, as the majority of
solid tumors are protected from apoptosis by Bcl-2. Bcl-2
overexpression also contributes to resistance of cancer cells to
chemotherapeutic drugs and .gamma.-radiation therapy. Results
presented here demonstrated that relatively short Nur77-peptides
derived from the Bcl-2-interacting domain in the Nur77 mimic the
effect of Nur77 by their ability to induce cancer cell apoptosis
through their binding to Bcl-2, targeting mitochondria, and
inducing Bcl-2 conformational change. Nur77 was also found to bind
other anti-apoptotic Bcl-2 family members, such as Bcl-B and Bfl-1.
Nur77 peptides also interacted with these anti-apoptotic
proteins.
[0067] Nur77-derived peptides induced cellular apoptosis at micro
molar concentrations, which is comparable to the currently
available peptides derived from the Bcl-2-family protein.
Significantly, a peptide with only 9 amino acid residues (NuBCP-9)
was sufficient to retain the pro-apoptotic Nur77 function in tumor
cells. In addition, the D-enantiomer of NuBCP-9, D-NuBCP-9, was
also found to mimic the function of Nur77. Because the NuBCP-9 and
D-NuBCP-9 peptides effectively regressed tumor growth in animal
models of cancer, they are useful agents for treating
Bcl-2-overexpressing cancers.
[0068] NuBCP-9 was found to bind to Bcl-2, inducing a Bcl-2
conformational change and extensive apoptosis of cancer cells in
vitro and in an in vivo mouse xenograft model. The apoptotic effect
of NuBCP-9 was not inhibited but rather potentiated by Bcl-2
overexpression. Furthermore, the functional activities of NuBCP-9
are retained in its all D-amino acid analog. These properties
distinguish NuBCP peptides from BH3 peptides and small molecules
whose activities are attenuated by Bcl-2, identifying a new
approach to target Bcl-2 for cancer treatment.
[0069] Several pieces of evidence show that the short Nur77 peptide
mimics Nur77 activities. First, a green fluorescent protein
(GFP)-fusion Nur77 peptide migrates to mitochondria as visualized
by fluorescent spectroscopy. Second, GFP-Nur77 fusion peptide but
not GFP binds Bcl-2 directly or indirectly in vivo as shown using
immunoprecipitation assays. Third, the cell-permeable Nur77 peptide
induces a conformational change in Bcl-2 in vivo as detected by
exposure of the Bcl-2 BH3 domain to BH3 antibodies. It was also
shown that Nur77 peptide induced apoptosis was Bcl-2 dependent
utilizing Bcl-2 siRNA and by comparing activities in matched pairs
of cells that either express or do not express Bcl-2.
[0070] The Nur77 9-mer peptide was scanned for critical amino acids
by substituting each amino acid with alanine and assessing the
effect on peptide activity. The critical amino acids mapped to the
hydrophobic face of a putative amphipathic alpha helix on Nurr 1.
This implies that Nur77 can undergo a conformational change while
converting Bcl-2 into a proapoptotic form. Based on the Nur77 9-mer
motif, additional Nur77 9-mer peptides, peptides based on
homologous Nur-family proteins and additional human cancer related
proteins such as p53 were identified that also induce apoptosis in
breast cancer cells.
[0071] According to one embodiment of the invention, the
pro-apoptotic modulator of Bcl-2 is a non-naturally occurring
peptide. The peptide can be attached to a carrier group through an
amino group of a native amino acid in the peptide, or is attached
through the side chain of a lysine amino acid added onto the
peptide. It can be appreciated that by providing a linking group
--NH--, the carrier can be attached through the carboxylic acid
side chain of an aspartic acid or glutamic acid residue by
formation of an amide bond; by providing an oxygen linking group on
the carrier, the carrier can be attached to the peptide through the
carboxylic acid side chain of an aspartic acid or glutamic acid
residue, forming an ester bond. Also, the peptides can be
terminated with Cys and linked by forming a disulfide bond.
[0072] By "non-naturally occurring", it is meant that the compound
and/or peptide is artificially produced by chemical synthesis,
genetic recombinant methods or enzymatic digestion of isolated
polypeptides, and that the compound does not comprise a full length
Nur77 polypeptide. The non-naturally occurring peptide may be
modified, wherein such modifications include glycosylation,
lipidation, amidation, phosphorylation, acetylation, PEGylation
(the addition of polyethylene glycol to stabilize the peptide) and
albumination (the conjugation of an albumin moiety to increase the
biological half-life of the peptide).
[0073] According to this embodiment, the amino acid sequence of the
peptide inhibitor of Bcl-2 function is identical to the native
amino acid sequence of a segment of an endogenous polypeptide
inhibitor of Bcl-2, which segment has inhibitory activity to Bcl-2.
Wherein said endogenous inhibitor of Bcl-2 has a sequence of e.g.,
SEQ ID NO: 55. Alternatively, one or more positions of the
corresponding native amino acid sequence of the inhibitory peptide
can be substituted with other amino acids. The substitutions may
include conservative amino acid substitutions. A conservative amino
acid substitution is a substitution made within a group of amino
acids which are categorized based upon the nature of the amino acid
side chain. The seven groups are as follows: (1) non-polar M, I, L
and V; (2) aromatic: F, Y and W; (3) basic: K, R and H; (4)
non-polar A and G; (5) polar with aliphatic side chains: S and T;
(6) polar Q and N; (7) acidic: E and D. According to one embodiment
of the invention, each segment has at least 50%, preferably at
least 70%, more preferably at least 80%, most preferably at least
90%, sequence identity with the corresponding native segment of the
same length. By "sequence identity" is meant the same amino acids
in the same relative positions.
[0074] In one embodiment, the pro-apoptotic modulator of Bcl-2 has
the following general formula: F Xaa.sub.n L Xaa.sub.n L Xaa.sub.n
L, wherein n is 0-3, and Xaa is any amino acid. The amino acids can
be substituted for synthetic or non-naturally occurring amino acids
as described herein. For example, any L-amino acid can be replaced
with an equivalent D-amino acid. Alanine can replace any interior
(Xaa) amino acid. Any alanine substitutable interior amino acid
(Xaa) can be removed, i.e. SRS and HS can be deleted one by one or
in any combination thereof. Alanine-substitutable amino acids can
be replaced with spatially equivalent linkers.
[0075] The pro-apoptotic modulator of Bcl-2 can be linked to
cell-penetrating peptide sequences such as penetratin, transportan
or tat either directly or though intervening sequences. In
addition, they can be linked to peptides that target cancer cells
direction or through intervening sequences. For example, the
pro-apoptotic modulator of Bcl-2 can be linked through GX to a
cyclic disulfide loop peptide, Lyp-1, that binds specifically to
breast cancer cells. Alternatively, the pro-apoptotic modulator of
Bcl-2 can be linked through GX to F3, a 31-residue peptide that
binds specifically to breast cancer cells. The additional examples
of cell-permeability enhancers that can be conjugated to the
pro-apoptotic modulator of Bcl-2 are provided below. The peptides
can also have the inverso-configuration.
[0076] A compound of the invention may have the following length
prior to being conjugated to a cell-permeability enhancer: 4-597
amino acids, preferably 4-400 amino acids, preferably 4-300 amino
acids, preferably 4-200 amino acids, preferably 4-100 amino acids,
preferably 4-50 amino acids, preferably 4-40 amino acids,
preferably 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122,
121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109,
108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95,
94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78,
77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61,
60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,
43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,
26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, or 4 amino acids. In one embodiment, the compound
has fewer than 30, 25, 20, 15 or 10 amino acids.
[0077] Exemplary pro-apoptotic modulators of Bcl-2 as well as the
previous nomenclature of the peptides are listed below. As shown in
Table 1, the peptide labels have changed over time. Table 1 lists
the previous names of each peptide, and the current name of the
same peptide.
TABLE-US-00001 TABLE 1 Exemplary pro-apoptotic modulators of Bcl-2
Pro-apoptotic modulator of Bcl-2 SEQ Previous Current ID Name Name
Sequence NO: TR3 peptide; NuBCP-20-r8
acetyl---GDWIDSILAFSRSLHSLLVDKKC-X-rrrrrrrr 1 Nur77 peptide
Nur77-15 NuBCP-15 acetyl--------SILAFSRSLHSLLVDGXrrrrrrrr-amide 2
Nur77-14 NuBCP-14 acetyl---------ILAFSRSLHSLLVDGXrrrrrrrr-amide 3
Nur77-13 NuBCP-13 acetyl----------LAFSRSLHSLLVDGXrrrrrrrr-amide 4
Nur77-12 NuBCP-12 acetyl-----------AFSRSLHSLLVDGXrrrrrrrr-amide 5
Nur77-11 NuBCP-1 1 acetyl------------FSRSLHSLLVDGXrrrrrrrr-amide 6
Nur77-10 NuBCP-10 acetyl-------------SRSLHSLLVDGXrrrrrrrr-amide 7
Nur77-N10 NuBCP-N10 acetyl------------FSRSLHSLLVGXrrrrrrrr-amide 8
TR3/1; NuBCP-9-r8, acetyl------------FSRSLHSLLGXrrrrrrrr-amide 9
Nur77-9 Nur77/1 Nur77-8 NuBCP-8
acetyl------------FSRSLHSLGXrrrrrrrr-amide 10 Nur77-7 NuBCP-7
acetyl------------FSRSLHSGXrrrrrrrr-amide 11 Nur77-9/AA NuBCP-9/AA
acetyl------------ASRSLHSLAGXrrrrrrrr-amide 12 TR3/1(D)
D-NuBCP-9-r8 acetyl------------fsrslhsllGXrrrrrrrr-amide 13 TR3/1
Nur77/1 acetyl------------LLSHLSRSFGXrrrrrrrr-amide 14 (inverso)
(inverso) TR3/1(retro- Nur77/1
acetyl------------llshlsrsfGXrrrrrrrr-amide 15 inverso)
(retro-inverso) TR3/2 Nur77/2
acetyl------------FGDWIDSILGXrrrrrrrr-amide 16 TR3/3 Nur77/3
acetyl------------FAALSALVLGXrrrrrrrr-amide 17 TR3/4 Nur77/4
acetyl------------FYLKLEDLVGXrrrrrrrr-amide 18 NOR1 peptide Nor1
peptide acetyl--------SIKDFSLNLQSLNLDG rrrrrrrr-amide 19 NUIT 1 NOT
peptide acetyl---SIVEFSSNLQNMNIDG rrrrrrrr-amide 20 TR3/1 Nur77/1
acetyl----rrrFrrrLrrLL-amide 21 (embedded) (embedded) TR3/1:
Nur77/1 acetyl----rrrfrrrlrrll-amide 22 (D/embedded) (D/embedded)
acetyl-------fSrSlHsLlGXrrrrrrrr-amide 23
acetyl-------fSRslHSllGXrrrrrrrr-amide 24
acetyl-------FARSLHSLLGXrrrrrrrr-amide 25
acetyl-------FSASLHSLLGXrrrrrrrr-amide 26
acetyl-------FSRALHSLLGXrrrrrrrr-amide 27
acetyl-------FSRSLASLLGXrrrrrrrr-amide 28
acetyl-------FSRSLHALLGXrrrrrrrr-amide 29 acetyl-------F
RSLHSLLGXrrrrrrrr-amide 30 acetyl-------FS SLHSLLGXrrrrrrrr-amide
31 acetyl-------FSR LHSLLGXrrrrrrrr-amide 32 acetyl-------FSRSL
SLLGXrrrrrrrr-amide 33 acetyl-------FSRSLH LLGXrrrrrrrr-amide 34
acetyl-------F SLHSLLGXrrrrrrrr-amide 35 acetyl-------f
rslhsllGXrrrrrrrr-amide 36 acetyl-------FXSLHSLLGXrrrrrrrr-amide 37
acetyl-------FSRSLHSLLGX(CGNKRTAC)-amide 38
acetyl---FSRSLHSLLGXAKVKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK- 39 amide
Nur77/short acetyl---F L LGXrrrrrrrr-amide 40 Nur77/short D
acetyl---f l llGXrrrrrrrr-amide 41 Nur77/1 Ant
acetyl---FSRSLHSLLC-CRQIKIWFQNRRMKWKK-amide 42 Nur77/ Ant (D)
acetyl---fsrslhsllc-crqvkvwfqnrrmkwkk-amide 43 p53 peptide
acetyl---FSD_LWKLL-GXrrrrrrrr-amide 44 Nur77 (embedded2) acetyl-
NFQHALQEVLQALKQVQAR-C-C-rrrrrrrr-amide 45 Nur77/1 (D/L)
acetyl---fSrSlHsLl-GXrrrrrrrr-amide 46 Nur77/1 (DD/LL)
acetyl---fSRslHSll-GXrrrrrrrr-amide 47 Nur77/1 NuBCP-9-
acetyl---FSRSLHSLL-(C-C)RQIKIWFQNRRMKWKK-amide 48 Penetratin
Penetratin Nur77/1 acetyl---fsrslhsll-(c-c)rqvkvwfqnrrmkwkk-amide
49 (D)Penetratin(D) Nur77/1
acetyl---FSRSLHSLL-(C-C)AGYLLGKINLKALAALAKKIL-amide 50
Transportan10 Nur77/1(D)
acetyl---fsrslhsll-(c-c)agyllqkvnlkalaalakkvl-amide 51
Transportan10(D) Nur77/1(L/D)
acetyl---FsRsLhSlL-(c-C)aGyLlGkInLkAlAaLaKkIl-amide 52
Transportan10 (L/D): Nur77/1 (LLDD)
acetyl---FSrsLHslL-(C-c)aGYllGKvnLKalAalaKkvi-amide 53
Transportan10 (LLDD) NuBCP-9-
acetyl---FSRSLHSLL-CCGWTLNSAGYLLGKINKALAALAKKIL-amide 54
Transportan
[0078] Single letter code is used for L-amino acids (capitalized),
while D-amino acids are lower case. Substituted and added amino
acids are in bold. r is aminoacid Arginine. X is 6-aminohexanoic
acid. The C--X bond is formed from the reaction of a C-terminal Cys
thiol group with a chloracetylated aminohexanoyl group. C--C bond
is formed by the oxidation of two cysteine amino acids to form a
disulfide bond. Brackets ( ) indicate that the two cysteines
oxidize to form a disulfide loop.
[0079] In one embodiment, the analog shares at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, sequence identity with the peptides listed above.
[0080] Peptides which bind the Bcl-2 pocket can be identified by a
Bcl-2 competition binding assay. The assay is based on fluorescence
polarization. The assay can rapidly measure Bcl-2 receptor-ligand
interaction without using filter binding, electrophoresis, or
precipitation steps. Fluorescence polarization gives a direct,
instantaneous equilibrium measure of the bound/free ratio between
ligand and receptor molecules.
[0081] Peptides, peptide analogs, peptidomimetics and small
molecules which bind Bcl-2 and induce a Bcl-2 conformational change
that is indicative of its pro-apoptotic form can be identified
using CD spectroscopy.
[0082] The cell permeability of a conjugate can be verified by
directly or indirectly labeling the conjugate with a detectable
label which can be visualized inside a cell with the aid of
microscopy. For example, a fluorescein derivative of the conjugate
can be made by methods well known to those skilled in the art for
conjugating fluorescein molecules to peptides. The fluorescein
conjugate is incubated with the relevant target cells in vitro. The
cells are harvested and fixed, then stained with
Streptavidin-fluorescein and observed in the dark under confocal
microscopy. Internalization of the exogenous molecule to which the
carrier is conjugated is apparent by fluorescence.
[0083] For peptide conjugates, the peptide portion can be a
recombinant peptide, a natural peptide, or a synthetic peptide. The
peptide can also be chemically synthesized, using, for example,
solid phase synthesis methods.
I. Definitions and General Parameters
[0084] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0085] As used herein, "pharmaceutically or therapeutically
acceptable carrier" refers to a carrier medium which does not
interfere with the effectiveness of the biological activity of the
active ingredients and which is not toxic to the host or
patient.
[0086] As used herein, "stereoisomer" refers to a chemical compound
having the same molecular weight, chemical composition, and
constitution as another, but with the atoms grouped differently.
That is, certain identical chemical moieties are at different
orientations in space and, therefore, when pure, have the ability
to rotate the plane of polarized light. However, some pure
stereoisomers can have an optical rotation that is so slight that
it is undetectable with present instrumentation. The compounds
described herein can have one or more asymmetrical carbon atoms and
therefore include various stereoisomers. All stereoisomers are
included within the scope of the present invention.
[0087] As used herein, "therapeutically- or
pharmaceutically-effective amount" as applied to the disclosed
compositions refers to the amount of composition sufficient to
induce a desired biological result. That result can be alleviation
of the signs, symptoms, or causes of a disease, or any other
desired alteration of a biological system. For example, the result
can involve a decrease and/or reversal of cancerous cell
growth.
[0088] As used herein, "homology" or "identity" or "similarity"
refers to sequence similarity between two peptides or between two
nucleic acid molecules. Homology can be determined by comparing a
position in each sequence which can be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same base or amino acid, then the molecules are identical at
that position. A degree of homology or similarity or identity
between nucleic acid sequences is a function of the number of
identical or matching nucleotides at positions shared by the
nucleic acid sequences. An "unrelated" or "non-homologous" sequence
shares less than about 40% identity, though preferably less than
about 25% identity, with one of the sequences described herein.
[0089] As used herein, the term "inhibitor" is interchangeably used
to denote "antagonist". Both these terms define compositions which
have the capability of decreasing certain enzyme activity or
competing with the activity or function of a substrate of said
enzyme.
[0090] As used herein "peptide" indicates a sequence of amino acids
linked by peptide bonds.
[0091] The term "peptidomimetic" means that a peptide according to
the invention is modified in such a way that it includes at least
one non-coded residue or non-peptidic bond.
[0092] Such modifications include, e.g., alkylation and, more
specifically, methylation of one or more residues, insertion of or
replacement of natural amino acid by non-natural amino acids, and
replacement of an amide bond with other covalent bond. A
peptidomimetic can optionally comprise at least one bond which is
an amide-replacement bond such as urea bond, carbamate bond,
sulfonamide bond, hydrazine bond, or any other covalent bond. The
design of appropriate "peptidomimetic" can be computer
assisted.
[0093] The term "spacer" denotes a chemical moiety whose purpose is
to link, covalently, a cell-permeability moiety and a peptide or
peptidomimetic. The spacer can be used to allow distance between
the cell-permeability moiety and the peptide, or it is a chemical
bond of any type. Linker denotes a direct chemical bond or a
spacer.
[0094] The term "core" refers to the peptidic segment or moiety of
the pro-apoptotic modulator of Bcl-2 which comprises peptide or
peptidomimetic and is optionally attached to a cell-permeability
enhancer.
[0095] The term "permeability" refers to the ability of an agent or
substance to penetrate, pervade, or diffuse through a barrier,
membrane, or a skin layer. "Cell permeability" or a
"cell-penetration" moiety refers to any molecule known in the art
which is able to facilitate or enhance penetration of molecules
through membranes. Non-limiting examples include: hydrophobic
moieties such as lipids, fatty acids, steroids and bulky aromatic
or aliphatic compounds; moieties which can have cell-membrane
receptors or carriers, such as steroids, vitamins and sugars,
natural and non-natural amino acids and transporter peptides.
Examples for lipid moieties which can be used are: Lipofectamine;
Transfectace; Transfectam; Cytofectin; DMRIE; DLRIE; GAP-DLRIE;
DOTAP; DOPE; DMEAP; DODMP; DOPC; DDAB; DOSPA; EDLPC; EDMPC; DPH;
TMADPH; CTAB; lysyl-PE; DC-Cho; -alanyl cholesterol; DCGS; DPPES;
DCPE; DMAP; DMPE; DOGS; DOHME; DPEPC; Pluronic; Tween; BRIJ;
plasmalogen; phosphatidylethanolamine; phosphatidylcholine;
glycerol-3-ethylphosphatidylcholine; dimethyl ammonium propane;
trimethyl ammonium propane; diethylammonium propane;
triethylammonium propane; dimethyldioctadecylammonium bromide; a
sphingolipid; sphingomyelin; a lysolipid; a glycolipid; a
sulfatide; a glycosphingolipid; cholesterol; cholesterol ester;
cholesterol salt; oil; N-succinyldioleoylphosphatidylethanolamine;
1,2-dioleoyl-sn-glycerol; 1,3-dipalmitoyl-2 succinylglycerol;
1,2-dipalmitoyl-sn-3-succinylglycerol;
1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine;
palmitoylhomocysteine;
N,N'-Bis(do-decyaminocarbonylmethylene)-N,N'-bis((-N,N,N-trimethylammoniu-
methyl-aminocarbonyl-methylene)ethylenediamine tetraiodide; N,N''
Bis(hexadecylaminocarbonylmethylene)-N,N',N''-tris((-N,N,N-trimethylammon-
ium-ethylaminocarbonylmethylenediethylenetri-aminehexaiodide;
N,N'-Bis(dodecylaminocarbonylmethylene)-N,N''-bis((-N,N,N-trimethylammoni-
umethylamino-carbonylmethylene)cy-clohexylene-1,4-diaminetetra-iodide;
1,7,7-tetra-((N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3--
hexadecylaminocarbonyl methylene-1,3,7-triaazaheptaneheptaiodide;
N,N,N',N-tetra((-N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N'--
(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetria-
mine tetraiodide; dioleoylphosphatidyl ethanolamine; a fatty acid;
a lysolipid; phosphatidylcholine; phosphatidylethanolamine;
phosphatidylserine; phosphatidylglycerol; phosphatidylinositol; a
sphingolipid; a glycolipid; a glucolipid; a sulfatide; a
glycosphingolipid; phosphatidic acid; palmitic acid; stearic acid;
arachidonic acid; oleic acid; a lipid bearing a polymer; a lipid
bearing a sulfonated saccharide; cholesterol; tocopherol
hemisuccinate; a lipid with an ether-linked fatty acid; a lipid
with an ester-linked fatty acid; a polymerized lipid; diacetyl
phosphate; stearylamine; cardiolipin; a phospholipid with a fatty
acid of 6-8 carbons in length; a phospholipid with asymmetric acyl
chains; 6-(5cholesten-3b-yloxy)-1-thio-b-D-galactopyranoside;
digalactosyldiglyceride;
6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy-1-thio-b-D-galactopyranosid-
e;
6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy-1-thio-a-D-mannopyranosid-
e;
12-(((7'-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoic
acid;
N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadec-
anoyl];-2-aminopalmitic acid;
cholesteryl(4'-trimethyl-ammonio)butanoate;
N-succinyldioleoyl-phosphatidylethanolamine;
1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinyl-glycerol;
1,3-dipalmitoyl-2-succinylglycerol;
1-hexadecyl-2-palmitoylglycero-phosphoethanolamine;
palmitoylhomocysteine; cyclic 9-amino-acid peptide as described in
Laakkonen et al. 2002 Nature Med 8:751-755; a peptide described in
Porkka et al. 2002 PNAS USA 99:7444-7449; and polymers of L- or
D-arginine as described in Mitchell et al. 2000 J Peptide Res
56:318-325.
[0096] As used herein, "cancer" and "cancerous" refer to any
malignant proliferation of cells in a mammal.
[0097] As used herein, "neurodegenerative disease" is a condition
which affects brain function and is a result of deterioration of
neurons. The neurodegenerative diseases are divided into two
groups: a) conditions causing problems with movements, and
conditions affecting memory and conditions related to dementia.
Neurodegenerative diseases include, for example, Huntington's
disease, spinocerebellar ataxias, Machado-Joseph disease, Spinal
and Bulbar muscular atrophy (SBMA or Kennedy's disease),
Dentatorubral Pallidoluysian Atrophy (DRPLA), Fragile X syndrome,
Fragile XE mental retardation, Friedreich ataxia, myotonic
dystrophy, Spinocerebellar ataxias (types 8, 10 and 12), spinal
muscular atrophy (Werdnig-Hoffman disease, Kugelberg-Welander
disease), Alzheimer's disease, amyotrophic lateral sclerosis,
Parkinson's disease, Pick's disease, and spongiform
encephalopathies. Additional neurodegenerative diseases include,
for example, age-related memory impairment, agyrophilic grain
dementia, Parkinsonism-dementia complex of Guam, auto-immune
conditions (e.g., Guillain-Barre syndrome, Lupus), Biswanger's
disease, brain and spinal tumors (including neurofibromatosis),
cerebral amyloid angiopathies, cerebral palsy, chronic fatigue
syndrome, corticobasal degeneration, conditions due to
developmental dysfunction of the CNS parenchyma, conditions due to
developmental dysfunction of the cerebrovasculature,
dementia--multi infarct, dementia--subcortical, dementia with Lewy
bodies, dementia of human immunodeficiency virus (HIV), dementia
lacking distinct histology, Dementia Pugilistica, diseases of the
eye, ear and vestibular systems involving neurodegeneration
(including macular degeneration and glaucoma), Down's syndrome,
dyskinesias (Paroxysmal), dystonias, essential tremor, Fahr's
syndrome, fronto-temporal dementia and Parkinsonism linked to
chromosome 17 (FTDP-17), frontotemporal lobar degeneration, frontal
lobe dementia, hepatic encephalopathy, hereditary spastic
paraplegia, hydrocephalus, pseudotumor cerebri and other conditions
involving CSF dysffinction, Gaucher's disease, Hallervorden-Spatz
disease, Korsakoff s syndrome, mild cognitive impairment, monomeric
amyotrophy, motor neuron diseases, multiple system atrophy,
multiple sclerosis and other demyelinating conditions (e.g.,
leukodystrophies), myalgic encephalomyelitis, myoclonus,
neurodegeneration induced by chemicals, drugs and toxins,
neurological manifestations of AIDS including AIDS dementia,
neurological/cognitive manifestations and consequences of bacterial
and/or viral infections, including but not restricted to
enteroviruses, Niemann-Pick disease, non-Guamanian motor neuron
disease with neurofibrillary tangles, non-ketotic hyperglycinemia,
olivo-ponto cerebellar atrophy, oculopharyugeal muscular dystrophy,
neurological manifestations of Polio myelitis including
non-paralytic polio and post-polio-syndrome, primary lateral
sclerosis, prion diseases including Creutzfeldt-Jakob disease
(including variant form), kuru, fatal familial insomnia,
Gerstmann-Straussler-Scheinker disease and other transmissible
spongiform encephalopathies, prion protein cerebral amyloid
angiopathy, postencephalitic Parkinsonism, progressive muscular
atrophy, progressive bulbar palsy, progressive subcortical gliosis,
progressive supranuclear palsy, restless leg syndrome, Rett
syndrome, Sandhoff disease, spasticity, sporadic fronto-temporal
dementias, striatonigral degeneration, subacute sclerosing
panencephalitis, sulphite oxidase deficiency, Sydenham's chorea,
tangle only dementia, Tay-Sach's disease, Tourette's syndrome,
vascular dementia, Wilson disease, Alexander disease, Alper's
disease, ataxia telangiectasia, Canavan disease, Cockayne syndrome,
Krabbe disease, multiple system atrophy, Pelizaeus-Merzbacher
Disease, primary lateral sclerosis, Refsum's disease, Sandhoff
disease, Schilder's disease, Steele-Richardson-Olszewski disease,
tabes dorsalis.
[0098] When two compounds are administered in combination or used
in combination therapy according to the invention the term
"combination" in this context means that the drugs are given
contemporaneously, either simultaneously or sequentially. This term
is exchangeable with the term "coadministration" which in the
context of this invention is defined to mean the administration of
more than one therapeutic in the course of a coordinated treatment
to achieve an improved clinical outcome. Such coadministration can
also be coextensive, that is, occurring during overlapping periods
of time.
[0099] In addition to peptides consisting only of
naturally-occurring amino acids, peptidomimetics or peptide analogs
are also considered. Peptide analogs are commonly used in the
pharmaceutical industry as non-peptide drugs with properties
analogous to those of the template peptide. These types of
non-peptide compounds are termed "peptide mimetics" or
"peptidomimetics" (see, e.g., Luthman et al. 1996 A Textbook of
Drug Design and Development, 14:386-406, 2nd Ed., Harwood Academic
Publishers; Grante 1994 Angew Chem Int Ed Engl 33:1699-1720;
Fauchere 1986 Adv Drug Res 15:29; Evans et al. 1987 J Med Chem
30:229). Peptide mimetics that are structurally similar to
therapeutically useful peptides can be used to produce an
equivalent or enhanced therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as naturally-occurring
receptor-binding polypeptide, but have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting
of: --CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--, by methods known in the art and further
described in the following references: Spatola, 1983, In: Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins, B.
Weinstein, eds., Marcel Dekker, New York, p. 267; Hudson et al.
1979 Int J Pept Prot Res 14:177-185 (1979) (--CH.sub.2NH--,
CH.sub.2CH.sub.2--); Spatola et al. 1986 Life Sci 38:1243-1249
(--CH.sub.2--S); Hann 1982 J Chem Soc Perkins Trans I, 307-314
(--CH--CH--, cis and trans); Almquist et al. 1980 J Med Chem
23:1392-1398 (--COCH.sub.2--); Jennings-White et al. 1982
Tetrahedron Lett 23:2533 (--COCH.sub.2--); Szelke, et al. European
Appln. EP 45665 (1982) (--CH(OH)CH.sub.2--); Holladay et al. 1983
Tetrahedron Lett 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby, 1982
Life Sci 31:189-199 (--CH.sub.2--S--); each of which is
incorporated herein by reference. A particularly preferred
non-peptide linkage is --CH.sub.2NH--. Such peptide mimetics can
have significant advantages over polypeptide embodiments,
including, for example: more economical production, greater
chemical stability, enhanced pharmacological properties (half-life,
absorption, potency, efficacy, etc.), altered specificity (e.g., a
broad-spectrum of biological activities), reduced antigenicity, and
others. Labeling of peptidomimetics usually involves covalent
attachment of one or more labels, directly or through a spacer
(e.g., an amide group), to non-interfering position(s) on the
peptidomimetic that are predicted by quantitative
structure-activity data and/or molecular modeling. Such
non-interfering positions generally are positions that do not form
direct contacts with the macromolecules(s) (e.g., immunoglobulin
superfamily molecules) to which the peptidomimetic binds to produce
the therapeutic effect. Derivatization (e.g., labeling) of
peptidomimetics should not substantially interfere with the desired
biological or pharmacological activity of the peptidomimetic.
Generally, peptidomimetics of receptor-binding peptides bind to the
receptor with high affinity and possess detectable biological
activity (i.e., are agonistic or antagonistic to one or more
receptor-mediated phenotypic changes).
[0100] Systematic substitution of one or more amino acids of a
consensus sequence with a D-amino acid of the same type (e.g.,
D-lysine in place of L-lysine) can be used to generate more stable
peptides. In addition, constrained peptides comprising a consensus
sequence or a substantially identical consensus sequence variation
can be generated by methods known in the art (Rizo, et al. 1992
Annu Rev Biochem 61:387, incorporated herein by reference); for
example, by adding internal cysteine residues capable of forming
intramolecular disulfide bridges which cyclize the peptide.
[0101] Synthetic or non-naturally occurring amino acids refer to
amino acids which do not naturally occur in vivo but which,
nevertheless, can be incorporated into the peptide structures
described herein. Preferred synthetic amino acids are the
D-.alpha.-amino acids of naturally occurring L-.alpha.-amino acid
as well as non-naturally occurring D- and L-.alpha.-amino acids
represented by the formula H.sub.2NCHR.sup.5 COOH where R.sup.5 is
1) a lower alkyl group, 2) a cycloalkyl group of from 3 to 7 carbon
atoms, 3) a heterocycle of from 3 to 7 carbon atoms and 1 to 2
heteroatoms selected from the group consisting of oxygen, sulfur,
and nitrogen, 4) an aromatic residue of from 6 to 10 carbon atoms
optionally having from 1 to 3 substituents on the aromatic nucleus
selected from the group consisting of hydroxyl, lower alkoxy,
amino, and carboxyl, 5) -alkylene-Y where alkylene is an alkylene
group of from 1 to 7 carbon atoms and Y is selected from the group
consisting of (a) hydroxy, (b) amino, (c) cycloalkyl and
cycloalkenyl of from 3 to 7 carbon atoms, (d) aryl of from 6 to 10
carbon atoms optionally having from 1 to 3 substituents on the
aromatic nucleus selected from the group consisting of hydroxyl,
lower alkoxy, amino and carboxyl, (e) heterocyclic of from 3 to 7
carbon atoms and 1 to 2 heteroatoms selected from the group
consisting of oxygen, sulfur, and nitrogen, (f) --C(O)R.sup.2 where
R.sup.2 is selected from the group consisting of hydrogen, hydroxy,
lower alkyl, lower alkoxy, and --NR.sup.3R.sup.4 where R.sup.3 and
R.sup.4 are independently selected from the group consisting of
hydrogen and lower alkyl, (g) --S(O).sub.nR.sup.6 where n is an
integer from 1 to 2 and R.sup.6 is lower alkyl and with the proviso
that R.sup.5 does not define a side chain of a naturally occurring
amino acid.
[0102] Other preferred synthetic amino acids include amino acids
wherein the amino group is separated from the carboxyl group by
more than one carbon atom such as beta (.beta.)-alanine, gamma
(.gamma.)-aminobutyric acid, and the like.
[0103] Particularly preferred synthetic amino acids include, by way
of example, the D-amino acids of naturally occurring L-amino acids,
L-(1-naphthyl)-alanine, L-(2-naphthyl)-alanine,
L-cyclohexylalanine, L-2-aminoisobutyric acid, the sulfoxide and
sulfone derivatives of methionine (i.e.,
HOOC--(H.sub.2NCH)CH.sub.2CH.sub.2--S(O).sub.nR.sup.6) where n and
R.sup.6 are as defined above as well as the lower alkoxy derivative
of methionine (i.e., HOOC--(H.sub.2NCH)CH.sub.2CH.sub.2--OR.sup.6
where R.sup.6 is as defined above).
II. Overview
[0104] Compounds that bind to Bcl-2-family members and alter their
function in apoptosis are also provided. These compounds include
"lead" peptide compounds and "derivative" compounds constructed so
as to have the same or similar molecular structure or shape as the
lead compounds but that differ from the lead compounds either with
respect to susceptibility to hydrolysis or proteolysis and/or with
respect to other biological properties, such as increased affinity
for the receptor.
[0105] The examples described herein demonstrate that the
nuclear-to-mitochondrial pathway of Nur77 can be extended to lung
and breast cancer cells. In addition, it is shown that Bcl-2 acts
as a mitochondrial receptor of Nur77 through their physical
interaction. In response to various apoptotic stimuli, the
expression of the Nur77 protein is increased and its localization
is altered from nuclear to cytoplasmic, more specifically to the
outer member of mitochondria. This association with the
mitochondria is the result of binding to the Bcl-2, whose normal
function is the inhibition of apoptosis, particularly the
inhibition of the release of cytochrome c from the mitochondria.
High expression of Nur77/.DELTA.DBD (a form of Nur77 without its
DNA-binding domain) induced cytochrome c release and apoptosis only
in cells expressing Bcl-2, indicating that Nur77 modulates the
function of Bcl-2 from anti-apoptotic to pro-apoptotic, without
cleaving the Bcl-2 protein.
[0106] Further data show that Nur77 induces a conformational change
in Bcl-2 which can cause the function of Bcl-2 to be modified from
an anti-apoptotic to a pro-apoptotic protein. Mutational analysis
indicated that the C-terminal domain of Nur77, which contains
several .alpha.-helices, is responsible for interacting with Bcl-2.
The C-terminal domain, DC3, and a shortened C-terminal domain, DC1,
was sufficient for interacting with Bcl-2 and inducing apoptotic
potential of Bcl-2. When analyzing the Bcl-2 domains involved in
the interaction, it was observed that mutations in the hydrophobic
pocket of Bcl-2 did not affect its interaction with Nur77.
Moreover, the N-terminal domain of Bcl-2, containing the loop
region and BH4 domain, was able to interact with Nur77/.DELTA.DBD.
Deletion of BH4 domain from Bcl-2 did not affect the interaction of
Nur77/.DELTA.DBD with Bcl-2, implying that the loop region of Bcl-2
was responsible for interaction. In addition, DC1 and BH3-only
Bcl-Gs did not compete for binding to Bcl-2. Instead, Bcl-Gs
enhanced the binding of DC1 to Bcl-2. Thus, Bcl-2 was found to
interact with Nur77 in a manner that is different from its
interaction with Bcl-2-family proteins containing only the BH3
domain.
[0107] Specific interaction of Nur77 with Bcl-2 is essential for
Nur77 to target mitochondria and results in conversion of Bcl-2
from an anti-apoptotic to a pro-apoptotic molecule. Concomitantly,
the conformation of Bcl-2 is changed by Nur77 resulting in the
exposure of the otherwise hidden BH3 domain. Thus, embodiments of
the invention include peptides derived from the specific
Bcl-2-interacting domain of Nur77, such as DC3, which mimic its
effect. Embodiments also include homologous sequences from
functionally related proteins. These embodiments include peptide
analogs, peptidomimetics and small molecules designed to mimic the
binding properties of these peptides. Peptides, peptide analogs,
peptidomimetics, and small molecules that specifically interact
with Bcl-2 will effectively induce apoptosis of cancer cells, thus
restricting tumor growth. In addition, the results provide a
molecular basis for developing various agents for treating cancers
and other therapeutic applications.
III. Preparation of Peptides and Peptide Mimetics
[0108] Preferred peptides can be synthesized using any method known
in the art, including peptidomimetic methodologies. These methods
include solid phase as well as solution phase synthesis methods.
The conjugation of the peptidic and permeability moieties can be
performed using any methods known in the art, either by solid phase
or solution phase chemistry. Non-limiting examples for these
methods are described herein. Some of the preferred compounds
disclosed herein can conveniently be prepared using solution phase
synthesis methods. Other methods known in the art to prepare
compounds like those described herein, can be used and are within
the scope of the present invention.
[0109] The amino acids used are those which are available
commercially or are available by routine synthetic methods. Certain
residues can require special methods for incorporation into the
peptide, and either sequential, divergent or convergent synthetic
approaches to the peptide sequence are useful in this invention.
Natural coded amino acids and their derivatives are represented by
three-letter codes according to IUPAC conventions.
[0110] When there is no indication, the L isomer was used. The D
isomers are indicated by lower case font.
[0111] Conservative substitutions of amino acids as known to those
skilled in the art are within the scope of the present invention.
Conservative amino acid substitutions includes replacement of one
amino acid with another having the same type of functional group or
side chain e.g., aliphatic, aromatic, positively charged,
negatively charged. These substitutions can enhance oral
bioavailability, penetration into the central nervous system,
targeting to specific cell populations and the like. One of skill
will recognize that individual substitutions, deletions or
additions to peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" where the alteration results in the substitution
of an amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art.
[0112] The following six groups each contain amino acids that are
conservative substitutions for one another:
TABLE-US-00002 Aromatic Phenylalanine Tryptophan Tyrosine
Ionizable: Acidic Aspartic acid Glutamic Acid Ionizable: Basic
Arginine Histidine Lysine Nonionizable Polar Asparagine Glutamine
Serine Cysteine Threonine Nonpolar (Hydrophobic) Alanine Glycine
Isoleucine Leucine Methionine Proline Valine Sulfur Containing
Cysteine Methionine
[0113] The following is a list of non limiting examples of
non-coded amino acids: Abu refers to 2-aminobutyric acid, Ahx6
refers to aminohexanoic acid, Ape5 refers to aminopentanoic acid,
ArgOl refers to argininol, .beta.Ala refers to .beta.-Alanine, Bpa
refers to 4-Benzoylphenylalanine, Bip refers to Beta
(4-biphenyl)-alanine, Dab refers to diaminobutyric acid, Dap refers
to Diaminopropionic acid, Dim refers to Dimethoxyphenylalanine, Dpr
refers to Diaminopropionic acid, Hol refers to homoleucine, HPhe
refers to Homophenylalanine, GABA refers to gamma aminobutyric
acid, GlyNH.sub.2 refers to Aminoglycine, Nle refers to Norleucine,
Nva refers to Norvaline, Orn refers to Ornithine, PheCarboxy refers
to para carboxy Phenylalanine, PheCl refers to para chloro
Phenylalanine, PheF refers to para fluoro Phenylalanine, PheMe
refers to pare methyl Phenylalanine, PheNH2 refers to pare amino
Phenylalanine, PheNO2 refers to para nitro Phenylalanine, Phg
refers to Phenylglycine, Thi refers to Thienylalanine.
[0114] In conventional solution phase peptide synthesis, the
peptide chain can be prepared by a series of coupling reactions in
which the constituent amino acids are added to the growing peptide
chain in the desired sequence. The use of various N-protecting
groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl
group, various coupling reagents (e.g., dicyclohexylcarbodiimide or
carbonyldiimidazole, various active esters, e.g., esters of
N-hydroxyphthalimide or N-hydroxy-succinimide, and the various
cleavage reagents, e.g., trifluoroacetic acid (TEA), HCl in
dioxane, boron tris-(trifluoracetate) and cyanogen bromide, and
reaction in solution with isolation and purification of
intermediates is well-known classical peptide methodology. The
preferred peptide synthesis method follows conventional Merrifield
solid-phase procedures (see Merrifield, 1963 J Amer Chem Soc
85:2149-54; and 1965 Science 50:178-85). Additional information
about the solid phase synthesis procedure can be had by reference
to the treatise by Steward and Young (Solid Phase Peptide
Synthesis, W. H. Freeman & Co., San Francisco, 1969, and the
review chapter by Merrifield in Advances in Enzymology 32:221-296,
F. F. Nold, Ed., Interscience Publishers, New York, 1969; and
Erickson and Merrifield, The Proteins 2:255 et seq. (ea. Neurath
and Hill), Academic Press, New York, 1976. The synthesis of
peptides by solution methods is described in Neurath et al., eds.
(The Proteins, Vol. II, 3d Ed., Academic Press, NY (1976)).
[0115] Crude peptides can be purified using preparative high
performance liquid chromatography. The amino terminus can be
blocked according, for example, to the methods described by Yang et
al. 1990 FEBS Lett 272:61-64.
[0116] Peptide synthesis includes both manual and automated
techniques employing commercially available peptide synthesizers.
The peptides described herein can be prepared by chemical synthesis
and biological activity can be tested using the methods disclosed
herein.
[0117] The peptides described herein can be synthesized in a manner
such that one or more of the bonds linking amino acid residues are
non-peptide bonds. These non-peptide bonds can be formed by
chemical reactions well known to those skilled in the art. In yet
another embodiment of the invention, peptides comprising the
sequences described above can be synthesized with additional
chemical groups present at their amino and/or carboxy termini, such
that, for example, the stability, bio-availability, and/or
inhibitory activity of the peptides is enhanced. For example,
hydrophobic groups such as carbobenzoxyl, dansyl, or
t-butyloxycarbonyl groups, can be added to the peptides' amino
terminus. Likewise, an acetyl group or a
9-fluorenylmethoxy-carbonyl group can be placed at the peptides'
amino terminus. Additionally, the hydrophobic group,
t-butyloxycarbonyl, or an amido group, can be added to the
peptides' carboxy terminus.
[0118] In addition, the peptides can be engineered to contain
additional functional groups to promote cell uptake. For example,
carbohydrate moieties such as glucose or xylose can be attached to
the peptide, such as by attachment to the hydroxyl function of a
serine or threonine amino acid of the peptide.
[0119] Further, the peptides of the invention can be synthesized
such that their stearic configuration is altered. For example, the
D-isomer of one or more of the amino acid residues of the peptide
can be used, rather than the usual L-isomer. Still further, at
least one of the amino acid residues of the peptide can be
substituted by one of the well known non-naturally occurring amino
acid residues. Alterations such as these can serve to increase the
stability, bioavailability and/or inhibitory action of the
peptides.
A. Solid Phase Synthesis
[0120] The peptides disclosed herein can be prepared by classical
methods known in the art, for example, by using standard solid
phase techniques. The standard methods include exclusive solid
phase synthesis, partial solid phase synthesis methods, fragment
condensation, classical solution synthesis, and even recombinant
DNA technology (see, e.g., Merrifield 1963 J Am Chem Soc 85:2149).
On solid phase, the synthesis is typically commenced from the
C-terminal end of the peptide using an alpha-amino protected resin.
A suitable starting material can be prepared, for instance, by
attaching the required alpha-amino acid to a chloromethylated
resin, a hydroxymethyl resin, or a benzhydrylamine resin. One such
chloromethylated resin is sold under the trade name BIO-BEADS
SX-1.TM. by Bio Rad Laboratories (Richmond, Calif.) and the
preparation of the hydroxymethyl resin is described by Bodonszky et
al. 1966 Chem Ind (London) 38:1597. The benzhydrylamine (BHA) resin
has been described by Pietta and Marshall 1970 Chem Comm 650, and
is commercially available from Beckman Instruments, Inc. (Palo
Alto, Calif.) in the hydrochloride form.
[0121] Thus, the compounds disclosed herein can be prepared by
coupling an alpha-amino protected amino acid to the
chloromethylated resin with the aid of, for example, a cesium
bicarbonate catalyst, according to the method described by Gisin,
1973 Helv Chim Acta 56:1467. After the initial coupling, the
alpha-amino protecting group is removed by a choice of reagents
including trifluoroacetic acid (TFA) or hydrochloric acid (HCl)
solutions in organic solvents at room temperature.
[0122] The alpha-amino protecting groups are those known to be
useful in the art of stepwise synthesis of peptides. Included are
acyl type protecting groups (e.g., formyl, trifluoroacetyl,
acetyl), aromatic urethane type protecting groups (e.g.,
benzyloxycarboyl (Cbz) and substituted Cbz), aliphatic urethane
protecting groups (e.g., t-butyloxycarbonyl (Boc),
isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type
protecting groups (e.g., benzyl, triphenylmethyl). Boc and Fmoc are
preferred protecting groups. The side-chain protecting group
remains intact during coupling and is not split off during the
deprotection of the amino-terminus protecting group or during
coupling. The side-chain protecting group must be removable upon
the completion of the synthesis of the final peptide and under
reaction conditions that will not alter the target peptide.
[0123] The side-chain protecting groups for Tyr include
tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z--Br-Cbz, and
2,5-dichlorobenzyl. The side-chain protecting groups for Asp
include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl.
The side-chain protecting groups for Thr and Ser include acetyl,
benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and
Cbz. The side-chain protecting group for Thr and Ser is benzyl. The
side-chain protecting groups for Arg include nitro, Tosyl (Tos),
Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc. The
side-chain protecting groups for Lys include Cbz,
2-chlorobenzyloxycarbonyl (2Cl-Cbz), 2-bromobenzyloxycarbonyl
(2-BrCbz), Tos, or Boc.
[0124] After removal of the alpha-amino protecting group, the
remaining protected amino acids are coupled stepwise in the desired
order. An excess of each protected amino acid is generally used
with an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in
methylene chloride (CH.sub.2Cl.sub.2), dimethyl formamide (DMF)
mixtures.
[0125] After the desired amino acid sequence has been completed,
the desired peptide is decoupled from the resin support by
treatment with a reagent such as trifluoroacetic acid or hydrogen
fluoride (HF), which not only cleaves the peptide from the resin,
but also cleaves all remaining side chain protecting groups. When
the chloromethylated resin is used, hydrogen fluoride treatment
results in the formation of the free peptide acids. When the
benzhydrylamine resin is used, hydrogen fluoride treatment results
directly in the free peptide amide. Alternatively, when the
chloromethylated resin is employed, the side chain protected
peptide can be decoupled by treatment of the peptide resin with
ammonia to give the desired side chain protected amide or with an
alkylamine to give a side chain protected alkylamide or
dialkylamide. Side chain protection is then removed in the usual
fashion by treatment with hydrogen fluoride to give the free
amides, alkylamides, or dialkylamides.
[0126] These solid phase peptide synthesis procedures are well
known in the art and further described by Stewart and Young, Solid
Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company,
1984).
B. Synthetic Amino Acids
[0127] These procedures can also be used to synthesize peptides in
which amino acids other than the 20 naturally occurring,
genetically encoded amino acids are substituted at one, two, or
more positions of any of the compounds described herein. For
instance, naphthylalanine can be substituted for tryptophan,
facilitating synthesis. Other synthetic amino acids that can be
substituted into the peptides include L-hydroxypropyl,
L-3,4-dihydroxy-phenylalanyl, amino acids such as
L-.alpha.-hydroxylysyl and D-.alpha.-methylalanyl,
L-.alpha.-methylalanyl, .beta. amino acids, and isoquinolyl. D
amino acids and non-naturally occurring synthetic amino acids can
also be incorporated into the peptides.
[0128] One can replace the naturally occurring side chains of the
20 genetically encoded amino acids (or D amino acids) with other
side chains, for instance with groups such as alkyl, lower alkyl,
cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl,
amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower
ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered
hetereocyclic. In particular, proline analogs in which the ring
size of the proline residue is changed from 5 members to 4, 6, or 7
members can be employed. Cyclic groups can be saturated or
unsaturated, and if unsaturated, can be aromatic or non-aromatic.
Heterocyclic groups preferably contain one or more nitrogen,
oxygen, and/or sulphur heteroatoms. Examples of such groups include
the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl,
isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl,
piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl,
piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,
pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g.,
1-pyrrolinyl), pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These
heterocyclic groups can be substituted or unsubstituted. Where a
group is substituted, the substituent can be alkyl, alkoxy,
halogen, oxygen, or substituted or unsubstituted phenyl.
[0129] One can also readily modify the peptides by phosphorylation
(see, e.g., Bannwarth et al. 1996 Bioorg Med Chem Letters
6:2141-2146), and other methods for making peptide derivatives of
the compounds disclosed herein are described in Hruby et al. 1990
Biochem J 268:249-262. Thus, the peptide compounds can also serve
as a basis to prepare peptide mimetics with similar biological
activity.
C. Terminal Modifications
[0130] Those of skill in the art recognize that a variety of
techniques are available for constructing peptide mimetics with the
same or similar desired biological activity as the corresponding
peptide compound but with more favorable activity than the peptide
with respect to solubility, stability, and susceptibility to
hydrolysis and proteolysis (see, e.g., Morgan et al. 1989 Ann Rep
Med Chem 24:243-252). The following describes methods for preparing
peptide mimetics modified at the N-terminal amino group, the
C-terminal carboxyl group, and/or changing one or more of the amido
linkages in the peptide to a non-amido linkage. It being understood
that two or more such modifications can be coupled in one peptide
mimetic structure (e.g., modification at the C-terminal carboxyl
group and inclusion of a --CH.sub.2-carbamate linkage between two
amino acids in the peptide).
[0131] 1. N-Terminal Modifications
[0132] The peptides typically are synthesized as the free acid but,
as noted above, could be readily prepared as the amide or ester.
One can also modify the amino and/or carboxy terminus of the
peptide compounds to produce other compounds. Amino terminus
modifications include methylation (i.e., --NHCH.sub.3 or
--NH(CH.sub.3).sub.2), acetylation, adding a benzyloxycarbonyl
group, or blocking the amino terminus with any blocking group
containing a carboxylate functionality defined by RCOO--, where R
is selected from the group consisting of naphthyl, acridinyl,
steroidyl, and similar groups. Carboxy terminus modifications
include replacing the free acid with a carboxamide group or forming
a cyclic lactam at the carboxy terminus to introduce structural
constraints.
[0133] Amino terminus modifications are as recited above and
include alkylating, acetylating, adding a carbobenzoyl group,
forming a succinimide group, etc. (see, e.g., Murray et al. 1995
Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., Vol. 1,
Wolf, ed., John Wiley and Sons, Inc.). Specifically, the N-terminal
amino group can then be reacted as follows:
[0134] a) to form an amide group of the formula RC(O)NH--where R is
as defined above by reaction with an acid halide (e.g., RC(O)Cl) or
symmetric anhydride. Typically, the reaction can be conducted by
contacting about equimolar or excess amounts (e.g., about 5
equivalents) of an acid halide to the peptide in an inert diluent
(e.g., dichloromethane) preferably containing an excess (e.g.,
about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine, to scavenge the acid generated during
reaction. Reaction conditions are otherwise conventional (e.g.,
room temperature for 30 minutes). Alkylation of the terminal amino
to provide for a lower alkyl N-substitution followed by reaction
with an acid halide as described above will provide for N-alkyl
amide group of the formula RC(O)NR--; or
[0135] b) to form a succinimide group by reaction with succinic
anhydride. As before, an approximately equimolar amount or an
excess of succinic anhydride (e.g., about 5 equivalents) can be
employed and the amino group is converted to the succinimide by
methods well known in the art including the use of an excess (e.g.,
ten equivalents) of a tertiary amine such as diisopropylethylamine
in a suitable inert solvent (e.g., dichloromethane) (see, for
example, Wollenberg et al., U.S. Pat. No. 4,612,132 which is
incorporated herein by reference in its entirety). It is understood
that the succinic group can be substituted with, for example,
C.sub.2-C.sub.6 alkyl or --SR substituents which are prepared in a
conventional manner to provide for substituted succinimide at the
N-terminus of the peptide. Such alkyl substituents are prepared by
reaction of a lower olefin (C.sub.2--C) with maleic anhydride in
the manner described by Wollenberg et al., supra and --SR
substituents are prepared by reaction of RSH with maleic anhydride
where R is as defined above; or
[0136] c) to form a benzyloxycarbonyl-NH-- or a substituted
benzyloxycarbonyl-NH-- group by reaction with approximately an
equivalent amount or an excess of CBZ--Cl (i.e., benzyloxycarbonyl
chloride) or a substituted CBZ--Cl in a suitable inert diluent
(e.g., dichloromethane) preferably containing a tertiary amine to
scavenge the acid generated during the reaction; or
[0137] d) to form a sulfonamide group by reaction with an
equivalent amount or an excess (e.g., 5 equivalents) of
R--S(O).sub.2Cl in a suitable inert diluent (dichloromethane) to
convert the terminal amine into a sulfonamide where R is as defined
above. Preferably, the inert diluent contains excess tertiary amine
(e.g., ten equivalents) such as diisopropylethylamine, to scavenge
the acid generated during reaction. Reaction conditions are
otherwise conventional (e.g., room temperature for 30 minutes);
or
[0138] e) to form a carbamate group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R--OC(O)Cl or
R--OC(O)OC.sub.6H.sub.4-p-NO.sub.2 in a suitable inert diluent
(e.g., dichloromethane) to convert the terminal amine into a
carbamate where R is as defined above. Preferably, the inert
diluent contains an excess (e.g., about 10 equivalents) of a
tertiary amine, such as diisopropylethylamine, to scavenge any acid
generated during reaction. Reaction conditions are otherwise
conventional (e.g., room temperature for 30 minutes); or
[0139] f) to form a urea group by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R--N.dbd.C.dbd.O in a
suitable inert diluent (e.g., dichloromethane) to convert the
terminal amine into a urea (i.e., RNHC(O)NH--) group where R is as
defined above. Preferably, the inert diluent contains an excess
(e.g., about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine. Reaction conditions are otherwise
conventional (e.g., room temperature for about 30 minutes).
[0140] 2. C-Terminal Modifications
[0141] In preparing peptide mimetics wherein the C-terminal
carboxyl group is replaced by an ester (i.e., --C(O)OR where R is
as defined above), the resins used to prepare the peptide acids are
employed, and the side chain protected peptide is cleaved with base
and the appropriate alcohol, e.g., methanol. Side chain protecting
groups are then removed in the usual fashion by treatment with
hydrogen fluoride to obtain the desired ester.
[0142] In preparing peptide mimetics wherein the C-terminal
carboxyl group is replaced by the amide --C(O)NR.sup.3R.sup.4, a
benzhydrylamine resin is used as the solid support for peptide
synthesis. Upon completion of the synthesis, hydrogen fluoride
treatment to release the peptide from the support results directly
in the free peptide amide (i.e., the C-terminus is --C(O)NH.sub.2).
Alternatively, use of the chloromethylated resin during peptide
synthesis coupled with reaction with ammonia to cleave the side
chain protected peptide from the support yields the free peptide
amide and reaction with an alkylamine or a dialkylamine yields a
side chain protected alkylamide or dialkylamide (i.e., the
C-terminus is --C(O)NRR.sup.1 where R and R.sup.1 are as defined
above). Side chain protection is then removed in the usual fashion
by treatment with hydrogen fluoride to give the free amides,
alkylamides, or dialkylamides.
[0143] In another alternative embodiment, the C-terminal carboxyl
group or a C-terminal ester can be induced to cyclize by internal
displacement of the --OH or the ester (--OR) of the carboxyl group
or ester respectively with the N-terminal amino group to form a
cyclic peptide. For example, after synthesis and cleavage to give
the peptide acid, the free acid is converted to an activated ester
by an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in
methylene chloride (CH.sub.2Cl.sub.2), dimethyl formamide (DMF)
mixtures. The cyclic peptide is then formed by internal
displacement of the activated ester with the N-terminal amine.
Internal cyclization as opposed to polymerization can be enhanced
by use of very dilute solutions. Such methods are well known in the
art.
[0144] One can also cyclize the peptides herein, or incorporate a
desamino or descarboxy residue at the termini of the peptide, so
that there is no terminal amino or carboxyl group, to decrease
susceptibility to proteases or to restrict the conformation of the
peptide. C-terminal functional groups of the compounds include
amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,
hydroxy, and carboxy, and the lower ester derivatives thereof, and
the pharmaceutically acceptable salts thereof.
[0145] In addition to the foregoing N-terminal and C-terminal
modifications, the peptide compounds, including peptidomimetics,
can advantageously be modified with or covalently coupled to one or
more of a variety of hydrophilic polymers. It has been found that
when the peptide compounds are derivatized with a hydrophilic
polymer, their solubility and circulation half-lives are increased
and their immunogenicity is masked. Quite surprisingly, the
foregoing can be accomplished with little, if any, diminishment in
their binding activity. Nonproteinaceous polymers suitable for use
include, but are not limited to, polyalkylethers as exemplified by
polyethylene glycol and polypropylene glycol, polylactic acid,
polyglycolic acid, polyoxyalkenes, polyvinylalcohol,
polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran
and dextran derivatives, etc. Generally, such hydrophilic polymers
have an average molecular weight ranging from about 500 to about
100,000 daltons, more preferably from about 2,000 to about 40,000
daltons and, even more preferably, from about 5,000 to about 20,000
daltons. In preferred embodiments, such hydrophilic polymers have
an average molecular weights of about 5,000 daltons, 10,000 daltons
and 20,000 daltons.
[0146] The peptide compounds can be derivatized with or coupled to
such polymers using, but not limited to, any of the methods set
forth in Zallipsky, 1995 Bioconjugate Chem 6:150-165 and Monfardini
et al. 1995 Bioconjugate Chem 6:62-69, all of which are
incorporated by reference in their entirety herein.
[0147] In a presently preferred embodiment, the peptide compounds
are derivatized with polyethylene glycol (PEG). PEG is a linear,
water-soluble polymer of ethylene oxide repeating units with two
terminal hydroxyl groups. PEGs are classified by their molecular
weights which typically range from about 500 daltons to about
40,000 daltons. In a presently preferred embodiment, the PEGs
employed have molecular weights ranging from 5,000 daltons to about
20,000 daltons. PEGs coupled to the peptide compounds can be either
branched or unbranched. (see, e.g., Monfardini et al. 1995
Bioconjugate Chem 6:62-69). PEGs are commercially available from
Shearwater Polymers, Inc. (Huntsville, Ala.), Sigma Chemical Co.
and other companies. Such PEGs include, but are not limited to,
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene
glycol-succinate (MePEG-S), monomethoxypolyethylene
glycol-succinimidyl succinate (MePEG-S--NHS),
monomethoxypolyethylene glycol-amine (MePEG-NH.sub.2),
monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).
[0148] Briefly, in one exemplar embodiment, the hydrophilic polymer
which is employed, e.g., PEG, is preferably capped at one end by an
unreactive group such as a methoxy or ethoxy group. Thereafter, the
polymer is activated at the other end by reaction with a suitable
activating agent, such as cyanuric halides (e.g., cyanuric
chloride, bromide or fluoride), diimadozle, an anhydride reagent
(e.g., a dihalosuccinic anhydride, such as dibromosuccinic
anhydride), acyl azide, p-diazoiumbenzyl ether,
3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The
activated polymer is then reacted with a peptide compound disclosed
or taught herein to produce a peptide compound derivatized with a
polymer. Alternatively, a functional group in the peptide compounds
can be activated for reaction with the polymer, or the two groups
can be joined in a concerted coupling reaction using known coupling
methods. It will be readily appreciated that the peptide compounds
can be derivatized with PEG using a myriad of reaction schemes
known to and used by those of skill in the art.
[0149] In addition to derivatizing the peptide compounds with a
hydrophilic polymer (e.g., PEG), it has been discovered that other
small peptides, e.g., other peptides or ligands that bind to a
receptor, can also be derivatized with such hydrophilic polymers
with little, if any, loss in biological activity (e.g., binding
activity, agonist activity, antagonist activity, etc.). It has been
found that when these small peptides are derivatized with a
hydrophilic polymer, their solubility and circulation half-lives
are increased and their immunogenicity is decreased. Again, quite
surprisingly, the foregoing can be accomplished with little, if
any, loss in biological activity. In fact, in preferred
embodiments, the derivatized peptides have an activity that is 0.1
to 0.01-fold that of the unmodified peptides. In more preferred
embodiments, the derivatized peptides have an activity that is 0.1
to 1-fold that of the unmodified peptides. In even more preferred
embodiments, the derivatized peptides have an activity that is
greater than the unmodified peptides.
[0150] Peptides suitable for use in this embodiment generally
include those peptides, i.e., ligands that bind to members of the
Bcl-2 receptor family. Such peptides typically comprise about 150
amino acid residues or less and, more preferably, about 100 amino
acid residues or less (e.g., about 10-12 kDa), even more preferably
about 10 amino acids or less. Hydrophilic polymers suitable for use
herein include, but are not limited to, polyalkylethers as
exemplified by polyethylene glycol and polypropylene glycol,
polylactic acid, polyglycolic acid, polyoxyalkenes,
polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose
derivatives, dextran and dextran derivatives, etc. Generally, such
hydrophilic polymers have an average molecular weight ranging from
about 500 to about 100,000 daltons, more preferably from about
2,000 to about 40,000 daltons and, even more preferably, from about
5,000 to about 20,000 daltons. In preferred embodiments, such
hydrophilic polymers have average molecular weights of about 5,000
daltons, 10,000 daltons and 20,000 daltons. The peptide compounds
can be derivatized with using the methods described above and in
the cited references.
D. Backbone Modifications
[0151] Other methods for making peptide derivatives of the
compounds described herein are described in Hruby et al. 1990
Biochem J 268:249-262, incorporated herein by reference. Thus, the
peptide compounds also serve as structural models for non-peptidic
compounds with similar biological activity. Those of skill in the
art recognize that a variety of techniques are available for
constructing compounds with the same or similar desired biological
activity as the lead peptide compound but with more favorable
activity than the lead with respect to solubility, stability, and
susceptibility to hydrolysis and proteolysis (see Morgan et al.
1989 Ann Rep Med Chem 24:243-252, incorporated herein by
reference). These techniques include replacing the peptide backbone
with a backbone composed of phosphonates, amidates, carbamates,
sulfonamides, secondary amines, and N-methylamino acids.
[0152] Peptide mimetics wherein one or more of the peptidyl
linkages [--C(O)NH--] have been replaced by such linkages as a
--CH.sub.2-carbamate linkage, a phosphonate linkage, a
--CH.sub.2-sulfonamide linkage, a urea linkage, a secondary amine
(--CH.sub.2NH--) linkage, and an alkylated peptidyl linkage
[--C(O)NR.sup.6-- where R.sup.6 is lower alkyl] are prepared during
conventional peptide synthesis by merely substituting a suitably
protected amino acid analogue for the amino acid reagent at the
appropriate point during synthesis.
[0153] Suitable reagents include, for example, amino acid analogues
wherein the carboxyl group of the amino acid has been replaced with
a moiety suitable for forming one of the above linkages. For
example, if one desires to replace a --C(O)NR-- linkage in the
peptide with a --CH.sub.2-carbamate linkage (--CH.sub.2OC(O)NR--),
then the carboxyl (--COOH) group of a suitably protected amino acid
is first reduced to the --CH.sub.2OH group which is then converted
by conventional methods to a --OC(O)Cl functionality or a
para-nitrocarbonate --OC(O)O--C.sub.6H.sub.4-p-NO.sub.2
functionality. Reaction of either of such functional groups with
the free amine or an alkylated amine on the N-terminus of the
partially fabricated peptide found on the solid support leads to
the formation of a --CH.sub.2OC(O)NR-- linkage. For a more detailed
description of the formation of such --CH.sub.2-carbamate linkages,
see Cho et al., 1993, Science 261:1303-1305.
[0154] Similarly, replacement of an amido linkage in the peptide
with a phosphonate linkage can be achieved in the manner set forth
in U.S. patent application Ser. Nos. 07/943,805, 08/081,577 and
08/119,700, the disclosures of which are incorporated herein by
reference in their entirety.
[0155] Replacement of an amido linkage in the peptide with a
--CH.sub.2-sulfonamide linkage can be achieved by reducing the
carboxyl (--COOH) group of a suitably protected amino acid to the
--CH.sub.2 OH group and the hydroxyl group is then converted to a
suitable leaving group such as a tosyl group by conventional
methods. Reaction of the tosylated derivative with, for example,
thioacetic acid followed by hydrolysis and oxidative chlorination
will provide for the --CH.sub.2--S(O).sub.2Cl functional group
which replaces the carboxyl group of the otherwise suitably
protected amino acid. Use of this suitably protected amino acid
analogue in peptide synthesis provides for inclusion of an
--CH.sub.2S(O).sub.2NR-- linkage which replaces the amido linkage
in the peptide thereby providing a peptide mimetic. For a more
complete description on the conversion of the carboxyl group of the
amino acid to a --CH.sub.2S(O).sub.2Cl group, see, for example,
Weinstein, Chemistry & Biochemistry of Amino Acids, Peptides
and Proteins, Vol. 7, pp. 267-357, Marcel Dekker, Inc., New York
(1983) which is incorporated herein by reference.
[0156] Replacement of an amido linkage in the peptide with a urea
linkage can be achieved in the manner set forth in U.S. patent
application Ser. No. 08/147,805, which is incorporated herein by
reference.
[0157] Secondary amine linkages wherein a CH.sub.2NH linkage
replaces the amido linkage in the peptide can be prepared by
employing, for example, a suitably protected dipeptide analogue
wherein the carbonyl bond of the amido linkage has been reduced to
a CH.sub.2 group by conventional methods. For example, in the case
of diglycine, reduction of the amide to the amine will yield after
deprotection H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2COOH which is then
used in N-protected form in the next coupling reaction. The
preparation of such analogues by reduction of the carbonyl group of
the amido linkage in the dipeptide is well known in the art (see,
e.g., Remington, 1994 Meth Mol Bio 35:241-247).
[0158] The suitably protected amino acid analogue is employed in
the conventional peptide synthesis in the same manner as would the
corresponding amino acid. For example, typically about 3
equivalents of the protected amino acid analogue are employed in
this reaction. An inert organic diluent such as methylene chloride
or DMF is employed and, when an acid is generated as a reaction
by-product, the reaction solvent will typically contain an excess
amount of a tertiary amine to scavenge the acid generated during
the reaction. One particularly preferred tertiary amine is
diisopropylethylamine which is typically employed in about 10 fold
excess. The reaction results in incorporation into the peptide
mimetic of an amino acid analogue having a non-peptidyl linkage.
Such substitution can be repeated as desired such that from zero to
all of the amido bonds in the peptide have been replaced by
non-amido bonds.
[0159] One can also cyclize the peptides described herein, or
incorporate a desamino or descarboxy residue at the termini of the
peptide, so that there is no terminal amino or carboxyl group, to
decrease susceptibility to proteases or to restrict the
conformation of the peptide. C-terminal functional groups of the
compounds include amide, amide lower alkyl, amide di(lower alkyl),
lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives
thereof, and the pharmaceutically acceptable salts thereof.
E. Disulfide Bond Formation
[0160] The compounds described herein can exist in a cyclized form
with an intramolecular disulfide bond between the thiol groups of
the cysteines. Alternatively, an intermolecular disulfide bond
between the thiol groups of the cysteines can be produced to yield
a dimeric (or higher oligomeric) compound. One or more of the
cysteine residues can also be substituted with a homocysteine.
[0161] Other embodiments of this invention provide for analogs of
these disulfide derivatives in which one of the sulfurs has been
replaced by a CH.sub.2 group or other isostere for sulfur. These
analogs can be made via an intramolecular or intermolecular
displacement, using methods known in the art.
[0162] Alternatively, the amino-terminus of the peptide can be
capped with an alpha-substituted acetic acid, wherein the alpha
substituent is a leaving group, such as an .alpha.-haloacetic acid,
for example, .alpha.-chloroacetic acid, .alpha.-bromoacetic acid,
or .alpha.-iodoacetic acid. The compounds can be cyclized or
dimerized via displacement of the leaving group by the sulfur of
the cysteine or homocysteine residue. See, e.g., Andreu et al. 1994
Meth Mol Bio 35:91-169; Barker et al. 1992 J Med Chem 35:2040-2048;
and Or et al. 1991 J Org Chem 56:3146-3149, each of which is
incorporated herein by reference.
[0163] Alternatively, the peptides can be prepared utilizing
recombinant DNA technology, which comprises combining a nucleic
acid encoding the peptide thereof in a suitable vector, inserting
the resulting vector into a suitable host cell, recovering the
peptide produced by the resulting host cell, and purifying the
polypeptide recovered. The techniques of recombinant DNA technology
are known to those of ordinary skill in the art. General methods
for the cloning and expression of recombinant molecules are
described in Maniatis (Molecular Cloning, Cold Spring Harbor
Laboratories, 1982), and in Sambrook (Molecular Cloning, Cold
Spring Harbor Laboratories, Second Ed., 1989), and in Ausubel
(Current Protocols in Molecular Biology, Wiley and Sons, 1987),
which are incorporated by reference.
[0164] The peptides can be labeled, for further use as biomedical
reagents or clinical diagnostic reagents. For example, a peptide of
the invention can be conjugated with a fluorescent reagent, such as
a fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), or other fluorescent. The fluorescent
reagent can be coupled to the peptide through the peptide
N-terminus or free amine side chains by any one of the following
chemistries, where R is the fluorescent reagent:
[0165] Alternatively, the peptide can be radiolabeled by peptide
radiolabeling techniques well-known to those skilled in the
art.
IV. Methods for Screening Peptides, Analogs, and Small Molecules
That Modulate Bcl-2-Family Member Protein Activity
[0166] The assays described herein are designed to identify
compounds that interact with (e.g., bind to) Bcl-2 and other
members of the Bcl-2-family of proteins, and modify their ability
to regulate apoptosis. This regulation can be by mimicking Nur77,
by inducing an equivalent conformation change, by enhancing the
Nur77 effect or by inhibiting the Nur77 effect.
[0167] The compounds which can be screened include, but are not
limited to peptides, fragments thereof, and other organic compounds
(e.g., peptidomimetics) that bind to the Bcl-2-family member and
either mimic the activity triggered by the natural regulatory
ligand (e.g., Nur77), enhance the activity triggered by the natural
regulatory ligand or inhibit the activity triggered by the natural
ligand; as well as peptides, antibodies or fragments thereof, and
other organic compounds that mimic the binding domain of the
Bcl-2-family member and bind to and "neutralize" natural
ligand.
[0168] Such compounds include, but are not limited to, peptides
such as, for example, soluble peptides, including but not limited
to members of random peptide libraries; (see, e.g., Lam et al. 1991
Nature 354:82-84; Houghten et al. 1991 Nature 354:84-86), and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids, phosphopeptides (including, but
not limited to, members of random or partially degenerate, directed
phosphopeptide libraries; see, e.g., Songyang et al. 1993 Cell
72:767-778), antibodies including, but not limited to, polyclonal,
monoclonal, humanized, anti-idiotypic, chimeric or single chain
antibodies, and FAb, F(ab').sub.2 and FAb expression library
fragments, and epitope-binding fragments thereof, and small organic
or inorganic molecules.
[0169] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds that can modulate Bcl-2-family member
activity. Having identified such a compound or composition, the
active sites or regions are identified. The active site can be
identified using methods known in the art including, for example,
from the amino acid sequences of peptides, from the nucleotide
sequences of nucleic acids, or from study of complexes of the
relevant compound or composition with its natural ligand. In the
latter case, chemical or X-ray crystallographic methods can be used
to find the active site by finding where on the factor the
complexed ligand is found. Next, the three dimensional geometric
structure of the active site is determined. This can be done by
known methods, including X-ray crystallography, which can determine
a complete molecular structure. On the other hand, solid or liquid
phase NMR can be used to determine certain intra-molecular
distances. Any other experimental method of structure determination
can be used to obtain partial or complete geometric structures. The
geometric structures can be measured with a complexed ligand,
natural or artificial, which can increase the accuracy of the
active site structure determined.
[0170] If an incomplete or insufficiently accurate structure is
determined, the methods of computer based numerical modeling can be
used to complete the structure or improve its accuracy. Any
recognized modeling method can be used, including parameterized
models specific to particular biopolymers such as proteins or
nucleic acids, molecular dynamics models based on computing
molecular motions, statistical mechanics models based on thermal
ensembles, or combined models. For most types of models, standard
molecular force fields, representing the forces between constituent
atoms and groups, are necessary, and can be selected from force
fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete
and more accurate structures computed by these modeling
methods.
[0171] Finally, having determined the structure of the active site,
either experimentally, by modeling, or by a combination, candidate
modulating compounds can be identified by searching databases
containing compounds along with information on their molecular
structure. Such a search seeks compounds having structures that
match the determined active site structure and that interact with
the groups defining the active site. Such a search can be manual,
but is preferably computer assisted. These compounds found from
this search are potential Bcl-2-family member modulating
compounds.
[0172] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0173] Further experimental and computer modeling methods useful to
identify modulating compounds based upon identification of the
active sites of Bcl-2 and related proteins will be apparent to
those of skill in the art.
[0174] Examples of molecular modeling systems are the CHARMM and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0175] A number of articles review computer modeling of drugs
interactive with specific-proteins, such as Rotivinen et al. 1988
Acta Pharm Fennica 97:159-166; McKinaly and Rossmann 1989 Annu Rev
Pharmacol Toxicol 29:111-122; Perry and Davies, OSAR: Quantitative
Structure-Activity Relationships in Drug Design, pp. 189-193, Alan
R. Liss, Inc. (1989); Lewis and Dean 1989 Proc R Soc Lond
236:125-140 and 141-162; and, with respect to a model receptor for
nucleic acid components, Askew et al. 1989 J Am Chem Soc
111:1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,
Canada), and Hypercube, Inc. (Cambridge, Ontario).
[0176] One could also screen libraries of known compounds,
including natural products or synthetic chemicals, and biologically
active materials, including proteins, for compounds which exhibit
binding properties similar to those of Nur77.
[0177] One could also screen libraries of known compounds,
including natural products or synthetic chemicals, and biologically
active materials, including proteins, for compounds which enhance
or inhibit the activity of or binding of Nur77 to Bcl-22 or related
Bcl-2 family members.
[0178] Once identified, these compounds can be subjected to assays
such as those described in the examples to identify whether the
compounds increase apoptosis or decrease apoptosis in cells.
[0179] Compounds identified via assays such as those described
herein can be useful, for example, in inducing or inhibiting
apoptosis.
V. In Vitro Screening Assays for Compounds That Bind to
Bcl-2-Family Member Proteins
[0180] In vitro systems can be designed to identify compounds
capable of interacting with (e.g., binding to) Bcl-2-family
members. Compounds identified can be useful, for example, in
modulating the activity of wild type and/or mutant Bcl related
proteins; can be useful in elaborating the biological function of
the Bcl related proteins; can be utilized in screens for
identifying compounds that disrupt normal Bcl-2-family member
interactions; or can in themselves disrupt such interactions.
[0181] The principle of the assays used to identify compounds that
bind to the Bcl-2-family member involves preparing a reaction
mixture of the protein and the test compound under conditions and
for a time sufficient to allow the two components to interact and
bind, thus forming a complex which can be removed and/or detected
in the reaction mixture.
[0182] The screening assays can be conducted in a variety of ways.
For example, one approach would involve anchoring the Bcl-2 related
protein, polypeptide, peptide or fusion protein or the test
substance to a solid phase, and detecting complexes of Bcl-2
related protein bound to test compounds anchored on the solid
phase. In one embodiment, the Bcl-2 related reactant can be
anchored to a solid surface and the test compound, which is not
anchored, can be labeled, either directly or indirectly. Bound
compound(s) could then be detected by various methods such as mass
spectrometry after elution from the bound protein. In another
embodiment, the binding specificity of test compounds can be tested
using a competition assay as follows: a) Bcl-2 or related Bcl-2
family member is anchored to a solid phase; b) immobilized Bcl-2 or
related Bcl-2 family member is incubated with Nur77 peptide labeled
with a fluorescent tag or other reporter molecule, in the presence
or absence of compounds being tested; c) after incubation under
suitable conditions, the solid phase is washed to remove unbound
reactants; d) the amount of labeled Nur77 peptide bound to the
solid phase is measured for each reaction; and e) the amount of
labeled Nur77 peptide bound in the presence of various test
compounds is compared with the amount of labeled Nur77 peptide
bound in the absence of test compounds, and the ability of each
test compound to compete for Bcl-2 or related Bcl-2 family member
binding sites is determined.
[0183] In practice, microtiter plates can conveniently be utilized
as the solid phase. The anchored component can be immobilized by
non-covalent or covalent attachments. Non-covalent attachment can
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized can be used to anchor the protein to the solid surface.
The surfaces can be prepared in advance and stored.
[0184] In order to conduct the assay, the nonimmobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously nonimmobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously nonimmobilized
component (the antibody, in turn, can be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0185] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected, e.g., using an immobilized antibody
specific for the Bcl related protein, polypeptide, peptide or
fusion protein or the test compound to anchor any complexes formed
in solution, and a labeled antibody specific for the other
component of the possible complex to detect anchored complexes.
[0186] Alternatively, cell-based assays can be used to identify
compounds that interact with Bcl-2-family members or compounds that
enhance or inhibit the interaction of Bcl-2 or related Bcl-2 family
members with Nur77 or Nur77 peptide. To this end, cell lines that
express Bcl related proteins, or cell lines (e.g., COS cells, CHO
cells, fibroblasts, etc.) that have been genetically engineered to
express Bcl-2 related proteins (e.g., by transfection or
transduction of DNA) can be used.
VI. Structure-Based Drug Design
[0187] To aid in the characterization and optimization of compounds
which can alter the activity of Bcl-2-family proteins,
structure-based drug design has become a useful tool. Solution
nuclear magnetic resonance (NMR) techniques can be used to map the
interactions between the BH3 domain of the Bcl-2-family protein and
chemical compounds that target these anti-apoptotic proteins. NMR
chemical shift perturbation is an efficient tool for rapid mapping
of interaction interfaces on proteins. Structure-activity
relationships (SAR) can be obtained by using nuclear magnetic
resonance (NMR), using the method known as "SAR by NMR" (Shuker et
al. 1996 Science 274:1531; Lugovskoy et al. 2002 J Am Chem Soc
124:1234). SAR by NMR can be used to identify, optimize and link
together small organic molecules that bind to proximal subsites of
a protein to produce high-affinity ligands.
[0188] In using NMR to structurally characterize protein-protein
and ligand-protein interactions, isotope labeling can result in
increased sensitivity and resolution, and in reduced complexity of
the NMR spectra. The three most commonly used stable isotopes for
macromolecular NMR are .sup.13C, .sup.15N and .sup.2H. Isotope
labeling has enabled the efficient use of heteronuclear
multi-dimensional NMR experiments, providing alternative approaches
to the spectral assignment process and additional structural
constraints from spin-spin coupling. Uniform isotope labeling of
the protein enables the assignment process through sequential
assignment with multidimensional triple-resonance experiments and
supports the collection of conformational constraints in de novo
protein structure determinations (Kay et al. 1990 J Magn Reson
89:496; Kay et al. 1997 Curr Opin Struct Biol 7:722). These
assignments can be used to map the interactions of a ligand by
following chemical-shift changes upon ligand binding. In addition,
intermolecular NOE (nuclear Overhauser effect) derived
inter-molecular distances can be obtained to structurally
characterize protein-ligand complexes.
[0189] In addition to uniform labeling, selective labeling of
individual amino acids or labeling of only certain types of amino
acids in proteins can result in a dramatic simplification of the
spectrum and, in certain cases, enable the study of significantly
larger macromolecules. For example, the methyl groups of certain
amino acids can be specifically labeled with .sup.13C and .sup.1H
in an otherwise fully .sup.2H-labeled protein. This results in well
resolved heteronuclear [.sup.13C,.sup.1H]-correlation spectra,
which enables straightforward ligand-binding studies either by
chemical shift mapping or by protein methyl-ligand inter-molecular
NOEs, thus providing key information for structure-based drug
design in proteins as large as 170 kDa (Pellecchia et al. 2002
Nature Rev Drug Discovery 1:211). 2D [.sup.13C,.sup.1H]-HMQC
(heteronuclear multiple quantum coherence) and .sup.13C-edited
[.sup.1H,.sup.1H]-NOESY NMR experiments on a ligand-receptor
complex can be used to detect binding, determine the dissociation
constant for the complex, and provide a low-resolution model based
on the available three-dimensional structure of the target, thus
revealing the relative position of the ligand with respect to
labeled side-chains.
[0190] Thus, NMR can be used to identify molecules that induce
apoptosis. Compounds can be screened for binding to labeled
Bcl-X.sub.L, for example. Such labels include .sup.15N and
.sup.13C. The interaction between the compound and Bcl-X.sub.L, and
therefore its ability to induce apoptosis, are determined via
NMR.
VII. Gene Therapy
[0191] Nucleic acid encoding Nur77, and deletions, truncations and
variations thereof, as well as any other peptides identified by the
methods above can be used in gene therapy. In gene therapy
applications, genes are introduced into cells in order to achieve
in vivo synthesis of a therapeutically effective product, for
example the replacement of a defective gene. "Gene therapy"
includes both conventional gene therapy where a lasting effect is
achieved by a single treatment, and the administration of gene
therapeutic agents, which involve the one time or repeated
administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for
blocking the expression of certain genes in vivo. It has been shown
that short antisense oligonucleotides can be imported into cells
where they act as inhibitors, despite their low intracellular
concentrations caused by their restricted uptake by the cell
membrane (Zamecnik et al. 1986 PNAS USA 83:4143-4146). The
oligonucleotides can be modified, e.g., by substituting their
negatively charged phosphodiester groups by uncharged groups.
[0192] There are a variety of techniques available for inducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include, but are not limited to, the use of liposomes,
electroporation, microinjection, cell fusion. DEAE-dextran, and
calcium phosphate precipitation method. The currently preferred in
vivo gene transfer techniques include transfection with viral
(typically retroviral) vectors and viral coat protein-liposome
mediated transfection (Dzau et al., 1993, Trends in Biotechnology
11, 205-210). In some situations it is desirable to provide the
nucleic acid source with an agent that targets the target cell,
such as an antibody specific for a cell surface membrane protein or
the target cell, or a ligand for a receptor on the target cell.
Where liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis can be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof that are tropic for a particular cell type,
antibodies for proteins which undergo internalization in cycling,
and proteins that target intracellular localizations and enhance
intracellular half-life. The technique of receptor-mediated
endocytosis is described, e.g., by Wu et al. 1987 J Biol Chem
262:4429-4432 and Wagner et al. 1990 PNAS USA 87:3410-3414. For
review of gene therapy protocols see Anderson et al. 1992 Science
256:808-813.
[0193] Given the teachings set forth herein, the skilled artisan
can select among various vectors and other expression/delivery
elements depending on such factors as the site and route of
administration and the desired level and duration of
expression.
[0194] For example, naked plasmid DNA can be introduced into muscle
cells, for example, by direct injection into the tissue. (Wolff et
al., 1989, Science 247:1465).
[0195] DNA-Lipid Complexes--Lipid carriers can be associated with
naked DNA (e.g., plasmid DNA) to facilitate passage through
cellular membranes. Cationic, anionic, or neutral lipids can be
used for this purpose. However, cationic lipids are preferred
because they have been shown to associate better with DNA which,
generally, has a negative charge. Cationic lipids have also been
shown to mediate intracellular delivery of plasmid DNA (Felgner and
Ringold 1989 Nature 337:387). Intravenous injection of cationic
lipid-plasmid complexes into mice has been shown to result in
expression of the DNA in lung (Brigham et al. 1989 Am J Med Sci
298:278). See also, Osaka et al. 1996 J Pharm Sci 85:612-618; San
et al. 1993 Human Gene Therapy 4:781-788; Senior et al. 1991
Biochim Biophys Acta 1070:173-179; Kabanov and Kabanov 1995
Bioconjugate Chem 6:7-20; Remy et al. 1994 Bioconjugate Chem
5:647-654; Behr 1994 Bioconjugate Chem 5:382-389; Behr et al. 1989
PNAS USA 86:6982-6986; and Wyman et al. 1977 Biochem
36:3008-3017.
[0196] Adenovirus-based vectors for the delivery of transgenes are
well known in the art and can be obtained commercially or
constructed by standard molecular biological methods. Recombinant
adenoviral vectors containing exogenous genes for transfer are,
generally, derived from adenovirus type 2 (Ad2) and adenovirus type
5 (Ad5). They can also be derived from other non-oncogenic
serotypes. See, e.g., Horowitz, "Adenoviridae and their
Replication" in Virology, 2d Ed., Fields et al. eds., Raven Press
Ltd., New York (1990) incorporated herein by reference.
[0197] It has been shown that Nur77 peptides specifically interact
with Bcl-2-family receptors, resulting in the conversion of these
molecules from anti-apoptotic to pro-apoptotic. These studies
provide a molecular basis for developing various anti-cancer drugs
and other therapeutic agents.
VIII. Pharmacology
[0198] In one embodiment, methods for treating cancer by inducing
apoptosis of cancer cells in an afflicted individual are provided.
Accordingly, one or more inducers of apoptosis of the invention,
targeting an intracellular death antagonist (e.g., Nur77 peptide or
peptidomimetic) is administered to a patient in need of such
treatment. A therapeutically effective amount of the drug can be
administered as a composition in combination with a pharmaceutical
vehicle. In other embodiments of the invention the apoptosis
modulator targets a death antagonist associated with virally
infected cells or self-reacting lymphocytes to comprise a treatment
for viral infection or autoimmune disease.
[0199] For a review of apoptosis in the pathogenesis of disease,
see Thompson, 1995 Science 267:1456-1462.
[0200] In particular, pro-apoptotic modulators of Bcl-2 or related
Bcl-2 family members can be used to treat any condition
characterized by the accumulation of cells which are regulated by
Bcl-2 or related Bcl-2 family members. By "regulated by Bcl-2" with
respect to the condition of a cell is meant that the balance
between cell proliferation and apoptotic cell death is controlled,
at least in part, by Bcl-2 or related Bcl-2 family members. For the
most part, the cells express or overexpress Bcl-2 or related Bcl-2
family members. Enhancement of Bcl-2 or related Bcl-2 family
members expression has been demonstrated to increase the resistance
of cells to almost any apoptotic signal (Hockenbery et al. 1990
Nature 348:334; Nunez et al. 1990 Immunol 144:3602; Vaux et al.
1988 Nature 335:440; Hockenbery et al. 1993 Cell 75:241; Ohmori et
al. 1993 Res Commun 192:30; Lotem et al. 1993 Cell Growth Differ
4:41; Miyashita et al. 1993 Blood 81:115). Principally, the
proliferative disorders associated with the inhibition of cell
apoptosis include cancer, autoimmune disorders and viral
infections. Overexpression of Bcl-2 or related Bcl-2 family members
specifically prevents cells from initiating apoptosis in response
to a number of stimuli (Hockenbery et al. 1990 Nature 348:334;
Nunez et al. 1990 J Immunol 144:3602; Vaux et al. 1988 Nature
335:440; Hockenbery et al. 1993 Cell 75:241). The induction of
genes that inhibit Bcl-2 or related Bcl-2 family members can induce
apoptosis in a wide variety of tumor types, suggesting that many
tumors continually rely on Bcl-2 or related gene products to
prevent cell death. Expression of Bcl-2 or related Bcl-2 family
members has been associated with a poor prognosis in at least
prostatic cancer, colon cancer and neuroblastoma (McDonnell et al.
1992 Cancer Res 52:6940; Hague et al. 1994 Oncogene 9:3367; Castle
et al. 1993 Am J Pathol 143: 1543). Bcl-2 or the related gene has
been found to confer resistance to cell death in response to
several chemotherapeutic agents (Ohmon et al. 1993 Res Commun
192:30; Lotem et al. 1993 Cell Growth Differ 4:41; Miyashita et al.
1993 Blood 81:115).
[0201] Physiologic cell death is important for the removal of
potentially autoreactive lymphocytes during development and for the
removal of excess cells after the completion of an immune response.
Failure to remove these cells can result in autoimmune disease. A
lupus-like autoimmune disease has been reported in transgenic mice
constitutively overexpressing Bcl-2 or related Bcl-2 family members
in their B cells (Strasser et al. 1991 PNAS USA 88:8661). Linkage
analysis has established an association between the Bcl-2 locus and
autoimmune diabetes in non-obese diabetic mice (Garchon et al. 1994
Eur J Immunol 24:380). The compositions described herein which
comprise inhibitors of Bcl-2 function can be used to induce
apoptosis of self-reactive lymphocytes. By "self-reactive" is meant
a lymphocyte which participates in an immune response against
antigens of host cells or host tissues.
[0202] Compositions comprising pro-apoptotic modulators of Bcl-2 or
related Bcl-2 family members can be used in the treatment of viral
infection, to induce apoptosis of virally infected cells. Viruses
have developed mechanisms to circumvent the normal regulation of
apoptosis in virus-infected cells, and these mechanisms have
implicated Bcl-2 or related Bcl-2 family members. For example, the
E1B 19-kDa protein is instrumental in the establishment of
effective adenoviral infection. The apoptosis-blocking ability of
E1B can be replaced in adenoviruses by Bcl-2 (Boyd et al. 1994 Cell
79:341). Genes of certain other viruses have been shown to have
sequence and functional homology to Bcl-2 (Neilan et al. 1993 J
Virol 67:4391; Henderson et al. 1993 PNAS USA 90:8479). The viral
gene LMP-1 specifically upregulates Bcl-2 providing a survival
advantage over latently infected cells (Henderson et al. 1991 Cell
65:1107). Sindbis infection is dependent on the host cell's
expression of Bcl-2 (Levine et al. 1993 Nature 361:739).
[0203] Apart from other considerations, the fact that the novel
active ingredients of the compositions described herein are
peptides, peptide analogs or peptidomimetics, dictates that the
formulation be suitable for delivery of these type of compounds.
Clearly, peptides are less suitable for oral administration due to
susceptibility to digestion by gastric acids or intestinal enzymes.
The preferred routes of administration of peptides are
intraarticular, intravenous, intramuscular, subcutaneous,
intradermal, or intrathecal. A more preferred route is by direct
injection at or near the site of disorder or disease. However, some
of the compounds disclosed herein were proved to be highly
resistance to metabolic degradation in addition to having the
ability to cross cell membrane. These properties make them
potentially suitable for oral administration. Pharmaceutical
compositions as described herein can be manufactured by processes
well known in the art, e.g., by means of conventional mixing,
dissolving, granulating, grinding, pulverizing, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0204] Toxicity and therapeutic efficacy of the pro-apoptotic
modulators of Bcl-2 described herein can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., by determining the IC.sub.50 (the concentration which
provides 50% inhibition) and the LD.sub.50 (lethal dose causing
death in 50% of the tested animals) for a subject compound. The
data obtained from these cell culture assays and animal studies can
be used in formulating a range of dosage for use in humans. The
dosage can vary depending upon the dosage form employed and the
route of administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition (e.g., Fingl et al. 1975, in
"The Pharmacological Basis of Therapeutics", Ch. 1 A1; and
Remington's Pharmaceutical Sciences, by Joseph P. Remington, Mack
Pub. Co. 1985).
[0205] Depending on the severity and responsiveness of the
condition to be treated, dosing can also be a single administration
of a slow release composition, with course of treatment lasting
from several days to several weeks or until cure is effected or
diminution of the disease state is achieved. The amount of a
composition to be administered will, of course, be dependent on the
subject being treated, the severity of the affliction, the manner
of administration, the judgment of the prescribing physician, and
all other relevant factors.
[0206] The targeted cell can be solitary and isolated from other
like cells (such as a single cell in culture or a metastatic or
disseminated neoplastic cell in viva), or the targeted cell can be
a member of a collection of cells (e.g., within a tumor).
Preferably, the cell is a neoplastic cell (e.g., a type of cell
exhibiting uncontrolled proliferation, such as cancerous or
transformed cells). Neoplastic cells can be isolated (e.g., a
single cell in culture or a metastatic or disseminated neoplastic
cell in vivo) or present in an agglomeration, either homogeneously
or, in heterogeneous combination with other cell types (neoplastic
or otherwise) in a tumor or other collection of cells. Where the
cell is within a tumor, some embodiments described herein provide a
method of retarding the growth of the tumor by administering
pro-apoptotic modulator of Bcl-2 to the tumor and subsequently
administering a cytotoxic agent to the tumor.
[0207] By virtue of the cytopathic effect on individual cells, the
inventive method can reduce or substantially eliminate the number
of cells added to the tumor mass over time. Preferably, the
inventive method effects a reduction in the number of cells within
a tumor, and, most preferably, the method leads to the partial or
complete destruction of the tumor (e.g., via killing a portion or
substantially all of the cells within the tumor).
[0208] Where the targeted cell is associated with a neoplastic
disorder within a patient (e.g., a human), some embodiments of the
invention provide a method of treating the patient by first
administering a pro-apoptotic modulator of Bcl-2 or related Bcl-2
family members to the patient ("pretreatment") and subsequently
administering a cytotoxic agent to the patient. This approach is
effective in treating mammals bearing intact or disseminated
cancer. For example, where the cells are disseminated cells (e.g.,
metastatic neoplasia), the cytopathic effects of the inventive
method can reduce or substantially eliminate the potential for
further spread of neoplastic cells throughout the patient, thereby
also reducing or minimizing the probability that such cells will
proliferate to form novel tumors within the patient. Furthermore,
by retarding the growth of tumors including neoplastic cells, the
inventive method reduces the likelihood that cells from such tumors
will eventually metastasize or disseminate. Of course, when the
inventive method achieves actual reduction in tumor size (and
especially elimination of the tumor), the method attenuates the
pathogenic effects of such tumors within the patient. Another
application is in high-dose chemotherapy requiring bone marrow
transplant or reconstruction (e.g., to treat leukemic disorders) to
reduce the likelihood that neoplastic cells will persist or
successfully regrow.
[0209] In many instances, the pretreatment of cells or tumors with
pro-apoptotic modulator of Bcl-2 or related Bcl-2 family members
before treatment with the cytotoxic agent effects an additive and
often synergistic degree of cell death. In this context, if the
effect of two compounds administered together in vitro (at a given
concentration) is greater than the sum of the effects of each
compound administered individually (at the same concentration),
then the two compounds are considered to act synergistically. Such
synergy is often achieved with cytotoxic agents able to act against
cells in the Go-Go phase of the cell cycle.
[0210] Any period of pretreatment can be employed. For example, in
therapeutic applications, such pretreatment can be for as little as
about a day to as long as about 5 days or more; the pretreatment
period can be between about 2 and about 4 days (e.g., about 3
days). Following pretreatment, a cytotoxic agent is administered.
However, in other embodiments, a glucocorticoid (e.g., cortisol,
dexamethasone, hydrocortisone, methylprednisolone, prednisolone,
prednisone, etc.), diphenhydramine, rantidine,
antiemetic-ondasteron, or ganistron can be adjunctively
administered, and such agents can be administered with the
pro-apoptotic modulator of Bcl-2 or related Bcl-2 family members.
The cytotoxic agent can be administered either alone or in
combination with continued administration of the pro-apoptotic
modulator of Bcl-2 or related Bcl-2 family members following
pretreatment. While, according to certain embodiments, treatment
ceases upon administration of the cytotoxic agent, it can be
administered continuously for a period of time (e.g., periodically
over several days) as desired.
[0211] Any cytotoxic agent can be employed in the context of the
invention, and as mentioned, many cytotoxic agents suitable for
chemotherapy are known in the art. Such an agent can be, for
example, any compound mediating cell death by any mechanism
including, but not limited; to, inhibition of metabolism or DNA
synthesis, interference with cytoskeletal organization,
destabilization or chemical modification of DNA, apoptosis, etc.
For example, the cytotoxic agent can be an antimetabolite (e.g.,
5-fiourouricil (5-FU), methotrexate (MTX), fludarabine, etc.), an
anti-microtubule agent (e.g., vincristine, vinblastine, taxanes
(such as paclitaxel and docetaxel), etc.), an alkylating agent
(e.g., cyclophasphamide, melphalan, bischloroethylnitrosurea
(BCNU), etc.), platinum agents (e.g., cisplatin (also termed cDDP),
carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines
(e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g.,
mitomycin-C), topoisomerase inhibitors (e.g., etoposide,
camptothecins, etc.), or other cytotoxic agents (e.g.,
dexamethasone). The choice of cytotoxic agent depends upon the
application of the inventive method. For research, any potential
cytotoxic agent (even a novel cytotoxic agent) can be employed to
study the effect of the toxin on cells or tumors pretreated with
vitamin D (or a derivative). For therapeutic applications, the
selection of a suitable cytotoxic agent will often depend upon
parameters unique to a patient; however, selecting a regimen of
cytotoxins for a given chemotherapeutic protocol is within the
skill of the art.
[0212] For in vivo application, the appropriate dose of a given
cytotoxic agent depends on the agent and its formulation, and it is
well within the ordinary skill of the art to optimize dosage and
formulation for a given patient. Thus, for example, such agents can
be formulated for administration via oral, subcutaneous,
parenteral, submucosal, intravenous, or other suitable routes using
standard methods of formulation. For example, carboplatin can be
administered at daily dosages calculated to achieve an AUC ("area
under the curve") of from about 4 to about 15 (such as from about 5
to about 12), or even from about 6 to about 10. Typically, AUC is
calculated using the Calvert formula, based on the glomerular
filtration rate of creatinine (e.g., assessed by analyzing a plasma
sample) (see, e.g., Martino et al. 1999 Anticancer Res 19:5587-91).
Paclitaxel can be employed at concentrations ranging from about 50
mg/ml to about 100 mg/ml (e.g., about 80 mg/ml). Where
dexamethasone is employed, it can be used in patients at doses
ranging between about 1 mg to about 10 mg (e.g., from about 2 mg to
about 8 mg), and more particularly from about 4 mg to about 6 mg,
particularly where the patient is human. The dosage of the tyrosine
kinase inhibitor is from 1 g/kg to 1 g/kg of body weight per day.
According to one embodiment, the dosage of the tyrosine kinase
inhibitor is from 0.01 mg/kg to 100 mg/kg of body weight per day.
The optimal dosage of the tyrosine kinase inhibitor will vary,
depending on factors such as type; and extent of progression of the
cancer, the overall health status of the patient, the potency of
the tyrosine kinase inhibitor, and route of administration.
Optimization of the tyrosine kinase dosage is within ordinary skill
in the art.
[0213] The pharmaceutical compositions disclosed herein can be most
preferably used for prevention and treatment of malignancies
selected from the group of hormone-refractory-prostate cancer;
prostate cancer (Zin et al 2001 Clin Cancer Res 7:2475-9); breast
cancer (Perez-Tenorio and Stal 2002 Brit J Cancer 86:540-45, Salh
et al. 2002 Int J Cancer 98:148-54); ovarian cancer (Liu et al.
1998 Cancer Res 15:2973-7); colon cancer (Semba at al. 2002 Clin
Cancer Res 8:1957-63); melanoma and skin cancer (Walderman, Wecker
and Diechmann 2002 Melanoma Res 12:45-50); lung cancer (Zin et al.
2001 Clin Cancer Res 7:2475-9); and hepatocarcinoma (Fang et al.
2001 Eur J Biochem 268:45 1 3-9).
[0214] Additional specific types of cancers that can be treated
using this invention include acute myelogenous leukemia, bladder,
cervical, cholangiocarcinoma, chronic myelogenous leukemia,
colorectal, gastric sarcoma, glioma, leukemia, lymphoma, multiple
myeloma, osteosarcoma, pancreatic, stomach, or tumors at localized
sites including inoperable tumors or in tumors where localized
treatment of tumors would be beneficial, and solid tumors.
[0215] According to one preferred embodiment, the pro-apoptotic
modulators of Bcl-2 can be administered in circumstances where the
underlying cancer resists treatment with other chemotherapeutics or
irradiation, due to the action of Bcl-2 blocking apoptosis.
[0216] Another embodiment of the invention provides a method of
treating prostate cancer within a patient by administrating
pro-apoptotic modulator of Bcl-2 or related Bcl-2 family members,
and possibly a glucocorticoid, to the patient. Any pro-apoptotic
modulator of Bcl-2 and glucocorticoid can be employed in accordance
with this aspect of the invention, many of which are discussed
elsewhere herein and others are generally known in the art.
Moreover, pro-apoptotic modulator of Bcl-2 or related Bcl-2 family
members and the glucocorticoid are delivered to the patient by any
appropriate method, some of which are set forth herein. Thus, they
can be formulated into suitable preparations and delivered
subcutaneously, intravenously, orally, etc., as appropriate. Also,
for example, the glucocorticoid is administered to the patient
concurrently, prior to, or after the administration of the
pro-apoptotic modulator of Bcl-2 or related Bcl-2 family members.
One effective dosing schedule is to deliver between about 5 .mu.g
and about 25 g/kg, pro-apoptotic modulator of Bcl-2 or related
Bcl-2 family members daily on alternative days (e.g., between 2 and
4 days a week, such as Mon-Wed-Fri or Tues-Thus-Sat, etc.), and
also between about 1 mg/kg and 20 mg/kg dexamethasone to a human
patient also on alternative days. In such a regimen, the
alternative days on which pro-apoptotic modulator of Bcl-2 or
related Bcl-2 family members and on which the glucocorticoid are
administered can be different, although preferably they are
administered on the same days. Even more preferably, the
glucocorticoid is administered once, by itself, prior to concurrent
treatment. Of course, the treatment can continue for any desirable
length of time, and it can be repeated, as appropriate to achieve
the desired end results. Such results can include the attenuation
of the progression of the prostate cancer, shrinkage of such
tumors, or, desirably, remission of all symptoms. However, any
degree of effect is considered a successful application of this
method. A convenient method of assessing the efficacy of the method
is to note the change in the concentration of prostate-specific
antigen (PSA) within a patient. Typically, such a response is
gauged by measuring the PSA levels over a period of time of about 6
weeks.
[0217] Desirably, the method results in at least about a 50%
decrease in PSA levels after 6 weeks of application, and more
desirably at least about 80% reduction in PSA. Of course, the most
desirable outcome is for the PSA levels to decrease to about normal
levels.
[0218] Another embodiment of the invention provides a method of
treating breast cancer within a patient by administrating the
non-naturally occurring pro-apoptotic modulator of Bcl-2 or related
Bcl-2 family members alone or in combination with any other
treatment regimen for breast cancer. Treatments for breast cancer
are well known in the art and continue to be developed. Treatments
include but are not limited to surgery, including axillary
dissection, sentinel lymph node biopsy, reconstructive surgery,
surgery to relieve symptoms of advanced cancer, lumpectomy (also
called breast conservation therapy), partial (segmental)
mastectomy, simple or total mastectomy, modified radical
mastectomy, and radical mastectomy; hormone therapy using a drug
such as tamoxifen, which blocks the effects of estrogen; aromatase
inhibitors, which stop the body from making estrogen;
immunotherapy, e.g., using HERCEPTIN.TM. (trastuzumab), an
anti-HER2 humanized monoclonal antibody developed to block the HER2
receptor; bone marrow transplantation; peripheral blood stem cell
therapy; bisphosphonates; additional chemotherapy agents; radiation
therapy; acupressure; and acupuncture. Particularly preferred
chemotherapy agents for use in combination with the
non-naturally-occurring compounds or peptides of the present
invention include doxorubicin, paclitaxel, fluorouracil,
cyclophosphamide, and tamoxifen. Any combination of therapies may
be used in conjunction with the present invention.
[0219] In some embodiments, the pro-apoptotic modulators of Bcl-2
or related Bcl-2 family members can be used in the form of a
pharmaceutically acceptable salt.
[0220] Suitable acids which are capable of forming salts include
inorganic acids such as hydrochloric acid, hydrobromic acid,
perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,
phosphoric acid and the like; and organic acids such as formic
acid, acetic acid, propionic acid, glycolic acid, lactic acid,
pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic
acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene
sulfonic acid, sulfanilic acid and the like.
[0221] Suitable bases capable of forming salts include inorganic
bases such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide and the like; and organic bases such as mono-, di- and
tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine,
methyl amine, dimethyl amine and the like) and optionally
substituted ethanol-amines (e.g., ethanolamine, diethanolamine and
the like).
[0222] Pharmaceutically acceptable vehicles for delivery of the
pro-apoptotic modulators of Bcl-2 or related Bcl-2 family members
include physiologically tolerable or acceptable diluents,
excipients, solvents, or adjuvants, for parenteral injection, for
intranasal or sublingual delivery, for oral administration, for
rectal or topical administration or the like. The compositions are
preferably sterile and nonpyrogenic. Examples of suitable carriers
include but are not limited to water, saline, dextrose, mannitol,
lactose, or other sugars, lecithin, albumin, sodium glutamate
cysteine hydrochloride, ethanol, polyols (propyleneglycol,
ethylene, polyethyleneglycol, glycerol, and the like), vegetable
oils (such as olive oil), injectable organic esters such as ethyl
oleate, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol
and sorbitan esters, microcrystalline cellulose, aluminum
methahydroxide, bentonite, agar-agar and tragacanth, or mixtures of
these substances, and the like.
[0223] The pharmaceutical compositions can also contain minor
amounts of nontoxic auxiliary substances such as wetting agents,
emulsifying agents, pH buffering agents, antibacterial and
antifungal agents (such as parabens, chlorobutanol, phenol, sorbic
acid, and the like). If desired, absorption enhancing or delaying
agents (such as liposomes, aluminum monostearate, or gelatin) can
be used. The compositions can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions.
[0224] Compositions containing the pro-apoptotic modulators of
Bcl-2 or related Bcl-2 family members can be administered by any
convenient route which will result in delivery of the conjugate to
cells expressing the intracellular target. Modes of administration
include, for example, orally, rectally, parenterally
(intravenously, intramuscularly, intraarterially, or
subcutaneously), intracisternally, intravaginally,
intraperitoneally, locally (powders, ointments or drops), or as a
buccal or nasal spray or aerosol.
[0225] The pharmaceutical compositions are most effectively
administered parenterally, preferably intravenously or
subcutaneously. For intravenous administration, they can be
dissolved in any appropriate intravenous delivery vehicle
containing physiologically compatible substances, such as sodium
chloride, glycine, and the like, having a buffered pH compatible
with physiologic conditions. Such intravenous delivery vehicles are
known to those skilled in the art. In a preferred embodiment, the
vehicle is a sterile saline solution. If the peptides are
sufficiently small, other preferred routes of administration are
intranasal, sublingual, and the like. Intravenous or subcutaneous
administration can comprise, for example, injection or
infusion.
[0226] The effective amount and method of administration of the
pro-apoptotic modulators of Bcl-2 or related Bcl-2 family members
will vary based upon the sex, age, weight and disease stage of the
patient, whether the administration is therapeutic or prophylactic,
and other factors apparent to those skilled in the art. Based upon
the in vitro studies described herein, a suitable dosage is a
dosage which will attain a tissue concentration of from about 1 to
about 100 .mu.M, more preferably from about 10 to about 75 .mu.M.
It is contemplated that lower or higher concentrations would also
be effective. The tissue concentration can be derived from peptide
conjugate blood levels. Such a dosage can comprise, for example,
from about 0.1 to about 100 mg/kg.
[0227] Those skilled in the art will derive appropriate dosages and
schedules of administration to suit the specific circumstances and
needs of the patient. Doses are contemplated on the order of from
about 1 to about 500, preferably from about 10 to about 100, most
preferably from about 30 to about 80, mg/kg of body weight. The
pro-apoptotic modulator of Bcl-2 or related Bcl-2 family members
can be administered by injection daily, over a course of therapy
lasting two to three weeks, for example. Alternatively, the agent
can be administered by continuous infusion, such as via an
implanted subcutaneous pumps, as is well-known in cancer
therapy.
[0228] The pro-apoptotic modulators of Bcl-2 or related Bcl-2
family members according described herein can be labeled with a
fluorescent, radiographic or other visually detectable label and
utilized in in vitro studies to identify cells expressing an
intracellular target, or to identify the location of the target
inside of such cells. For example, a pro-apoptotic modulator of
Bcl-2 or related Bcl-2 family members can be synthesized with an
attached biotin molecule and incubated with cells suspected of
expressing the target. The cells are then incubated with
streptavidin-fluorescein. Cells expressing the intracellular target
will bind the biotin conjugate, and the streptavidin-fluorescein
complex. The result is a pattern of fluorescence inside the cell.
In particular, a pro-apoptotic modulator of Bcl-2 or related Bcl-2
family members which binds the Bcl-2 protein or related Bcl-2
family members can be utilized to identify tumor cells which
express Bcl-2 or related Bcl-2 family members. Assessment of Bcl-2
expression has prognostic value, as tumors expressing high levels
of Bcl-2 or related Bcl-2 family members are likely to be
chemoresistant and/or radiation resistant.
[0229] Selected compounds described herein are peptide-based
substrate mimetic pro-apoptotic modulators of Bcl-2 that are stable
in plasma for 6-24 hours, slowly metabolized by hepatic cells and
are membrane permeable. The pro-apoptotic modulators of Bcl-2
induce apoptosis in cancer cells in the same concentrations that
cell death is induced, while no cytotoxic death is observed at
these concentrations by cell cycle analysis.
[0230] In addition, additional indications that can be treated
using the pharmaceutical compositions described herein include any
condition involving undesirable or uncontrolled cell proliferation
Such indications include restenosis, benign tumors, abnormal
stimulation of endothelial cells (atherosclerosis), insults to body
tissue due to surgery, abnormal wound healing, abnormal
angiogenesis, diseases that produce fibrosis of tissue, repetitive
motion disorders, disorders of tissues that are not highly
vascularized, and proliferative responses associated with organ
transplants.
[0231] Specific types of restenotic lesions that can be treated
include coronary, carotid, and cerebral lesions. Specific types of
benign tumors that can be treated include hemangiomas, acoustic
neuromas, neurofibroma, trachomas and pyogenic granulomas.
[0232] Treatment of cell proliferation due to insults to body
tissue during surgery can be possible for a variety of surgical
procedures, including joint surgery, bowel surgery, and keloid
scarring. Diseases that produce fibrotic tissue include emphysema.
Repetitive motion disorders that can be treated include carpal
tunnel syndrome. An example of cell proliferative disorders that
can be treated is a bone tumor.
[0233] Abnormal angiogenesis that can be treated include those
abnormal angiogenesis accompanying rheumatoid arthritis, psoriasis,
diabetic retinopathy, and other ocular angiogenic diseases such as
retinopathy of prematurity (detrimental fibroplastic), macular
degeneration, corneal graft rejection, neuromuscular glaucoma and
Ouster Webber syndrome.
[0234] The proliferative responses associated with organ
transplantation that can be treated include those proliferative
responses contributing to potential organ rejections or associated
complications. Specifically, these proliferative responses can
occur during transplantation of the heart, lung, liver, kidney, and
other body organs or organ systems.
IX. Assays for Detection of Apoptosis:
[0235] The compounds described herein can be tested in cells for
induction of apoptosis of cancer cell lines. Apoptosis is assayed
at least by two methods in each cell line. Cells are seeded at the
appropriate plates for each method, treated with or without the
inhibitory compounds for different time points and analyzed by one
of the methods described below.
[0236] a. Annexin-V Staining
[0237] This assay identifies the early event of phosphatidyl-serine
presentation on cell membrane. Cells are assayed for apoptosis
using the Annexin-V.
[0238] Cells are seeded in 6-well plates (0.3.times.10.sup.6/well),
and washed twice with PBS, 24 hrs after treatment with the
inhibitory compounds, and resuspended in Annexin-V binding buffer
(10 mM HEPES/NaOH pH 7.4, 140 mM NaCl and 2.5 mM CaCl.sub.2).
Annexin-V is diluted 1:40 and added to each sample with 0.2 nM
Propidium Iodide (PI). 0.5.times.10.sup.6 cells are taken per
sample and analyzed by FACS.
[0239] b. Caspases Activity.
[0240] This assay indicates very early events of apoptosis.
Caspases (1, 8, 9, 5, 7, 3, 6, 4, and 2) activity is assayed
according to the manufacturer's instructions using the CaspaTag
Caspase activity kit (Intergene), 24 hrs after treatment with the
inhibitory compounds of the invention. Briefly, 10.sup.6 of
suspended cells/sample are labeled with 10 .mu.l of 30.times.
working dilution FAM peptide-FMK-Fluorescein and incubated for 1 hr
at 37.degree. C. under 5% CO.sub.2. Samples are washed 3 times with
1.times. working dilution wash buffer and the cell pellets are
resuspended with 700 .mu.l of the same buffer. 2 .mu.l of 0.2 nM
propidium iodide solution is added and caspases activity is
determined by FACS analysis.
[0241] c. DNA Fragmentation Measurement.
[0242] DNA fragmentation is a late event in the apoptosis cascade.
DNA fragmentation is measured according to the manufacturer's
instructions using the In situ cell death detection kit (Roche), 72
hrs after treatment with the compounds of the invention. Briefly,
2.times.10.sup.6 of adherent cells/sample are trypsinized, washed
twice with PBS, and replated in 96 well plates. Then, the samples
are fixed with 2% paraformaldehyde in PBS at room temperature for 1
hr, washed with PBS and resuspended with permeabilization solution
for 2 min on ice. Cells are washed twice with PBS, and labeled with
TUNEL reaction mixture containing labeling solution and TdT enzyme
solution for 1 hr at 37.degree. C. Samples are washed again with
PBS and analyzed by FACS.
X. Growth Inhibition Assays:
[0243] Selected compounds, which were found active in the
enzyme-inhibition assays are screened for their ability to inhibit
growth of tumor cell lines. Screening for inhibitory compounds is
done, initially, at concentration of 50 .mu.M. Active compounds
from the first screening are further tested at different
concentrations (50, 25, 12.5, 6.25, 3.125 and 1.56 .mu.M) in order
to determine their IC.sub.50. Growth inhibition is tested using two
methods: A. staining of viable cells with methylene blue, B.
incorporation of .sup.3H-thymidine. For both methods cells are
grown in 96-well plates for 24 hours, before tested compounds are
added. The assays are done in triplicate for one to six days.
[0244] a. Staining Viable Cells With Methylene Blue
[0245] Cells are fixed by 0.5% glutardialdehyde followed by
staining with 1% methylene blue in borate buffer (Sigma) for one
hour. Cells are then washed a few times with distilled water, air
dried and the color is extracted by adding 0.1 M HCl for one hour
at 37.degree. C. Quantitation of color intensity is performed by
measurement of the optical density at 600 nm by ELISA reader.
[0246] b. Incorporation of 3H-thymidine:
[0247] At the appropriate time in culture loci of .sup.3H-thymidine
(stock of 5 Ci/mmole, Amersham) is added to each well containing
100 .mu.l of medium for 5 hours. At the end of the incubation the
cells are washed a few times with PBS using a cell harvester
(Packard, USA), air dried for a few hours and 50 .mu.l of
scintillation liquid is added. The radioactivity is counted using a
microplate counter, for example, Packard Top Count.
[0248] Fifty percent inhibitory concentration (IC.sub.50) values
are calculated using nonlinear regression in one site competition
model with GraphPad Prism version 3.03 Windows (GraphPad Software,
San Diego, USA)
[0249] The following examples are intended to illustrate how to
make and use the compounds and methods of this invention and are in
no way to be construed as a limitation. Although the invention will
now be described in conjunction with specific embodiments thereof,
it is evident that many modifications and variations will be
apparent to those skilled in the art.
[0250] Accordingly, it is intended to embrace all such
modifications and variations that fall within the spirit and broad
scope of the appended claims.
Examples
Example 1
[0251] The following examples describe the processes used to
identify and characterize peptides that target Bcl-2-family members
and regulate their apoptotic functions.
[0252] Peptides from DC3 fragment of Nur77, known to interact with
Bcl-2, were synthesized and conjugated with the
cell-penetrating-peptide (CPP), D-Arg octamer (r8) (FIG. 1A) as
described below. Amongst these, a peptide with 20 amino acids (480
to 499) (NuBCP-20-r8) exhibited potent apoptotic effects in various
cancer cell lines. In ZR-75-1 breast cancer cells, significant
apoptosis (>80%) was observed by <10 .mu.M NuBCP-20, whereas
normal primary mammary epithelial cells were resistant (FIGS. 1B,
C, FIG. 5A, B).
[0253] To identify the minimal-length of NuBCP required for
inducing apoptosis, NuBCP-20 was subjected to serial deletion
analysis (FIG. 1D) as described below. A Nur77 peptide with only 9
amino acids, NuBCP-9-r8, was found to potently induce apoptosis.
NuBCP-9 is considerably shorter than the shortest BH3 peptide that
binds Bcl-2. Further deletion from either its N-terminal or
C-terminal end or substituting Ala for NuBCP-9 terminal amino acids
(NuBCP-9/AA-r8) completely abolished its apoptotic effect. NuBCP-9
linked to other CPPs, penetratin or transportan-10 (Jones, S. W. et
al. 2005 Br J Pharmacol 145:1093-102) as well as r8 by a disulfide
bond showed a similar degree of apoptosis (FIG. 1E). Since
disulfide bonds are rapidly reduced in cells, the apoptotic effect
of NuBCP-9 was not due to the conjugated CPPs. The NuBCP-20-r8 was
more potent than BH3 peptides derived from pro-apoptotic Bcl-2
family member t-Bid (FIG. 1F, 5C, D).
[0254] To probe the stereochemical requirements for NuBCP-induced
apoptosis, the L-amino acids were substituted with D-amino acids.
When H460 lung cancer cells were exposed to the D-analog of NuBCP-9
(D-NuBCP-9-r8), apoptosis was retained and even slightly enhanced
compared with NuBCP-9-r8 (FIG. 1F). In contrast, the D-enantiomer
of Bad-BH3 peptide did not bind Bcl-2 and was not apoptotic (FIG.
1F), demonstrating a clear difference in the mode of action of the
NuBCP and Bad-BH3 peptide.
[0255] To determine whether NuBCPs bound to Bcl-2, DNA sequences
encompassing NuBCP-9 (Nur77/489-497) were cloned into a vector
containing the green fluorescence protein (GFP). Like NuBCPs, the
GFP-Nur77/489-497 fusion was apoptotic. When transfected into
HEK293T cells, it was precipitated by anti-Bcl-2 antibody only when
Bcl-2 was co-expressed (FIG. 2A), which was inhibited by addition
of NuBCP, but not Smac-peptide-r8. To study whether D-NuBCP-9
interacted with Bcl-2, a competition assay was used as described
below. Nur77 lacking its DNA-binding-domain (DBD),
Nur77/.DELTA.DBD, bound strongly with Bcl-2, and the binding was
abrogated when NuBCP-9-r8 or D-NuBCP-9-r8 was present (FIG. 2B).
The GFP-Nur77/489-497 fusion colocalized extensively with RFP-Mito,
a red fluorescence protein (RFP) fused with a classical
mitochondria-targeting sequence. FITC-D-NuBCP-9-r8 also displayed
extensive colocalization with RFP-Mito. Similar results were
obtained with NuBCP-20 (FIG. 6). Thus, NuBCP-9 and its
D-enantiomer, similar to Nur77, bind Bcl-2 and target mitochondria,
which is a repository for Bcl-2 where apoptosis is triggered.
[0256] Fluorescence polarization (FP) analysis was used to
determine whether NuBCPs interacted directly with Bcl-2. Nur77 did
not bind Bcl-X.sub.L and served as a control. GST-Bcl-2, but not
GST-Bcl-X.sub.L or GST, induced a concentration-dependent FP of
FITC-NuBCP-9-r8 and FITC-D-NuBCP-9-r8, while FITC-NuBCP-9/AA-r8 was
little affected (FIG. 2C-E). In addition, unconjugated NuBCP-9 and
D-NuBCP-9 competed for binding of FITC-L-NuBCP-9-r8 or
FITC-D-NuBCP-9-r8, whereas NuBCP-9/AA did not (FIG. 2F-H). Thus,
NuBCP-9 and D-NuBCP-9 bind directly and competitively to Bcl-2. The
site on Bcl-2 targeted by NuBCPs is distinct from BH3 peptides, as
shown by the failure of either a BH3 peptide or a highly potent
chemical inhibiter (ABT-737) that targets the BH3-binding site to
reduce FITC-NuBCP-9-r8 binding (FIG. 2F-H).
[0257] A hallmark of Nur77 is its induction of apoptosis in a
Bcl-2-dependent manner. Therefore experiments were performed to
examine whether NuBCP-9-induced apoptosis was dependent on Bcl-2.
Transfection of Bcl-2 siRNA, but not control GFP siRNA, reduced
Bcl-2 levels (FIG. 7A) and inhibited the apoptotic effects of
NuBCP-9-r8 and D-NuBCP-9-r8 in H460 lung cancer cells (FIG. 3A).
Exposure of cells to Bcl-2 antisense oligonucleotides also
attenuated their apoptotic function (FIG. 3B). In contrast, stable
expression of Bcl-2 in Jurkat or CEM cells strongly enhanced
NuBCP-induced cell death (FIGS. 3C-E, 7A-C, 8A-D). For comparison,
apoptosis induced by Bad BH3 peptide or staurosporine was
attenuated by overexpression of Bcl-2 in these cells, demonstrating
the dual role of Bcl-2. The NuBCP-induced apoptosis requires Bax or
Bak, as the peptides similarly induced apoptosis of wild-type,
Bax.sup.-/-, and Bak.sup.-/- mouse embryonic fibroblasts (MEFs),
but lacked death activity in double knockout MEFs (FIG. 3F).
[0258] Nur77 binding to Bcl-2 resulted in exposure of the Bcl-2 BH3
domain, a characteristic of Bcl-2 pro-apoptotic state. Such a Bcl-2
conformation is recognized by an anti-Bcl-2 antibody raised against
the Bcl-2 BH3 domain. To determine whether NuBCP-9 induced a Bcl-2
conformational change, flow cytometric assays were conducted which
HEK293T cells transfected with Bcl-2 were exposed to NuBCPs and
subsequently stained with the anti-Bcl-2/BH3 antibody. Fluorescence
microscopy showed that the antibody failed to stain Bcl-2 in
control cells, reflecting the anti-apoptotic conformation of Bcl-2,
in which its BH3 domain epitope is buried. By contrast, cells
exposed to NuBCP-9-r8 or D-NuBCP-9-r8 were strongly stained. Thus,
both peptides were able to induce a pro-apoptotic Bcl-2
conformation. Consistent with its inability to induce apoptosis,
NuBCP-9/AA-r8 failed to induce Bcl-2 immunostaining.
[0259] Flow cytometry analysis also revealed a strong Bcl-2
immunofluorescence in cells exposed to L-NuBCP-9-r8 or
D-NuBCP-9-r8, but not NuBCP-9/AA-r8, when stained by the
anti-Bcl-2/BH3 antibody (FIG. 4A). In contrast, cells exposed to
apoptosis-inducing t-Bid-BH3 peptide or Smac-peptide-r8 did not
show any Bcl-2 immunofluorescence. NuBCP-9-r8-induced Bcl-2
immunofluorescence was observed in the presence of the caspase
inhibitor zVAD, excluding the involvement of caspases in the Bcl-2
conformational change (FIG. 8D). The effect of NuBCPs on Bcl-2
conformation was further examined by immunoprecipitation assays
(FIG. 4B), showing that the anti-Bcl-2/BH3 antibody precipitated
endogenous Bcl-2 in cells treated with L-NuBCP-9-r8 or
D-NuBCP-9-r8, but not NuBCP-9/AA-r8, t-Bid-BH3-peptide-r8, or
Bad-BH3-peptide-r8.
[0260] We then explored whether the Bcl-2 conformational change by
Bcl-2 was a direct consequence of Nur77 binding. Experiments were
performed to determine whether NuBCPs could induce a conformational
change of bacterially expressed Bcl-2. To this end, the ability of
purified GST-Bcl-2 protein to react with anti-Bcl-2/BH3 antibody in
the presence of NuBCP was analyzed by immunoprecipitation assay.
Incubation with NuBCP-9-r8 but not NuBCP-9/AA-r8, resulted in a
strong precipitation of GST-Bcl-2 protein by the anti-Bcl-2/BH3
antibody (FIG. 4C), demonstrating a direct role of NuBCP binding in
Bcl-2 conformational change. This was further supported by circular
dichroism (CD) analysis, revealing similar changes in GST-Bcl-2
protein spectra, when incubated with NuBCP-9 or D-NuBCP-9, but not
NuBCP-9/AA (FIG. 4D). Binding was saturating and stoichiometric
with a Kd=2.1.+-.0.2 .mu.M (FIG. 4E), in agreement with FP assays.
In contrast, NuBCP-9, D-NuBCP-9 and NuBCP-9/AA had no effect on CD
spectra for GST or Bcl-X.sub.L (FIGS. 9-11). Thus, the
NuBCP-induced Bcl-2 conformational change observed in cells can be
accounted for by direct binding of NuBCPs to Bcl-2.
[0261] The competitive FPA assays described above demonstrates how
the FITC-NuBCP-9-r8 peptide can be used to identify NuBCP peptide
analogs that compete with NuBCP-9-r8 for binding to Bcl-2 or Bcl-2
related proteins. This same assay can also be used to identify
peptidomimetics and small molecule mimics or antagonists of Nur77
or functionally related proteins such as Nor1 or Not (also Nurr1).
Mimics can be distinguished from antagonists using CD analysis
described above to detect the presence or absence of a
conformational change. Nur77 mimics would induce a conformational
change in Bcl-2 similar to that observed for NuBCP-9 while
antagonists would compete with NuBCP-9-r8 for binding to Bcl-2 but
not induce a conformational change. Mimics would identify compounds
that could act as pro-apoptic compounds whereas antagonists could
block the pro-apoptotic effects of Nur77 or functionally equivalent
proteins such as Nor1 and Not. Compounds that mimic Nur77 could be
used for treating cancers characterized by elevated levels of Bcl-2
while compounds that antagonize Nur77 or functionally related
proteins could be used to treat neurodegenerative diseases that are
characterized by converting Bcl-2 to its pro-apoptotic form.
[0262] To further evaluate NuBCPs, their effects on the growth of
tumors formed in SCID mice were examined by methods described
below. MDA-MB435 breast cancer cells, which are sensitive to NuBCPs
(FIG. 12) and rapidly form tumors in SCID mice, were used.
Comparison with control (NuBCP-9/AA-r8) peptide showed that the
injection of either L-NuBCP-9-r8 or D-NuBCP-9-r8 dramatically
suppressed the growth of tumors (FIG. 4F), and potently induced
apoptosis of tumor cells in vivo. Apoptosis of tumor cells was
associated with a Bcl-2 conformational change, as revealed by
extensive overlapping of TUNEL staining with Bcl-2
immunofluorescence stained by anti-Bcl-2/BH3 antibody. Together,
these results demonstrate that NuBCPs effectively inhibit tumor
growth in mice through apoptosis induction by inducing a Bcl-2
conformational change.
[0263] Alanine scan of Nur77/1 peptide using H460 lung cancer
cells. To characterize amino acid residues in the Nur77-9 peptide
critical for its activity, each amino acid was substituted with
alanine as follows: acetyl-ASRSLHSLLGXrrrrrrrr-amide (SEQ ID NO:
61); acetyl-FSRSAHSLLGXrrrrrrrr-amide (SEQ ID NO: 62);
acetyl-FSRSLHSALGXrrrrrrrr-amide (SEQ ID NO: 63);
acetyl-FSRSLHSLAGXrrrrrrrr-amide (SEQ ID NO: 64). Analysis of these
mutant Nur77-9 peptides showed that Phe489, Leu493, Leu497 and
Leu498 were critical. Replacement of these amino acids with alanine
largely impaired, while simultaneous substitutions of Phe489 and
Leu497 completely abolished, the apoptotic effect of Nur77-9 (FIG.
13).
[0264] Induction of apoptosis by reverse Nur77 peptides. To further
explore the sequence requirements for apoptosis, we reversed the
Nur77-9 sequence and also made the corresponding D-enantiomer of
the Nur77-9 reverse sequence. Both the reverse (inverso) sequence
and the corresponding D-enantiomer (retroinverso) sequence were
apoptotic (FIG. 14).
[0265] Induction of apoptosis by Nur77-DC3 derived peptides. A
Nur77 fragment, DC-3 (FIG. 15, SEQ ID NO: 60), which corresponds to
a portion of the Nur77 ligand-binding domain (LBD), binds Bcl-2,
resulting in its conformational change and apoptosis. The fragment
displays four sequences including one found in Nur77-9 (SEQ ID NO:
9) that share a common motif, F Xaa.sub.n L Xaa.sub.n L Xaa.sub.n
L, wherein n is between 0 and 3, Xaa is any amino acid, and Leu can
be replaced by a hydrophobic amino acid. Each of the additional
Nur77 DC3 sequences: FGDWIDSIL (SEQ ID NO: 16), FAALSALVL (SEQ ID
NO: 17), FYLKLEDLV (SEQ ID NO: 18) when extended with GXrrrrrrrr
are apoptotic (FIG. 16).
[0266] Induction of apoptosis by short Nur77 peptides. In addition,
any alanine substitutable interior amino acid (Xaa) can be removed,
i.e. SRS and HS can be deleted one by one or in any combination
thereof. For example, each of the alanine-substitutable amino acids
can be removed to give FLLL (SEQ ID NO: 40) which is apoptotic.
These studies identify a short Nur77 9-mer peptide that mimics the
converter activity of the Nur77 protein (FIG. 17). This
demonstrates that small molecule drugs would be useful for
converting Bcl-2 from a protector to a killer of breast cancer
cells which could reverse the drug and gamma-irradiation resistance
of breast cancers.
[0267] Nur77 peptide linked to various cell penetrating peptides
are apoptotic. The pro-apoptotic modulator of Bcl-2 can be linked
to various cell penetrating peptide sequences including the
penetratin sequence (RQIKIWFQNRRMKWKK, SEQ ID NO: 65, and the
transportan 10 sequence (AGYLLGKINLKALAALAKKIL, SEQ ID NO: 66).
Both the Nur77 sequence and the cell penetrating sequences can
exist in the L form, D form or mixed D/L or DDLL forms to induce
apoptosis (FIG. 18). Thus the Nur77 D-peptide can be linked to a
D-transportan peptide and is apoptotic.
[0268] All of the D-peptides were protease resistant, an important
property for in vivo use. FAM-labeled Nur77/1 (D) Transportan 10
(D) is proteolytically stable for up to one day in mouse serum and
upon intravenous injection in a mouse is proteolytically stable
during the course of its excretion.
[0269] Nur77 peptide binds anti-apoptotic Bcl-2 family members.
Nur77-peptide binds Bcl-B, Bfl-1, and Bcl-2, but not Mcl-1 and
Bcl-xL. DNA sequences corresponding with NuBCP-9 (Nur77/489-497)
were cloned as a GFP fusion. The expression vector for the
GFP-Nur77/489-497 fusion was transfected into HEK293T cells
together with or without the indicated expression vector for
myc-tagged Bcl-2 family member. Cell lysates were prepared and
analyzed for interaction of Nur77/489-497 with Bcl-family members
by co-immunoprecipitation assay using anti-Myc antibody.
Immunoprecipitates were then separated on SDS/PAGE and
immunoblotted using anti-GFP antibody. The results showed that
GFP-Nur77/489-497 interacts with Bcl-B, Bfl-1, and Bcl-2, but not
with Bcl-X.sub.L and Mcl-1 (FIG. 19).
Materials and Methods
[0270] Plasmids--Plasmids encoding Nur77 and Nur77/.DELTA.DBD (Li,
et al. 2000 Science 289:1159-1164), Bcl-2, Bcl-Gs, L216E-Bcl-Gs,
Bcl-2/.DELTA.loop (Cheng et al. 1997 Science 278:1966-1968), Bax,
and Bcl-X.sub.L (Guo, et al. 2001 J Biol Chem 276:2780-2785), have
been described previously. To construct plasmids encoding N168,
DC3, DC1, Nur77/.DELTA.DBD/DC1, Nur77/.DELTA.DBD/.DELTA.471-488,
and Bcl-2/1-80, appropriate Nur77 or Bcl-2 fragments were prepared
either by restriction enzyme digestion or amplified by polymerase
chain reaction (PCR) by well known methods. The resulting Nur77
fragments were then cloned into pGFP-N2 vector (Clontech, USA).
[0271] Nur77/.DELTA.DBD/L487A mutants were cloned by substituting
Leu487 with Ala by PCR site-directed mutagenesis on the
Nur77/.DELTA.DBD template. Bcl-2/Y108K, Bcl-2/L137A, Bcl-2/G145A,
and Bcl-2/R146 were constructed by substituting Tyr108, Leu137,
Gly145 and Arg146 with Lys, Ala, Ala, and Glu, respectively, by PCR
site-directed mutagenesis using the Bcl-2 cDNA as a template.
Bcl2/.DELTA.BH1, Bcl-2/.DELTA.BH2, Bcl-2/.DELTA.BH3,
Bcl-2/.DELTA.BH4, Bcl-2/.DELTA.TM are deletions of 132-160,
189-204, 90-114, 7-30, 205-239 amino acids in Bcl-2 (Hanada et al.
1995 J Biol Chem 270:11962-11969). All mutations were confirmed by
DNA sequencing.
[0272] Bcl-2 siRNAs and antisense oligonucleotides--The target
siRNA SMARTpools for Bcl-2 and Bak and the siRNA oligonucleotide
for Nur77 (5'-CAG UCC AGC CAU GCU CCU dTdT) (SEQ ID NO: 67) were
purchased from Dharmacon Research Inc. Target or control siRNA were
transfected at a final concentration of 200 nM into cells at 40%
confluency using Oligofectamine reagent (Invitrogen) according to
the manufacturer's recommendations. After 48 h, cells were
analyzed. Bcl-2 antisense oligonucleotide targeting Bcl-2 and
negative control oligonucleotides were obtained from Calbiochem.
They (2.5 .mu.M) were transfected into cells at 60% confluency for
36 h before analysis.
[0273] Peptide synthesis. Peptides were synthesized on MBHA resin
using Fmoc synthesis and DIC/HOBt coupling with an Advanced Chem
Tech 350 and 396 multiple peptide synthesizer. All peptides except
FITC-peptides were acetylated on their N-termini and all were
amidated on their C-termini. Standard deprotection conditions were
used for all peptides except those with Pbf-protected D-arginine
octamers which were treated for 6 hr. Peptides were purified by
HPLC on C18 columns and confirmed by MALDI mass analysis. Disulfide
linked peptides were prepared as described (Giriat, I. & Muir,
T. W. 2003 J Am Chem Soc 125:7180-1). Peptides with C-terminal
cysteines were covalently linked to chloroacetylated N-aminocaproic
acid in a displacement reaction.
[0274] Nur77/Bcl-2 interaction assays--Reporter gene assays using
NurRE-tk-CAT in CV-1 cells, and GST pull-down assay were described
previously (Li, et al. 2000 Science 289:1159-1164). For the
mammalian two-hybrid assays, CV-1 cells were co-transfected with
pcDNA-Ga14TAD-Nur77 or pcDNA-Gal4TAD-Nur77/.DELTA.DBD and
pcDNA-Gal4DBD-Bcl-2/.DELTA.TM along with a luciferase reporter gene
driven by four copies of the Gal4-binding site. The cells were
harvested and reporter gene activity was measured. For Co-IP
assays, HEK293T cells were transiently transfected with various
expression plasmids using a modified calcium phosphate
precipitation method (Wu et al, 1997) in the presence of caspase
inhibitors (zVAD-fmk) to prevent degradation of Nur77 protein due
to apoptosis. Cells were suspended in lysis buffer (50 mM Tris-HCl,
PH7.4; 150 mM NaCl; 20 mM EDTA; 1% NP-40; 1 mM PMSF; 50 .mu.g/ml
Leupeptin; 20 mg/ml Aprotinin; 0.1 mM Na.sub.3VO.sub.4; and 1 mM
DTT). Cells extracts were cleared by incubation with the Protein
A/G plus Agarose beads (Santa Cruz) and then incubated with
appropriate antibody and 30 .mu.l of Protein A or G plus Agarose
beads overnight at 4.degree. C. Beads were then washed and boiled
in Laemmli gel-loading solution before performing
SDS-PAGE/immunoblotting using the following polyclonal or
monoclonal antibodies: monoclonal mouse anti-GFP (Medical and
Biological Laboratories), monoclonal mouse anti-HA (Roche Molecular
Biochemicals), monoclonal mouse anti-FLAG (Sigma), monoclonal mouse
anti-Myc (Santa Cruz), polyclonal rabbit anti-Nur77 (Active Motif),
or monoclonal mouse anti-Bcl-2 (Santa Cruz). Immunoreactive
products were detected by chemiluminescence with an enhanced
chemiluminescence system (ECL) (Amersham).
[0275] Subcellular localization assays--Cells were seeded onto
cover-slips in 6-well plates overnight, then transiently
transfected with GFP-fusion expression plasmids. After 16 hours,
cells were washed with PBS and fixed in 4% paraformaldehyde. For
mitochondrial staining, cells were then incubated with anti-Hsp60
goat IgG (Santa Cruz, USA), followed by anti-goat IgG conjugated
with Cy3 (Sigma). For cyt c staining, cells were incubated with
monoclonal anti-cyt c IgG (PharMingen), followed by anti-mouse IgG
conjugated with Cy5 (Amersham). Fluorescent images were collected
and analyzed using a MRC-1024 MP laser-scanning confocal microscope
(Bio-Rad). Subcellular fractionation assays were performed as
described (Li, et al. 2000 Science 289:1159-1164). Briefly, cells
(1.times.10.sup.7 cells) suspended in 0.5 ml hypotonic buffer (5 mM
Tris-HCl, pH 7.4, 5 mM KCl, 1.5 mM MgCl.sub.2, 0.1 mM EGTA, pH 8.0,
and 1 mM DTT) were homogenized and cell extracts were centrifuged
at 500.times.g for 5 min. The resulting supernatant was centrifuged
at 10,000.times.g for 30 min at 4.degree. C. to obtain the HM
fraction. HM fraction was resuspended in 100 .mu.l lysis buffer (10
mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, pH 8.0)
for immunoblotting analysis.
[0276] Apoptosis assays--For nuclear morphological change analysis,
cells were trypsinized, washed with PBS, fixed with 3.7%
paraformaldehyde, and stained with DAPI
(4,6-diamidino-2-phenylindole) (50 .mu.g/ml) to visualize the
nuclei by UV-microscopy. The percentages of apoptotic cells were
determined by counting 300 GFP-positive cells, scoring cells having
nuclear fragmentation and/or chromatin condensation.
[0277] Fluorescence polarization (FP) assays. GST-Bcl-2,
GST-Bcl-X.sub.L, or GST protein was briefly incubated with
FITC-conjugated peptide with or without competitors in Greiner
Fluotrac 600 96-well microplates. Fluorescence polarization was
recorded using an Analyst HT 96-384 microplate reader (Molecular
Devices, Sunnyvale, Calif.) with excitation wavelength set at 485
nm and dynamic polarizer for emission at 530 nm.
[0278] Circular dichroism (CD) spectroscopy. Stock solutions of 3
mM peptide in 30% acetonitrile/water were added to 0.5 mL of 2
.mu.M purified GST-proteins in PBS, pH 7.6. CD spectra were
obtained in a 0.2 cm pathlength cell at 20.degree. C. using an AVIV
62 DS spectropolarimeter for a wavelength range from 200 to 260 nm
with a step size of 1 nm averaged for 5 sec. Three spectra were
corrected for background and averaged for each sample. The Kd was
determined using nonlinear regression analysis for a
one-site-binding model (.chi.2>0.98). Stoichiometry was
determined from a Zhou plot (Jones, G. et al. 2002 Tet. Let. 43:
6079-6082).
[0279] Cell culture. H460 lung cancer cells were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum (FBS). HEK293T
embryonic kidney cells were grown in DME medium supplemented with
10% FBS. Jurkat and Jurkat cells stably expressing Bcl-2 were
kindly provided by Dr. John Reed, and they were maintained in
RPMI1640 medium.
[0280] Confocal microscopy. Cells were seeded on chamber slides
overnight and treated with apoptotic agents in medium containing
0.5% FBS. After treatments cells were fixed in PBS containing 3.7%
paraformaldehyde for 10 min and washed twice with PBS. Cells were
then permeabilized with 0.1% triton X-100 in PBS for 5 min. Fixed
cells were pre-incubated for 30 min in PBS containing 5% BSA at
room temperature.
[0281] Transient transfection assays. Cells (1.times.10.sup.5
cells/well) seeded in 24-well plates were transiently transfected
using a modified calcium phosphate precipitation procedure.
[0282] Immunoblotting. Cell lysates were boiled in SDS sample
buffer, resolved by SDS-polyacrylamide gel electrophoresis, and
transferred to nitrocellulose. After transfer, the membranes were
blocked in 5% milk in TBST (10 mM Tris-HCl, pH.8.0, 150 mM NaCl,
0.05% Tween 20) containing antibody. The membranes were washed
three times with TBST, then incubated for 1 hr at room temperature
in 5% milk in TBST containing horseradish peroxide-linked
anti-immunoglobulin. After 3 washes in TBST, immunoreactive
products were detected by chemiluminescence with an enhanced
chemiluminescence system (ECL, Amersham).
[0283] Animal studies. Female SCID mice (6-week-old) (Tacomic) were
injected with 10.sup.6 MDA-MB435 breast carcinoma cells. Tumors
were palpable on day 7. On days 10 and 13, test peptides (620 .mu.g
in 50 .mu.L PBS) were injected into the tumor areas of 5 mice.
Tumor volumes (1.times.w.sup.2) were determined using calipers. No
weight changes were observed. Established tumors in control mice
were injected with test peptides and tumor tissues were excised and
sectioned after 3 days. Tissues were fixed (10% buffered formalin),
then rapidly paraffin-embedded. Apoptosis was detected by the TUNEL
assay.
Example 2
[0284] The FITC-NuBCP-9-r8 peptide is used in competitive FPA
assays to identify peptidomimetics and small molecule mimics or
antagonists of Nur77 or functionally related proteins such as Nor1
or Not (also Nurr1) that compete with NuBCP-9-r8 for binding to
Bcl-2 or Bcl-2 related proteins. Mimics are distinguished from
antagonists using CD analysis described above which detects the
presence or absence of a conformational change. Nur77 mimics induce
a conformational change in Bcl-2 similar to that observed for
NuBCP-9 while antagonists compete with NuBCP-9-r8 for binding to
Bcl-2 but not induce a conformational change. Mimics are used to
identify compounds that act as pro-apoptic compounds whereas
antagonists block the pro-apoptotic effects of Nur77 or
functionally equivalent proteins such as Nor1 and Not. Compounds
that antagonize Nur77 or functionally related proteins are used to
treat neurodegenerative diseases that are characterized by
converting Bcl-2 to its pro-apoptotic form.
Example 3
[0285] A patient diagnosed with breast cancer is selected for
treatment with the D-NuBCP-9-r8 peptide. The patient is given a
therapeutically effective intravenous dose of the peptide at
regular intervals over a six week period. Following the end of the
treatment period it is observed that the breast cancer has
regressed.
[0286] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
figures, tables, appendices, patents, patent applications and
publications, referred to above, are hereby incorporated by
reference.
Sequence CWU 1
1
67132PRTArtificial Sequencesynthetic peptide, NuBCP-20-r8 1Gly Asp
Trp Ile Asp Ser Ile Leu Ala Phe Ser Arg Ser Leu His Ser1 5 10 15Leu
Leu Val Asp Lys Lys Cys Xaa Arg Arg Arg Arg Arg Arg Arg Arg 20 25
30225PRTArtificial Sequencesynthetic peptide NuBCP-15 2Ser Ile Leu
Ala Phe Ser Arg Ser Leu His Ser Leu Leu Val Asp Gly1 5 10 15Xaa Arg
Arg Arg Arg Arg Arg Arg Arg 20 25324PRTArtificial Sequencesynthetic
peptide, NuBCP-14 3Ile Leu Ala Phe Ser Arg Ser Leu His Ser Leu Leu
Val Asp Gly Xaa1 5 10 15Arg Arg Arg Arg Arg Arg Arg Arg
20423PRTArtificial Sequencesynthetic peptide, NuBCP-13 4Leu Ala Phe
Ser Arg Ser Leu His Ser Leu Leu Val Asp Gly Xaa Arg1 5 10 15Arg Arg
Arg Arg Arg Arg Arg 20522PRTArtificial Sequencesynthetic peptide,
NuBCP-12 5Ala Phe Ser Arg Ser Leu His Ser Leu Leu Val Asp Gly Xaa
Arg Arg1 5 10 15Arg Arg Arg Arg Arg Arg 20621PRTArtificial
Sequencesynthetic peptide, NuBCP-11 6Phe Ser Arg Ser Leu His Ser
Leu Leu Val Asp Gly Xaa Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg
20720PRTArtificial Sequencesynthetic peptide, NuBCP-10 7Ser Arg Ser
Leu His Ser Leu Leu Val Asp Gly Xaa Arg Arg Arg Arg1 5 10 15Arg Arg
Arg Arg 20820PRTArtificial Sequencesynthetic peptide, NuBCP-N10
8Phe Ser Arg Ser Leu His Ser Leu Leu Val Gly Xaa Arg Arg Arg Arg1 5
10 15Arg Arg Arg Arg 20919PRTArtificial Sequencesynthetic peptide,
NuBCP-9-r8 9Phe Ser Arg Ser Leu His Ser Leu Leu Gly Xaa Arg Arg Arg
Arg Arg1 5 10 15Arg Arg Arg1018PRTArtificial Sequencesynthetic
peptide, NuBCP-8 10Phe Ser Arg Ser Leu His Ser Leu Gly Xaa Arg Arg
Arg Arg Arg Arg1 5 10 15Arg Arg1117PRTArtificial Sequencesynthetic
peptide, NuBCP-7 11Phe Ser Arg Ser Leu His Ser Gly Xaa Arg Arg Arg
Arg Arg Arg Arg1 5 10 15Arg1219PRTArtificial Sequencesynthetic
peptide, NuBCP-9/AA 12Ala Ser Arg Ser Leu His Ser Leu Ala Gly Xaa
Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg1319PRTArtificial
Sequencesynthetic peptide, D-NuBCP-9-r8 13Phe Ser Arg Ser Leu His
Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg1419PRTArtificial SequenceSynthetic peptide, Nur77/1 (inverso)
14Leu Leu Ser His Leu Ser Arg Ser Phe Gly Xaa Arg Arg Arg Arg Arg1
5 10 15Arg Arg Arg1519PRTArtificial SequenceSynthetic peptide,
Nur77/1 (retroinverso) 15Leu Leu Ser His Leu Ser Arg Ser Phe Gly
Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg1619PRTArtificial
SequenceSynthetic peptide, Nur77/2 16Phe Gly Asp Trp Ile Asp Ser
Ile Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg1719PRTArtificial SequenceSynthetic peptide, Nur77/3 17Phe Ala
Ala Leu Ser Ala Leu Val Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg
Arg Arg1819PRTArtificial SequenceSynthetic peptide, Nur77/4 18Phe
Tyr Leu Lys Leu Glu Asp Leu Val Gly Xaa Arg Arg Arg Arg Arg1 5 10
15Arg Arg Arg1924PRTArtificial SequenceSynthetic peptide, Nor1
19Ser Ile Lys Asp Phe Ser Leu Asn Leu Gln Ser Leu Asn Leu Asp Gly1
5 10 15Arg Arg Arg Arg Arg Arg Arg Arg 202024PRTArtificial
SequenceSynthetic peptide, NOT 20Ser Ile Val Glu Phe Ser Ser Asn
Leu Gln Asn Met Asn Ile Asp Gly1 5 10 15Arg Arg Arg Arg Arg Arg Arg
Arg 202112PRTArtificial SequenceSynthetic peptide, Nur77/1
(embedded) 21Arg Arg Arg Phe Arg Arg Arg Leu Arg Arg Leu Leu1 5
102212PRTArtificial SequenceSynthetic peptide, Nur77/1 (D/embedded)
22Arg Arg Arg Phe Arg Arg Arg Leu Arg Arg Leu Leu1 5
102319PRTArtificial SequenceSynthetic peptide 23Phe Ser Arg Ser Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2419PRTArtificial SequenceSynthetic peptide 24Phe Ser Arg Ser
Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2519PRTArtificial SequenceSynthetic peptide 25Phe Ala Arg Ser
Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2619PRTArtificial SequenceSynthetic peptide 26Phe Ser Ala Ser
Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2719PRTArtificial SequenceSynthetic peptide 27Phe Ser Arg Ala
Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2819PRTArtificial SequenceSynthetic peptide 28Phe Ser Arg Ser
Leu Ala Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg2919PRTArtificial SequenceSynthetic peptide 29Phe Ser Arg Ser
Leu His Ala Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg3018PRTArtificial SequenceSynthetic peptide 30Phe Arg Ser Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3118PRTArtificial SequenceSynthetic peptide 31Phe Ser Ser Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3218PRTArtificial SequenceSynthetic peptide 32Phe Ser Arg Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3318PRTArtificial SequenceSynthetic peptide 33Phe Ser Arg Ser
Leu Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3418PRTArtificial SequenceSynthetic peptide 34Phe Ser Arg Ser
Leu His Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3517PRTArtificial SequenceSynthetic peptide 35Phe Ser Leu His
Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg Arg1 5 10
15Arg3618PRTArtificial SequenceSynthetic peptide 36Phe Arg Ser Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3718PRTArtificial SequenceSynthetic peptide 37Phe Xaa Ser Leu
His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg3819PRTArtificial SequenceSynthetic peptide 38Phe Ser Arg Ser
Leu His Ser Leu Leu Gly Xaa Cys Gly Asn Lys Arg1 5 10 15Thr Ala
Cys3945PRTArtificial SequenceSynthetic peptide 39Phe Ser Arg Ser
Leu His Ser Leu Leu Gly Xaa Ala Lys Val Lys Asp1 5 10 15Glu Pro Gln
Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro Ala Pro Pro 20 25 30Lys Pro
Glu Pro Lys Pro Lys Lys Ala Pro Ala Lys Lys 35 40
454014PRTArtificial SequenceSynthetic peptide, Nur77/short 40Phe
Leu Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg Arg Arg1 5
104114PRTArtificial SequenceSynthetic peptide, Nur77/short D 41Phe
Leu Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg Arg Arg1 5
104227PRTArtificial SequenceSynthetic peptide, Nur77/1 Ant 42Phe
Ser Arg Ser Leu His Ser Leu Leu Cys Cys Arg Gln Ile Lys Ile1 5 10
15Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 20
254327PRTArtificial SequenceSynthetic peptide, Nur77/1 Ant (D)
43Phe Ser Arg Ser Leu His Ser Leu Leu Cys Cys Arg Gln Ile Lys Ile1
5 10 15Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 20
254418PRTArtificial SequenceSynthetic p53 44Phe Ser Asp Leu Trp Lys
Leu Leu Gly Xaa Arg Arg Arg Arg Arg Arg1 5 10 15Arg
Arg4529PRTArtificial SequenceSynthetic peptide, Nur77 (embedded2)
45Asn Phe Gln His Ala Leu Gln Glu Val Leu Gln Ala Leu Lys Gln Val1
5 10 15Gln Ala Arg Cys Cys Arg Arg Arg Arg Arg Arg Arg Arg 20
254619PRTArtificial SequenceSynthetic peptide, Nur77/1 (D/L) 46Phe
Ser Arg Ser Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10
15Arg Arg Arg4719PRTArtificial SequenceSynthetic peptide, Nur77/1
(DD/LL) 47Phe Ser Arg Ser Leu His Ser Leu Leu Gly Xaa Arg Arg Arg
Arg Arg1 5 10 15Arg Arg Arg4827PRTArtificial SequenceSynthetic
peptide, NuBCP-9- Penetratin 48Phe Ser Arg Ser Leu His Ser Leu Leu
Cys Cys Arg Gln Ile Lys Ile1 5 10 15Trp Phe Gln Asn Arg Arg Met Lys
Trp Lys Lys 20 254927PRTArtificial SequenceSynthetic peptide,
Nu77/1 (D) Penetratin (D) 49Phe Ser Arg Ser Leu His Ser Leu Leu Cys
Cys Arg Gln Val Lys Ile1 5 10 15Trp Phe Gln Asn Arg Arg Met Lys Trp
Lys Lys 20 255032PRTArtificial SequenceSynthetic peptide, Nu77/1
Transportan10 50Phe Ser Arg Ser Leu His Ser Leu Leu Cys Cys Ala Gly
Tyr Leu Leu1 5 10 15Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala
Lys Lys Ile Leu 20 25 305132PRTArtificial SequenceSynthetic
peptide, Nu77/1 (D)Transportan10 (D) 51Phe Ser Arg Ser Leu His Ser
Leu Leu Cys Cys Ala Gly Tyr Leu Leu1 5 10 15Gly Lys Val Asn Leu Lys
Ala Leu Ala Ala Leu Ala Lys Lys Val Leu 20 25 305232PRTArtificial
SequenceSynthetic peptide, Nu77/1 (L/D)Transportan10 (L/D) 52Phe
Ser Arg Ser Leu His Ser Leu Leu Cys Cys Ala Gly Tyr Leu Leu1 5 10
15Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
20 25 305332PRTArtificial SequenceSynthetic peptide, Nu77/1
(LLDD)Transportan10 (LLDD) 53Phe Ser Arg Ser Leu His Ser Leu Leu
Cys Cys Ala Gly Tyr Leu Leu1 5 10 15Gly Lys Val Asn Leu Lys Ala Leu
Ala Ala Leu Ala Lys Lys Val Leu 20 25 305437PRTArtificial
SequenceSynthetic peptide, NuBCP-9-Transportan 54Phe Ser Arg Ser
Leu His Ser Leu Leu Cys Cys Gly Trp Thr Leu Asn1 5 10 15Ser Ala Gly
Tyr Leu Leu Gly Lys Ile Asn Lys Ala Leu Ala Ala Leu 20 25 30Ala Lys
Lys Ile Leu 3555598PRTHomo sapiens 55Met Pro Cys Ile Gln Ala Gln
Tyr Gly Thr Pro Ala Pro Ser Pro Gly1 5 10 15Pro Arg Asp His Leu Ala
Ser Asp Pro Leu Thr Pro Glu Phe Ile Lys 20 25 30Pro Thr Met Asp Leu
Ala Ser Pro Glu Ala Ala Pro Ala Ala Pro Thr 35 40 45Ala Leu Pro Ser
Phe Ser Thr Phe Met Asp Gly Tyr Thr Gly Glu Phe 50 55 60Asp Thr Phe
Leu Tyr Gln Leu Pro Gly Thr Val Gln Pro Cys Ser Ser65 70 75 80Ala
Ser Ser Ser Ala Ser Ser Thr Ser Ser Ser Ser Ala Thr Ser Pro 85 90
95Ala Ser Ala Ser Phe Lys Phe Glu Asp Phe Gln Val Tyr Gly Cys Tyr
100 105 110Pro Gly Pro Leu Ser Gly Pro Val Asp Glu Ala Leu Ser Ser
Ser Gly 115 120 125Ser Asp Tyr Tyr Gly Ser Pro Cys Ser Ala Pro Ser
Pro Ser Thr Pro 130 135 140Ser Phe Gln Pro Pro Gln Leu Ser Pro Trp
Asp Gly Ser Phe Gly His145 150 155 160Phe Ser Pro Ser Gln Thr Tyr
Glu Gly Leu Arg Ala Trp Thr Glu Gln 165 170 175Leu Pro Lys Ala Ser
Gly Pro Pro Gln Pro Pro Ala Phe Phe Ser Phe 180 185 190Ser Pro Pro
Thr Gly Pro Ser Pro Ser Leu Ala Gln Ser Pro Leu Lys 195 200 205Leu
Phe Pro Ser Gln Ala Thr His Gln Leu Gly Glu Gly Glu Ser Tyr 210 215
220Ser Met Pro Thr Ala Phe Pro Gly Leu Ala Pro Thr Ser Pro His
Leu225 230 235 240Glu Gly Ser Gly Ile Leu Asp Thr Pro Val Thr Ser
Thr Lys Ala Arg 245 250 255Ser Gly Ala Pro Gly Pro Ser Glu Gly Arg
Cys Ala Val Cys Gly Asp 260 265 270Asn Ala Ser Cys Gln His Tyr Gly
Val Arg Thr Cys Glu Gly Cys Lys 275 280 285Gly Phe Phe Lys Arg Thr
Val Gln Lys Asn Ala Lys Tyr Ile Cys Leu 290 295 300Ala Asn Lys Asp
Cys Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln305 310 315 320Phe
Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Val Lys Glu Val 325 330
335Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys
340 345 350Pro Lys Gln Pro Pro Asp Ala Ser Pro Ala Asn Leu Leu Thr
Ser Leu 355 360 365Val Leu Ala His Leu Asp Ser Gly Pro Ser Thr Ala
Lys Leu Asp Tyr 370 375 380Ser Lys Phe Gln Glu Leu Val Leu Pro His
Phe Gly Lys Glu Asp Ala385 390 395 400Gly Asp Val Gln Gln Phe Tyr
Asp Leu Leu Ser Gly Ser Leu Glu Val 405 410 415Ile Arg Lys Trp Ala
Glu Lys Ile Pro Gly Phe Ala Glu Leu Ser Pro 420 425 430Ala Asp Gln
Asp Leu Leu Leu Glu Ser Ala Phe Leu Glu Leu Phe Ile 435 440 445Leu
Arg Leu Ala Tyr Arg Ser Lys Pro Gly Glu Gly Lys Leu Ile Phe 450 455
460Cys Ser Gly Leu Val Leu His Arg Leu Gln Cys Ala Arg Gly Phe
Gly465 470 475 480Asp Trp Ile Asp Ser Ile Leu Ala Phe Ser Arg Ser
Leu His Ser Leu 485 490 495Leu Val Asp Val Pro Ala Phe Ala Cys Leu
Ser Ala Leu Val Leu Ile 500 505 510Thr Asp Arg His Gly Leu Gln Glu
Pro Arg Arg Val Glu Glu Leu Gln 515 520 525Asn Arg Ile Ala Ser Cys
Leu Lys Glu His Val Ala Ala Val Ala Gly 530 535 540Glu Pro Gln Pro
Ala Ser Cys Leu Ser Arg Leu Leu Gly Lys Leu Pro545 550 555 560Glu
Leu Arg Thr Leu Cys Thr Gln Gly Leu Gln Arg Ile Phe Tyr Leu 565 570
575Lys Leu Glu Asp Leu Val Pro Pro Pro Pro Ile Ile Asp Lys Ile Phe
580 585 590Met Asp Thr Leu Pro Phe 5955617PRTArtificial
SequenceSynthetic peptide, Smac-peptide-r8 56Ala Val Pro Ile Ala
Gln Lys Cys Xaa Arg Arg Arg Arg Arg Arg Arg1 5 10
15Arg5730PRTArtificial SequenceSynthetic peptide, t-Bid BH3
peptide-r8 57Glu Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Gln
Val Gly Asp1 5 10 15Ser Met Asp Arg Cys Xaa Arg Arg Arg Arg Arg Arg
Arg Arg 20 25 305843PRTArtificial SequenceSynthetic peptide, Bad
BH3 peptide-CC-Ant 58Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu
Leu Arg Arg Met Ala1 5 10 15Asp Glu Phe Val Asp Ala Phe Lys Lys Cys
Cys Arg Gln Ile Lys Ile 20 25 30Trp Phe Gln Asn Arg Arg Met Lys Trp
Lys Lys 35 405943PRTArtificial SequenceSynthetic peptide, D-Bad BH3
peptide-CC-Ant 59Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu
Arg Arg Met Ala1 5 10 15Asp Glu Phe Val Asp Ala Phe Lys Lys Cys Cys
Arg Gln Ile Lys Ile 20 25 30Trp Phe Gln Asn Arg Arg Met Lys Trp Lys
Lys 35 4060132PRTHomo sapiens 60Gly Leu Val Leu His Arg Leu Gln Cys
Ala Arg Gly Phe Gly Asp Trp1 5 10 15Ile Asp Ser Ile Leu Ala Phe Ser
Arg Ser Leu His Ser Leu Leu Val 20 25 30Asp Val Pro Ala Phe Ala Cys
Leu Ser Ala Leu Val Leu Ile Thr Asp 35 40 45Arg His Gly Leu Gln Glu
Pro Arg Arg Val Glu Glu Leu Gln Asn Arg 50 55 60Ile Ala Ser Cys Leu
Lys Glu His Val Ala Ala Val Ala Gly Glu Pro65 70 75 80Gln Pro Ala
Ser Cys Leu Ser Arg Leu Leu Gly Lys Leu Pro Glu Leu 85 90 95Arg Thr
Leu Cys Thr Gln Gly Leu Gln Arg Ile Phe Tyr Leu Lys Leu 100 105
110Glu Asp Leu Val Pro Pro Pro Pro Ile Ile Asp Lys Ile Phe Met Asp
115 120 125Thr Leu Pro Phe 1306119PRTArtificial SequenceSynthetic
peptide
61Ala Ser Arg Ser Leu His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1
5 10 15Arg Arg Arg6219PRTArtificial SequenceSynthetic peptide 62Phe
Ser Arg Ser Ala His Ser Leu Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10
15Arg Arg Arg6319PRTArtificial SequenceSynthetic peptide 63Phe Ser
Arg Ser Leu His Ser Ala Leu Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg
Arg Arg6419PRTArtificial SequenceSynthetic peptide 64Phe Ser Arg
Ser Leu His Ser Leu Ala Gly Xaa Arg Arg Arg Arg Arg1 5 10 15Arg Arg
Arg6516PRTArtificial SequenceSynthetic peptide, penetratin 65Arg
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
156621PRTArtificial SequenceSynthetic peptide, transportan10 66Ala
Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu1 5 10
15Ala Lys Lys Ile Leu 206718DNAArtificial SequenceSynthetic
oligonucleotide 67caguccagcc augcuccu 18
* * * * *