U.S. patent application number 10/352512 was filed with the patent office on 2004-05-20 for proteasome regulation of nf-kb activity.
This patent application is currently assigned to Harvard College. Invention is credited to Goldberg, Alfred L., Maniatis, Thomas P., Palombella, Vito J., Rando, Oliver.
Application Number | 20040097420 10/352512 |
Document ID | / |
Family ID | 22782682 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040097420 |
Kind Code |
A1 |
Palombella, Vito J. ; et
al. |
May 20, 2004 |
Proteasome regulation of NF-kB activity
Abstract
Disclosed herein is a method for regulating the activity of
NF-.kappa.B in an animal comprising contacting cells of the animal
with certain proteasome inhibitors.
Inventors: |
Palombella, Vito J.;
(Needham, MA) ; Goldberg, Alfred L.; (Brookline,
MA) ; Maniatis, Thomas P.; (Belmont, MA) ;
Rando, Oliver; (Newton, MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Assignee: |
Harvard College
Cambridge
MA
|
Family ID: |
22782682 |
Appl. No.: |
10/352512 |
Filed: |
January 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10352512 |
Jan 28, 2003 |
|
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08210381 |
Mar 18, 1994 |
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6660268 |
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Current U.S.
Class: |
435/5 ;
514/20.1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 31/12 20180101; A61P 31/00 20180101; C07K 5/06043 20130101;
A61P 37/00 20180101; A61K 38/06 20130101; A61P 29/00 20180101; C07K
5/06078 20130101; A61P 31/18 20180101; A61P 43/00 20180101; A61K
38/55 20130101; A61K 38/05 20130101 |
Class at
Publication: |
514/017 ;
514/018 |
International
Class: |
A61K 038/08; A61K
038/06 |
Claims
What is claimed is:
1. A method for reducing the cellular content and activity of
NF-.kappa.B in an animal comprising contacting cells of the animal
with inhibitors of proteasome function or ubiquitin
conjugation.
2. A method for reducing the cellular content and activity of
NF-.kappa.B in an animal comprising contacting cells of the animal
with a proteasome function or ubiquitin conjugation inhibitor of
the structure: 62where P is an amino-group-protecting moiety;
B.sup.1, B.sup.2, B.sup.3, and B.sup.4 are independently selected
from the group consisting of 63 X.sup.1, X.sup.2, and X.sup.3 are
independently selected from group consisting of 64R is a hydrogen,
alkyl, acyl, or carboxyl; R.sup.1, R.sup.2, R.sup.3, and R.sup.4
are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, and --CH.sub.2--R.sup.5,
where R.sup.5 is aryl, aralkyl, alkaryl, cycloalkyl or
--Y--R.sup.6, where Y is a chalcogen, and R.sup.6 is alkyl; and A
is 0 or 1.
3. The method of claim 2 wherein P is 65and R.sup.1 is alkyl, aryl,
alkaryl, aralkyl, alkoxy, aryloxy, alkaryloxy, or aralkoxy.
4. The method of claim 3 wherein X.sup.1, X.sup.2, and X.sup.3 are
66
5. The method of claim 4 wherein A is 0 and B.sup.1, B.sup.2, and
B.sup.3 are 67
6. The method of claim 5 wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of alkyl and
--CH.sub.2--R.sup.5, where R.sup.5 is cyclohexyl or naphthyl.
7. The method of claim 6 wherein R1 and R.sup.2 are isobutyl.
8. The method of claim 2 wherein the proteasome inhibitor is
selected from the group consisting of 68697071
9. The method of claim 2 wherein the proteasome inhibitor is
selected from the group consisting of 7273747576777879
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method for reducing the
cellular content and activity of NF-.kappa.B by use of inhibitors
of proteasome function or ubiquitin conjugation.
[0003] 2. Description of Related Art
[0004] The transcription factor NF-.kappa.B and other members of
the rel family of protein complexes play a central role in the
regulation of a remarkably diverse set of genes involved in the
immune and inflammatory responses (Grilli et al., International
Review of Cytology 143: 1-62 (1993)). For example, NF-.kappa.B is
required for the expression of a number of immune response genes,
including the Ig-.kappa. light chain immunoglobulin gene, the IL-2
receptor .alpha. chain gene, the T cell receptor .beta. chain gene,
and class I and II major histocompatibility genes. In addition,
NF-.kappa.B has been shown to be required for a number of genes
involved in the inflammatory response, such as the TNF-.alpha. gene
and the cell adhesion genes, E-selectin, I-cam, and V-cam.
NF-.kappa.B is also required for the expression of a large number
of cytokine genes such as IL-2, IL-6, G-CSF, and IFN-.beta..
Finally, NF-.kappa.B is essential for the expression of the human
immunodeficiency virus (HIV).
[0005] In the cytosol, there is a soluble proteolytic pathway that
requires ATP and involves covalent conjugation of the cellular
proteins with the small polypeptide ubiquitin ("Ub") (Hershko et
al., A. Rev. Biochem. 61:761-807 (1992); Rechsteiner et al., A.
Rev. Cell. Biol. 3:1-30 (1987)). Thereafter, the conjugated
proteins are hydrolyzed by a 26S proteolytic complex containing a
20S degradative particle called the proteasome (Goldberg, Eur. J.
Biochem. 203:9-23 (1992); Goldberg et al., Nature 357:375-379
(1992)). This multicomponent system is known to catalyze the
selective degradation of highly abnormal proteins and short-lived
regulatory proteins. However, the system also appears to be
responsible for the breakdown of most proteins in maturing
reticulocytes (Boches et al., Science 215:978-980 (1982); Spenser
et al., J. Biol. Chem. 257:14122-14127 (1985)), in growing
fibroblasts (Ciechanover et al., Cell 37:57-66 (1984); Gronostajski
et al., J. Biol. Chem. 260:3344-3349 (1985)), and in atrophying
skeletal muscle.
[0006] The first step in degradation of many proteins involves
their conjugation to Ub by an ATP-requiring process. The
ubiquitinated proteins are then degraded by an ATP-dependent
proteolytic complex, referred to above, known as the 26S proteasome
complex.
[0007] The precise nature of the 26S proteasome complex is unclear,
although it has been shown that the 1000-1500 kDa (26S) complex can
be formed in extracts of energy-depleted reticulocytes by an
ATP-dependent association of three components, referred to as CF-1,
CF-2, and CF-3 (Ganoth, D. et al., J. Biol. Chem. 263:12412-12419
(1988)). A large (.about.700 kDa) multimeric protease found in the
cytoplasm and nucleus of eukaryotic cells, referred to as the
proteasome, is a component (CF-3) (Driscoll et al., J. Biol. Chem.
265:4789-4792 (1992); Eytan et al., Proc. Natl. Acad. Sci. USA
86:7751-7755 (1989); Orlowski, Biochemistry 29:10289-10297 (1990)
and Rivett, Arch. Biochem. Biophys. 268:1-8 (1989)).
[0008] The proteasome is believed to make up the catalytic core of
the large 26S multisubunit cytoplasmic particle necessary for the
ubiquitin-dependent pathway of intracellular proteolysis (Driscoll
et al., J. Biol. Chem. 265:4789-4692 (1990); Eytan et al., Proc.
Natl. Acad. Sci. U.S.A. 86:7751-7755 (1989); Hough et al.,
Biochemistry 262:8303-8313 (1987); McGuire et al., Biochim.
Biophys. Acta 967: 195-203 (1988); Rechsteiner et al., A. Rev.
Cell. Biol. 3:1-30 (1987); Waxman et al., J. Biol. Chem.
262:2451-2457 (1987)). By itself, the proteasome is unable to
degrade ubiquitinated proteins, but provides most of the
proteolytic activity of the 26S proteasome complex.
[0009] There is another ATP-dependent protease that is involved in
degradation of ubiquitinated proteins, forms a complex with the
proteasome, and appears to be part of the 26S proteasome complex,
which rapidly degrades proteins conjugated to ubiquitin. This
protease, referred to as multipain, has been identified in muscle
and plays an essential role in the ATP-ubiquitin-dependent
pathway.
[0010] The complex formed between multipain and proteasome in vitro
appears very similar or identical to the 1500 kDa Ub-conjugate,
degrading enzyme; or 26S proteolytic complex, isolated from
reticulocytes and muscle. The complexes contain the characteristic
20-30 kDa proteasome subunits, plus a number of larger subunits,
including the six large polypeptides found in multipain. The
complex formed contains at least 10-12 polypeptides of 40-150
kDa.
[0011] A 40 kDa polypeptide regulator of the proteasome, which
inhibits the proteasome's proteolytic activities has been purified
from reticulocytes and shown to be an ATP-binding protein whose
release appears to activate proteolysis. The isolated regulator
exists as a 250 kDa multimer and is quite labile (at 42.degree.
C.). It can be stabilized by the addition of ATP or a
nonhydrolyzable ATP analog, although the purified regulator does
not require ATP to inhibit proteasome function and lacks ATPase
activity. The regulator has been shown to correspond to an
essential component of the 1500 kDa proteolytic complex. The
regulator appears identical to CF-2 by many criteria. These
findings suggest that the regulator plays a role in the
ATP-dependent mechanism of the 26S proteasome complex.
[0012] There is also a system in the cytosol that generates
antigenic particles from endogenously synthesized cellular and
viral proteins (Moore et al., Cell 54:777-785 (1988); Morrison et
al., J. Exp. Med. 163:903-921 (1986); Powis et al., Nature
354:529-531 (1991); Spies et al., Nature 351:323-324 (1991);
Townsend et al., Cell 42:457-467 (1985); Townsend et al., Nature
324:575-577 (1986); Monaco et al., Proc. Natl. Acad. Sci. U.S.A.
79:3001-3005 (1982); Monaco, Immun. Today 13:173-179 (1992);
Yewdell et al., Adv. Immun. 52:1-123 (1992); Townsend et al., J.
Exp. Med. 168:1211-1224 (1988)). Indirect evidence suggests a role
for proteolytic particles closely resembling and perhaps identical
to the proteasome (Goldberg et al., Nature 357:375-379 (1992);
Monaco, Immun. Today 13:173-179 (1992); Parham, Nature 348:674-675
(1990); Yang et al., Proc. Natl. Acad. Sci. U.S.A. 89:4928-4932
(1992); (Brown et al., Nature 353:355-357 (1991)). It has been
shown that the proteasome is responsible for cytoplasmic processing
of MHC class I antigen molecules.
[0013] The 20S proteasome is composed of about 15 distinct 20-30
kDa subunits. It contains at least three different peptidases that
cleave specifically on the carboxyl side of the hydrophobic, basic,
and acidic amino acids (Goldberg et al., Nature 357:375-379 (1992);
Goldberg, Eur. J. Biochem. 203:9-23 (1992); Orlowski, Biochemistry
29:10289-10297 (1990); Rivett et al., Archs. Biochem. Biophys.
218:1 (1989); Rivett et al., J. Biol. Chem. 264:12,215-12,219
(1989); Tanaka et al., New Biol. 4:1-11 (1992)). These peptidases
are referred to as the chymotrypsin-like peptidase, the
trypsin-like peptidase, and the peptidylglutamyl peptidase. Which
subunits are responsible for these activities is unknown, although
the cDNA's encoding several subunits have been cloned (Tanaka et
al., New Biol. 4:1-11 (1992)).
[0014] Recent studies have found that the 20S proteasomes resemble
in size and subunit composition the MHC-linked LMP particles
(Driscoll et al., Cell 68:823 (1992); Goldberg et al., Nature
357:375-379 (1992); Matthews et al., Proc. Natl. Acad. Sci. U.S.A.
86:2586 (1989); Monaco et al., Human Immunology 15:416 (1986);
Parham, Nature 348:674-675 (1990); Martinez et al., Nature 353:664
(1991); Oritz-Navarette et al., Nature 353:662 (1991); Glynne et
al., Nature 353:357 (1991); Kelly et al., Nature 353:667 (1991);
Monaco et al., Proc. Natl. Acad. Sci. U.S.A. 79:3001 (1982); Brown
et al., Nature 353:355 (1991); Goldberg, Eur. J. Biochem. 203:9-23
(1992); Tanaka et al., New Biol. 4:1-11 (1992)).
[0015] Various inhibitors of the peptidases of the proteasome have
been reported (Dick et al., Biochemistry 30:2725-2734 (1991);
Goldberg et al., Nature 357:375-379 (1992); Goldberg, Eur. J.
Biochem. 203:9-23 (1992); Orlowski, Biochemistry 29:10289-10297
(1990); Rivett et al., Archs. Biochem. Biophys. 218:1 (1989);
Rivett et al., J. Biol. Chem. 264:12,215-12,219 (1989); Tanaka et
al., New Biol. 4:1-11 (1992)). These include known inhibitors of
chymotrypsin-like and trypsin-like proteases, as well as inhibitors
of thiol (or cysteine) and serine proteases. In addition, some
endogenous inhibitors of proteasome activities have been isolated.
These include the 240 kDa and the 200 kDa inhibitors isolated from
human erythrocytes (Murakami et al., Proc. Natl. Acad. Sci. U.S.A.
83:7588-7592 (1986); Li et al., Biochemistry 30:9709-9715 (1991))
and purified CF-2 (Goldberg, Eur. J. Biochem. 203:9-23 (1992)). In
addition to antibiotic inhibitors originally isolated from
actinomycetes (Aoyagi et al., Proteases and Biological Control,
Cold Spring Harbor Laboratory Press, pp. 429-454 (1975)), a variety
of peptide aldehydes have been synthesized, such as the inhibitors
of chymotrypsin-like proteases described by Siman et al. (WO
91/13904).
[0016] Novel molecules can also be obtained and tested for
inhibitory activity. As illustrated by the above cited references,
various strategies are known in the art for obtaining the
inhibitors for a given protease. Compound or extract libraries can
be screened for inhibitors using peptidase assays. Alternatively,
peptide and peptidomimetic molecules can be designed based on
knowledge of the substrates of the protease. For example, substrate
analogs can be synthesized containing a reactive group likely to
interact with the catalytic site of the protease (see, e.g., Siman
et al., WO 91/13904; Powers et al. in Proteinase Inhibitors,
Barrett et al. (eds.), Elsevier, pp. 55-152 (1986)). The inhibitors
can be stable analogs of catalytic transition states (transition
state analog inhibitors), such as Z-Gly-Gly-Leu-H, which inhibits
the chymotrypsin-like activity of the proteasome (Orlowski,
Biochemistry 29:10289-10297 (1990); see also Kennedy and Schultz,
Biochemistry 18:349 (1979)).
[0017] Various natural and chemical protease inhibitors reported in
the literature, or molecules similar to them, include peptides
containing an .alpha.-diketone or an .alpha.-keto ester, peptide
chloromethyl ketones, isocoumarins, peptide sulfonyl fluorides,
peptidyl boronates, peptide epoxides, and peptidyl diazomethanes
(Angelastro et al., J. Med Chem. 33:11-13 (1990); Bey et al., EPO
363,284; Bey et al., EPO 364,344; Grubb et al., WO 88/10266;
Higuchi et al., EPO 393,457; Ewoldt et al., Molecular Immunology
29(6):713-721 (1992); Hernandez et al., Journal of Medicinal
Chemistry 35(6):1121-1129 (1992); Vlasak et al., Journal of
Virology 63(5):2056-2062 (1989); Hudig et al., Journal of
Immunology 147(4):1360-1368 (1991); Odakc et al., Biochemistry
30(8):2217-2227 (1991); Vijayalakshmi et al., Biochemistry
30(8):2175-2183 (1991); Kam et al., Thrombosis and Haemostasis
64(1): 133-137 (1990); Powers et al., Journal of Cellular
Biochemistry 39(1):33-46 (1989); Powers et al., Proteinase
Inhibitors, Barrett et al., Eds., Elsevier, pp. 55-152 (1986);
Powers et al., Biochemistry 29(12):3108-3118 (1990); Oweida et al.,
Thrombosis Research 58(2):391-397 (1990); Hudig et al., Molecular
Immunology 26(8):793-798 (1989); Orlowski et al., Archives of
Biochemistry and Biophysics 269(1):125-136 (1989); Zunino et al.,
Biochimica et BiophysicaActa. 967(3):331-340(1988); Kam et al.,
Biochemistry 27(7):2547-2557 (1988); Parkes et al., Biochem J.
230:509-516 (1985); Green et al., J. Biol. Chem. 256:1923-1928
(1981); Angliker et al., Biochem. J. 241:871-875 (1987); Puri et
al., Arch. Biochem. Biophys. 27:346-358 (1989); Hanada et al.,
Proteinase Inhibitors: Medical and Biological Aspects, Katunuma et
al., Eds., Springer-Verlag pp. 25-36 (1983); Kajiwara et al.,
Biochem. Int. 15:935-944 (1987); Rao et al., Thromb. Res.
47:635-637 (1987); Tsujinaka et al., Biochem. Biophys. Res. Commun.
153:1201-1208 (1988)).
[0018] Various inhibitors of ubiquitin conjugation to proteins are
also known (Wilkinson et al., Biochemistry 29:7373-7380
(1990)).
[0019] Certain peptide aldehydes and peptide .alpha.-keto esters
containing a hydrophobic residue in the P.sub.1 position were
tested by Vinitsky et al. (Biochemistry 31:9421-9428 (1992), see
also, Orlowski et al. Biochemistry 32:1563-1572 (1993)) as
potential inhibitors of the chymotrypsin-like activity of the
proteasome. Three peptide aldehydes,
(benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-H),
N-acetyl-Leu-Leu-Norleucinal (Ac-LLnL-H), and
N-acetyl-Leu-Leu-methionina- l (Ac-LLM-H) were found to be slow
binding inhibitors with K.sub.i values of 0.46, 5.7, and 33 .mu.M,
respectively. Of the several peptide .alpha.-keto ester inhibitors
tested, Z-Leu-Leu-Phe-COOEt was the most potent inhibitor of the
chymotrypsin-like activity with a K.sub.i of 53 .mu.M. Many such
compounds exist.
[0020] Other tripeptides that have been described in the literature
include Ac-Leu-Leu-Leu-H, Ac-Leu-Leu-Met-OR, Ac-Leu-Leu-Nle-OR,
Ac-Leu-Leu-Leu-OR, Ac-Leu-Leu-Arg-H, Z-Leu-Leu-Leu-H,
Z-Arg-Leu-Phe-H, and Z-Arg-Ile-Phe-H, where OR, along with the
carbonyl of the preceding amino acid residue, represents an ester
group.
[0021] Goldberg, in U.S. patent application Ser. No. 07/699,184,
filed May 13, 1991, discloses that the ATP-ubiquitin-dependent
process has been shown to be responsible for the excessive protein
degradation that occurs in conditions or disease states in which
there is severe loss of body mass and negative nitrogen balance. A
method of inhibiting the accelerated or enhanced proteolysis, a
method of identifying inhibitors of the process, multipain and
proteasome inhibitors are also disclosed.
[0022] Goldberg et al., in U.S. patent application Ser. No.
08/016,066, filed Feb. 10, 1993, disclose methods and drugs that
inhibit the processing of antigens for presentation by major
histocompatibility complex class I molecules. Specifically,
inhibitors of the ATP-ubiquitin-dependent proteolytic pathway are
described, which can inhibit MHC-I antigen presentation. These
methods and drugs may be useful for the treatment of autoimmune
diseases and for reducing rejection of organs and graft
transplants. See, also, Michalek et al., Nature 363:552-554
(1993).
[0023] Tsubuki et al., Biochem. and Biophys. Res. Commun.
196(3):1195-1201 (1993) reported that a tripeptide aldehyde
protease inhibitor, benzyloxycarbonyl(Z)-Leu-Leu-leucinal,
initiates neurite outgrowth in PC12 cells at an optimal
concentration of 30 nM. The following synthetic peptides are also
mentioned: Z-Leu-Leu-Gly-H, Z-Leu-Leu-Ala-H, Z-Leu-Leu-Ile-H,
Z-Leu-Leu-Val-H, Z-Leu-Leu-Nva-H, Z-Leu-Leu-Phe-H, Z-Leu-Leu-Leu-H,
Bz-Leu-Leu-Leu-H, Ac-Leu-Leu-Leu-H, Z-Leu-Leu-Leu.sc,
Z-Leu-Leu-Leu.ol, Z-Leu-Leu-Leu, Dns-Leu-Leu-Leu-H,
Dns-Leu-Leu-Leu-CH.sub.2Cl, and Leupeptin.
[0024] Siman et al. (WO 91/13904) disclose chymotrypsin-like
proteases and their inhibitors. The inhibitors have the formula
R-A4-A3-A2-Y. wherein
[0025] R is hydrogen, or a N-terminal blocking group;
[0026] A4 is a covalent bond, an amino acid or a peptide;
[0027] A3 is a covalent bond, a D-amino acid, Phe, Tyr, Val, or a
conservative amino acid substituent of Val;
[0028] A2 is a hydrophobic amino acid or lysine or a conservative
amino acid substituent thereof, or when A4 includes at least two
amino acids, A2 is any amino acid; and
[0029] Y is a group reactive with the active site of said
protease.
[0030] Powers (WO 92/12140) discloses peptide ketoamides,
ketoacids, and ketoesters and their use in inhibiting serine
proteases and cysteine proteases.
[0031] Bartus et al. (WO 92/1850) disclose uses for Calpain
inhibitor compounds and pharmaceutical compositions containing
them. One use of these compounds is in the treatment of a
neurodegenerative pathology in a human patient. The disclosure also
provides additional uses and pharmaceutical compositions containing
Calpain inhibitor compounds, such as peptide ketoamides, peptide
ketoacids, and peptide ketoesters.
SUMMARY OF THE INVENTION
[0032] The present invention relates to a method for reducing the
cellular content and activity of NF-.kappa.B.
[0033] In a preferred embodiment, the present invention relates to
a method for reducing the cellular content and activity of
NF-.kappa.B in an animal comprising contacting cells of the animal
with inhibitors of proteasome function or ubiquitin
conjugation.
[0034] More particularly, the present invention is directed to a
method for reducing the cellular content and activity of
NF-.kappa.B in an animal comprising contacting cells of the animal
with a proteasome function or ubiquitin conjugation inhibitor of
the structure (1): 1
[0035] where
[0036] P is an amino-group-protecting moiety;
[0037] B.sup.1, B.sup.2, B.sup.3, and B.sup.4 are independently
selected from the group consisting of 2
[0038] X.sup.1, X.sup.2, and X.sup.3 are independently selected
from the group consisting of 3
[0039] R is a hydrogen, alkyl, acyl, or carboxyl;
[0040] R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, and --CH.sub.2--R.sup.5,
[0041] where R.sup.5 is aryl, aralkyl, alkaryl, cycloalkyl or
--Y--R.sup.6,
[0042] where Y is a chalcogen, and R.sup.6 is alkyl; and
[0043] A is 0 or 1.
[0044] The "animals" referred to herein are preferably mammals.
Both terms are intended to include humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows that proteolytic processing of the p60Tth
precursor to p50 in vitro requires ATP.
[0046] FIG. 2 is a diagram showing the role of ubiquitin and
proteasome in the generation of active NF-.kappa.B.
[0047] FIG. 3 depicts processing of p105/p60Tth in
proteasome-depleted and proteasome-enriched extracts.
[0048] FIG. 4 shows that immunodepletion of the proteasome inhibits
the processing of NF-.kappa.B.
[0049] FIG. 5 shows that purified proteasomes stimulate the
processing of p60Tth.
[0050] FIG. 6 shows that the p60Tth precursor protein is
ubiquitinated.
[0051] FIG. 7 shows that ubiquitin is required for the processing
of NF-.kappa.B.
[0052] FIG. 8 shows that processing of p105 in Saccharomyces
cerevisiae requires the proteasome.
[0053] FIG. 9 shows that specific inhibitors of the proteasome
block the processing of p105 in vivo.
[0054] FIG. 10 shows that specific inhibitors of the proteasome
block activation of NF-.kappa.B.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0055] NF-.kappa.B exists in an inactive form in the cytosol
complexed with an inhibitor protein, I.kappa.B. In order for the
NF-.kappa.B to become active and perform its function, it must
enter the cell nucleus. It cannot do this, however, until the
I.kappa.B portion of the complex is removed, a process referred to
by those skilled in the art as the activation of, or processing of,
NF-.kappa.B. In some diseases, the normal performance of its
function by the NF-.kappa.B can be detrimental to the health of the
patient. For example, as mentioned above, NF-.kappa.B is essential
for the expression of the human immunodeficiency virus (HIV).
Accordingly, a process that would prevent the activation of the
NF-.kappa.B in patients suffering from such diseases could be
therapeutically beneficial. The inhibitors employed in the practice
of the present invention are capable of preventing this activation.
Thus, blocking NF-.kappa.B activity or production could have
important application in various areas of medicine, e.g.,
inflammation, sepsis, AIDS, and the like.
[0056] More specifically, the activity of NF-.kappa.B is highly
regulated (Grilli et al., International Review of Cytology 143:
1-62 (1993); Beg et al., Genes and Development 7:2064-2070 (1993)).
NF-.kappa.B comprises two subunits, p50 and an additional member of
the rel gene family, e.g., p65 (also known as Rel A). In most
cells, the p50 and p65 are present in an inactive precursor form in
the cytoplasm, bound to I.kappa.B. In addition, the p50 subunit of
NF-.kappa.B is generated by the proteolytic processing of a 105 kD
precursor protein NF-.kappa.B.sub.1 (p105), and this processing is
also regulated. The sequence of the N-terminal 50 kD portion of
p105 is similar to that of p65 and other members of the rel gene
family (the rel homology domain). By contrast, the C-terminal 55 kD
of p105 bears a striking resemblance to I.kappa.B-.alpha. (also
known as MAD3). Significantly, unprocessed p105 can associate with
p65 and other members of the rel family to form a p65/p105
heterodimer. Processing of p105 results in the production of p50,
which can form the transcriptionally active p50/p65 heterodimer.
The C-terminal I.kappa.B-.alpha.-homologous sequence of p105 is
rapidly degraded upon processing.
[0057] There is another rel-related protein, NF-.kappa.B.sub.2
(p100), that is similar to p105 in that it, too, is processed to a
DNA binding subunit, p52 (Neri et al., Cell 67:1075 (1991); Schmid
et al., Nature 352:733 (1991); Bours et al., Molecular and Cellular
Biology 12:685 (1992); Mercurio et al., DNA Cell Biology 11:523
(1992)). Many of the structural and regulatory features of p100 are
similar to p105. In addition, the p100 protein can also form a
heterodimer with p65 and other rel family members.
[0058] In summary, the transcriptional activity of heterodimers
consisting of p50 and one of the many rel family proteins, such as
p65, can be regulated by at least two mechanisms. First, the
heterodimers associate with I.kappa.B-.alpha. to form an inactive
ternary cytoplasmic complex. Second, the rel family members
associate with p105 and p100 to form inactive complexes. The
ternary complex can be activated by the dissociation and
destruction of I.kappa.B-.alpha., while the p65/p105 and p65/p100
heterodimer can be activated by processing p105 and p100,
respectively.
[0059] The dissociation of I.kappa.B-.alpha. can be induced by a
remarkably large number of extracellular signals, such as
lipopolysaccharides, phorbol esters, TNF-.alpha., and a variety of
cytokines. The I.kappa.B-.alpha. is then rapidly degraded. Recent
studies suggest that p105 and p100 processing can also be induced
by at least some of these extracellular signals. Neither the signal
transduction pathways leading to NF-.kappa.B activation, nor the
mechanisms of I.kappa.B-.alpha. inactivation or p105/p100
processing are understood. Accordingly, the inventors do not wish
to be limited by any theory of the mechanism(s) by which the
inhibitors employed in the practice of the present invention
achieve their useful effects. The inventors do have clear evidence
of effects with certain inhibitors that block the hydrophobic site
on the 20S (core) proteasome particle, but it must be realized that
other essential sites exist on this particle, which constitutes the
catalytic core on the 26S proteasome complex. Consequently, similar
effects would be anticipated with inhibitors of other essential
activities of the proteasome. Removal of the proteasome particle by
ultracentrifugation or immunoprecipitation prevents the activation
of NF-.kappa.B. There are many other catalytic functions on this
particle that, if blocked, would also be expected by those skilled
in the art to prevent its ability to process NF-.kappa.B and/or
destroy I.kappa.B. It is also possible that the effects achieved by
the inhibitors used in the process of the present invention may be
achieved by inhibition of the ubiquitination of NF-.kappa.B and/or
I.kappa.B. Indeed, the inventors have discovered that
ubiquitin-conjugation is necessary for their degradation.
Accordingly, there may be specific E-2s or E-3s involved in the
processing of NF-.kappa.B and/or I.kappa.B whereby one could
predict that any inhibitor of E-1, E-2, or E-3-dependent
Ub-conjugation would prevent NF-.kappa.B activation by blocking the
ubiquitin-proteasome pathway.
[0060] Studies have demonstrated that p105 or a truncated form of
p105 (p60Tth) can be processed to p50 in vitro (Fan et al., Nature
354:395-398 (1991)). Certain of the requirements and
characteristics of this in vitro processing reaction (e.g.,
ATP/Mg.sup.++ dependency) suggested to the present inventors that
the ATP-dependent protease complex of the ubiquitin-mediated
protein degradation pathway was involved (i.e. proteasome;
Rechsteiner, 1991, Goldberg, Eur. J. Biochem. 203:9-23 (1992),
Hershko et al., Annu Rev. Biochem. 61:761-807 (1992)). However,
this structure was only known to catalyze the complete degradation
of proteins to small acid-soluble peptides and was not believed
capable of processing precursors to generate active proteins, such
as p50 NF-.kappa.B.
[0061] Using a variety of experimental approaches, the present
inventors have proven that the proteasome is indeed required for
the processing of p105 to p50. First, it was found that the
p105/p60Tth proteins are not processed in mammalian cell
cytoplasmic extracts depleted of proteasome activity. However,
addition of purified 26S proteasomes to these depleted extracts
restores the processing activity. Second, specific inhibitors of
the proteasome block the formation of p50 in mammalian cell
extracts and in vivo. Third, mammalian p105 is processed to p50 in
Saccharomyces cerevisiae in vivo, and a mutant in the
chymotrypsin-like activity of the proteasome results in a
significant decrease in p105 processing. p60Tth is ubiquitinated in
vitro and this ubiquitination is a pre-requisite for p105
processing.
[0062] As mentioned above, the C-terminal half of the p105 (p105C')
is rapidly degraded during the formation of p50 and the sequence of
p105C' is remarkably similar to that of I.kappa.B. Because of the
similarity in the structures and activities of p105C' and
I.kappa.B-.alpha., the present inventors initiated studies to
determine whether the proteasome is also involved in the
inactivation of I.kappa.B-.alpha.. I.kappa.B-.alpha. is rapidly
degraded in response to NF-.kappa.B inducers and this degradation
has been shown to be necessary for the activation (Mellits et al.,
Nucleic Acids Research 21(22):5059-5066 (1993); Henkel et al.,
Nature 365: 182-185 (1993); Beg et al., Molecular and Cellular
Biology 13(6):3301-3310 (1993)). The present inventors have now
shown that I.kappa.B-.alpha. degradation and the activation of
NF-.kappa.B is indeed blocked by inhibitors of proteasome function
or ubiquitin conjugation.
[0063] Accordingly, the proteasome plays an essential role in the
regulation of NF-.kappa.B activity. First, the proteasome is
required for the processing of p105 and possibly p100.
The-degradation of the inhibitory C-terminus may also require the
proteasome. Second, the proteasome appears to be required for the
degradation of I.kappa.B-.alpha. in response to extracellular
inducers.
[0064] The present invention preferably relates to the use of
inhibitors of proteasome function or ubiquitin conjugation of the
structure (1): 4
[0065] where
[0066] P is an amino-group-protecting moiety;
[0067] B.sup.1, B.sup.2, B.sup.3, and B.sup.4 are independently
selected from the group consisting of 5
[0068] X.sup.1, X.sup.2, and X.sup.3 are independently selected
from the group consisting of 6
[0069] R is a hydrogen, alkyl, acyl, or carboxyl;
[0070] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, and --CH.sub.2--R.sup.5,
[0071] where R.sup.5 is aryl, aralkyl, alkaryl, cycloalkyl or
--Y--R.sup.6,
[0072] where Y is a chalcogen, and R.sup.6 is alkyl; and
[0073] A is 0 or 1.
[0074] In a preferred embodiment of the present invention, the P
moiety of the proteasome inhibitor is (2) 7
[0075] and R.sup.7 is alkyl, aryl, alkaryl, aralkyl, alkoxy,
aryloxy, alkaryloxy, or aralkoxy.
[0076] Where R.sup.7 is alkyl, it is preferably alkyl of from 1 to
4 carbon atoms, e.g., methyl, ethyl, propyl, butyl, or isomers
thereof. Additionally, where R.sup.7 is alkaryl, aralkyl, alkoxy,
alkaryloxy, or aralkoxy, the alkyl moiety thereof is also
preferably one having from 1 to 4 carbon atoms.
[0077] Where R.sup.7 is aryl, it is preferably aryl of from 6 to 10
carbon atoms, e.g., phenyl or naphthyl, which may, if desired, be
ring substituted. Additionally, where R.sup.7 is alkaryl, aralkyl,
aryloxy, alkaryloxy, or aralkoxy, the aryl moiety thereof is also
preferably one having from 6 to 10 carbon atoms.
[0078] It is more preferred that R.sup.7 be alkyl or aralkoxy, most
preferably methyl or benzyloxy, i.e., 8
[0079] In structure (1), X represents a peptide bond or an isostere
that can be used as a peptide bond replacement in the proteasome
inhibitors to increase bioavailability and reduce hydrolytic
metabolism. As noted above, X can be 9
[0080] Introduction of these moieties into the proteasome
inhibitors results in the following: 10
[0081] For example, if Z-Leu-Leu-Leu-H is found to undergo rapid
hydrolytic metabolism to produce Z-Leu-OH and H.sub.2N-Leu-Leu-H,
the hydroxyethylene isostere can be prepared to eliminate this
reaction: 11
[0082] Another isostere within the scope of the present invention
is the aza-peptide isostere. This is the result of the replacement
of the .alpha.-carbon atom of an amino acid with a nitrogen atom,
e.g., 12
[0083] As noted above, A in structure (1) can be either 0 or 1.
Thus, when A is 0, the amino acid residue within the brackets is
not present and the inhibitor is a tripeptide. Similarly, where A
is 1, the amino acid residue within the brackets is present and the
inhibitor is a tetrapeptide. It is preferred that A be 0.
[0084] It is preferred that R.sup.1 and R.sup.2 in structure (1) be
independently selected from the group consisting of alkyl and
--CH.sub.2--R.sup.5. More preferably, R.sup.1 and R.sup.2 are
independently selected from the group consisting of alkyl groups of
from 1 to 4 carbon atoms, e.g., methyl, ethyl, propyl, butyl, or
isomers thereof, e.g., isopropyl, isobutyl, sec-butyl, t-butyl, or
--CH.sub.2--R.sup.5, where R.sup.5 is cycloalkyl or naphthyl. It is
more preferred that at least one of R.sup.1 and R.sup.2 be
isobutyl, 13
[0085] and most preferred that both R.sup.1 and R.sup.2 be
isobutyl.
[0086] Where R.sup.3 is alkyl, it is preferably alkyl of from 1 to
4 carbon atoms, e.g., methyl, ethyl, propyl, butyl, or isomers
thereof, which groups may be substituted or unsubstituted.
[0087] Where R.sup.3 is aryl, it is preferably aryl of from 6 to 10
carbon atoms, e.g., phenyl or naphthyl, which groups may be
substituted or unsubstituted.
[0088] Where R.sup.3 is a substituted alkyl, it is preferably an
alkyl of from 1 to 4 carbon atoms substituted with at least one
aryl group of from 6 to 10 carbon atoms or at least one cycloalkyl
group, preferably a cycloalkyl group having 5 or 6 carbon atoms,
which groups may be substituted or unsubstituted.
[0089] Where R.sup.3 is substituted aryl, it is preferably
substituted with at least one alkyl group of from 1 to 4 carbon
atoms, which groups may be substituted or unsubstituted.
[0090] Where R.sup.3 is cycloalkyl, it is preferably cycloalkyl of
from 5 to 6 carbon atoms, e.g., cyclopentyl or cyclohexyl, which
groups may be substituted or unsubstituted.
[0091] Where R.sup.3 is substituted cycloalkyl, it is preferably
substituted with at least one aryl group of from 6 to 10 carbon
atoms or at least one alkyl group, preferably an alkyl group having
1 to 4 carbon atoms, which groups may be substituted or
unsubstituted.
[0092] Where R.sup.3 is --Y--R.sup.6, Y is a chalcogen, preferably
oxygen or sulfur, more preferably sulfur; and R.sup.6 is alkyl,
preferably alkyl of from 1 to 4 carbon atoms, e.g., methyl, ethyl,
propyl, butyl, or isomers thereof.
[0093] R in the structure shown above is hydrogen, alkyl, acyl, or
carboxyl.
[0094] Where R is alkyl, it is preferably alkyl of from 1 to 4
carbon atoms, e.g., methyl, ethyl, propyl, butyl, or isomers
thereof.
[0095] Where R is acyl, it preferably comprises a carbonyl moiety
covalently bonded to an alkyl moiety of from 1 to 4 carbon atoms,
e.g., methyl, ethyl, propyl, butyl, or isomers thereof, which may
be substituted or unsubstituted.
[0096] Where R is carboxyl, it is preferably a carboxyl of the
structure [R.sup.8].sub.n--COO--, where R.sup.8 is alkylene of from
1 to 4 carbon atoms, e.g., methylene, ethylene, propylene,
butylene, and isomers thereof, and n is 0 or 1, and where the final
0 is normally bound to an alkyl group of 1 to 4 carbon atoms, which
may be substituted or unsubstituted, thereby forming an ester.
[0097] R is preferably hydrogen, alkyl, or carboxyl; more
preferably, hydrogen.
[0098] Examples of suitable proteasome inhibitors include, without
limitation, the following compounds: 14151617
[0099] Most preferably, the proteasome inhibitors are:
1819202122232425
[0100] The present invention relates to a method for reducing the
cellular content and activity of NF-.kappa.B in an animal
comprising contacting cells of the animal with inhibitors of
proteasome function or ubiquitin conjugation. In the present
method, the accelerated proteolysis is inhibited by interfering
with the ATP-Ub-dependent pathway at one or more possible steps
(e.g., by interfering with activity of the 26S proteasome complex,
or by interfering with activity of one of its components).
[0101] A particularly useful approach to testing drug candidates
for their ability to inhibit the ATP-ubiquitin-dependent
degradative process is to do so in cultured cells in which a
short-lived protein whose degradation is ubiquitin-dependent is
produced. Inhibition of the process leads to accumulation of the
protein in the cytosol. The extent to which the protein accumulates
in the cytosol can be determined, using known methods. For example,
a potential inhibitor of the process can be introduced into
cultured cells producing a short-lived enzyme and the extent to
which the enzyme is present in the cytosol in the presence of the
potential inhibitor can be compared with the extent to which it
occurs in its absence. Accumulation of the enzyme in the presence
of the potential inhibitor is indicative of inhibition of the
ATP-ubiquitin-dependent processes by the potential inhibitor being
tested. Cultured cells, such as COS cells, which are stably
transformed with a gene encoding a short-lived protein whose
degradation is ubiquitin-dependent (e.g., a short-lived enzyme,
such as a mutant .beta.-galactosidase from E. coli, whose half-life
is about 15 minutes and whose degradation is ubiquitin-dependent)
can be used (Bachmair, A. et al., Science 234:179-186 (1986);
Gonda, D. K. et al., J. Biol. Chem. 264:16700-16712 (1989)). Other
mutant forms of enzymes that are rapidly degraded can also be used.
Accumulation of the mutant .beta.-galactosidase in COS cytosol in
the presence of a substance being assessed for its ability to
inhibit the process (a potential inhibitor) is indicative of
inhibition of the process. An appropriate control is COS cells
maintained under the same conditions, but in the absence of the
potential inhibitor. This approach can be used to screen for
effective inhibitors from microbial broths or chemical
libraries.
[0102] Tables I-III summarize results from kinetic experiments that
measured the inhibition of the 20S and 26S proteasomes, as well as
cathepsin B and calpain. In these tables, K.sub.i values are
reported, which are dissociation constants for the equilibrium that
is established when enzyme and inhibitor interact to form the
enzyme:inhibitor complex.
[0103] The substances and assay conditions are briefly summarized
in the footnotes to Table I. MG 101 and MG 102, also known as
Calpain Inhibitor I and II, were purchased from Calbiochem as
catalogue products.
1TABLE I PROTEASE SELECTIVITY OF N-ACETYL TRIPEPTIDE ALDEHYDES
K.sub.i (nM) Inhibitor 20 S.sup.a 26 S.sup.b Cat B.sup.c
Calpain.sup.d MG 101 26 140* 1,000 6 5 MG 102 27 1,000 28,000 94
120 .sup.(a)Rabbit muscle. SDS-activated. Substrate: Suc-LLVY-AMC.
.sup.(b)Rabbit muscle. Substrate: Suc-Suc-LLVY-AMC-AMC. [Mg:ATP] =
2 mM. .sup.(c)Bovine spleen. Substrate: Z-RR-AMC. [DTT] = 2 mM,
[EDTA] = 5 mM, pH = 5.5, T = 37.degree. C. .sup.(d)Rabbit muscle,
80 kD catalytic subunit. Substrate: Suc-LLVY-AMC. [CaCl.sub.2] = 1
mM, [DTT] = 2 mM, pH = 7.8, T = 20.degree. C.
[0104]
2TABLE II PROTEASE SELECTIVITY OF N-CARBOBENZOXY TRIPEPTIDE
ALDEHYDES.sup.(a) K.sub.i (nM) Inhibitor 20 S 26 S Cat B Calpain MG
118 28 3,800 28,000 94 120 MG 111 29 210 690 6 11 MG 119 30 50 280
18 15 MG 115 31 21 78 10 10 MG 120 32 94 560 36 14 MG 114 33 47 120
7 12 MG 110 34 48 180 100 62 MG 121 35 25 70 73 62 .sup.(a)Same
reaction conditions as listed in footnotes of Table I.
[0105]
3TABLE III PROTEASE SELECTIVITY OF CARBOXY-ACTIVATED
TRIPEPTIDES.sup.(a) K.sub.i (nM) Inhibitor 20 S 26 S Cat B Calpain
MG 113 36 690 1,300 <4 45 .sup.(a)Same reaction conditions as
listed in footnotes of Table I.
[0106] Key points to note from Tables I-III:
[0107] (1) Peptide chain length is important for inhibitory potency
against the 20S proteasome: compare the K.sub.i of 47 nM for
Z-Leu-Leu-Nle-H (MG 114) with the K.sub.i of 15,000 nM for
Z-Leu-Nle-H (MG 105; prepared by Calbiochem as catalogue product,
not shown in tables).
[0108] (2) Potency against the 20S proteasome is also increased
with increasing hydrophobicity of the N-terminal blocking group:
compare the K.sub.i of 47 nM for Z-Leu-Leu-Nle-H (MG 114) with the
K.sub.i of 140 nM for Ac-Leu-Leu-Nle-H (MG 101).
[0109] (3) In the series of compounds in Table II in which
unbranched alkyl chain length is increased monotonically at the
P.sub.1 position (MG 118 (hydrogen), MG 111 (methyl), MG 119
(ethyl), MG 115 (n-propyl), and MG 114 (n-butyl)), there is a
maximum of potency with Z-Leu-Leu-Nva-H (MG 115).
[0110] (4) Inhibitory potency against the 26S proteasome is always
less than potency against the 20S proteasome. The difference is
smallest for Z-Leu-Leu-Nva-H (MG 115; K.sub.i,20S=21 nM and
K.sub.i,26S=78 nM), Z-Leu-Leu-Nal-H (MG 121; K.sub.i,20S=25 nM and
K.sub.i,26S=70 nM), and Z-Leu-Leu-Phe-C(O)--OMe (Mg 113;
K.sub.i,20S=690 nM and K.sub.i,26S=1,300 nM).
[0111] (5) The peptide aldehydes that were examined more potently
inhibit cathepsin B and calpain than they inhibit the 20S and 26S
proteasome, except for the two inhibitors with large, hydrophobic
P.sub.1 residues, Z-Leu-Leu-Phe-H and Z-Leu-Leu-NaI-H (MG 110 and
MG 121, respectively).
[0112] Data also show that MG 101 is an inhibitor of the 26S
ATP-dependent protease and an inhibitor of the proteasome
(macropain, multi-catalytic protease) (Table IV).
4TABLE IV MG 101 Inhibits Different Forms of Muscle Proteasome No
Inhibitor Enzyme Relative Catalytic MG 101 Preparation Efficiency
K.sub.i (.mu.M) 20S Proteasome 1 7 26S Proteasome Complex 2 3 20S
Proteasome + 50 0.6 Muscle 180 kDa Activator 20S Proteasome .+-.
SDS 140 0.14
[0113] The inhibitors can be used in vitro or in vivo. They can be
administered by any number of known routes, including orally,
intravenously, intramuscularly, topically, and by infusion (Platt
et al., U.S. Pat. No. 4,510, 130; Badalamente et al., Proc. Natl.
Acad. Sci. U.S.A. 86:5983-5987 (1989); Staubli et al., Brain
Research 444:153-158 (1988)) and will generally be administered in
combination with a physiologically acceptable carrier (e.g.,
physiological saline). The effective quantity of inhibitor to be
given will be determined empirically and will be based on such
considerations as the particular inhibitor used, the condition of
the individual, and the size and weight of the individual. They can
be administered alone or in combination with another inhibitor or
an inhibitor of another pathway.
[0114] Table V summarizes data for the inhibition of the 20S
proteasome by various tripeptide aldehyde inhibitors.
5TABLE V Protease Selectivity of Miscellaneous Tripeptide Aldehydes
Compound K.sub.i (nM) MG 124 37 59 MG 129 38 60 MG 131 39 6 MG 132
40 9 MG 133 41 470 MG 134 42 100 MG 135 43 290 MG 136 44 1,000 MG
139 45 20 MG 140 46 28 MG 141 47 50 MG 142 48 0.3 MG 150 49 0.4 MG
151 50 10 MG 152 51 6 MG 153 52 280 MG 154 53 75 MG 155 54 54 MG
158 55 7 MG 160 56 51 MG 161 57 64 MG 165 58 0.24 MG 166 59 0.035
MG 167 60 0.015 MG 168 61 0.49
[0115] The present invention will now be illustrated by the
following examples, which are not intended to be limiting in any
way.
EXAMPLES 1-3
Preparation of Peptidyl Aldehydes
[0116] All peptidyl N,O-dimethylhydroxylamides were prepared by
solution phase method using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as
coupling reagent (Sheehan et al., J. Am. Chem. Soc. 87:2492-2493
(1965)). Reduction of the hydroxylamide with lithium aluminum
hydride provided peptidyl aldehyde (Fehrentz et al.,
Synthesis:676-678 (1983)); Fehrentz et al., Int. J. Peptide Protein
Res. 26:236-241 (1985)); All compounds are characterized by Proton
nuclei magnetic resonance (NMR) spectroscopy. The purity of the
products was verified by thin layer chromatography and, in some
cases, by high performance liquid chromatography (HPLC).
EXAMPLE 1
Preparation of Z-L-leucine-L-leucine-L-norvalinal
[0117] a) Boc-Lnorvaline N,O-dimethylhydroxylamide
[0118] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(443 mg) in one portion was added to a mixture of N-Boc-L-norvaline
dicyclohexylammonium salt (838 mg), N,O-dimethylhydroxylamine
hydrochloride (215 mg), 1-hydroxybenzotriazole monohydrate (340
mg), and N-methylmorpholine (0.28 ml) in dimethyl formamide (DMF,
20 ml) at 0.degree. C. The mixture was stirred at 0.degree. C. for
2 hours, then at room temperature for 40 hours. The reaction was
quenched with water (80 mL) and the mixture was extracted with
ethyl acetate (EtOAc, 3.times.100 mL). The combined organic layers
were washed with aqueous 10% hydrogen chloride (HCl), saturated
sodium bicarbonate (NaHCO.sub.3), and brine, then dried over
anhydrous magnesium sulfate (MgSO.sub.4), filtered, and evaporated
to afford the product (546 mg) as an oil.
[0119] b) Z-L-leucine-L-leucine-L-norvaline
N,O-dimethylhydroxylamide
[0120] A solution of N-Boc-L-norvaline N,O-dimethylhydroxylamide
(546 mg) and trifluoroacetic acid (8 mL) in methylene chloride (20
mL) was stirred at 0.degree. C. for 3 hours. The solvent was
evaporated under reduced pressure and the residue was dried under
vacuum. To this flask was added Z-L-leucine-L-leucine (794 mg),
1-hydroxybenzotriazole monohydrate (340 mg), N-methylmorpholine
(0.28 mL), and DMF (20 mL).
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (442
mg) was then added at 0.degree. C. The mixture was stirred at
0.degree. C. for 2 h, then at room temperature for 24 h. The
reaction was quenched with water (40 mL) and the mixture was
extracted with EtOAc (3.times.60 mL). The combined organic layers
were washed with aqueous 10% HCl, saturated NaHCO.sub.3, and brine,
then dried over anhydrous MgSO.sub.4, filtered, and evaporated to
afford the product (1.09 g) as a white solid.
[0121] c) Z-L-leucine-L-leucine-L-norvalinal
[0122] A solution of Z-L-leucine-L-leucine-L-norvaline
N,O-dimethylhydroxylamide (1.09 g) was dissolved in 20 mL dry
tetrahydrofuran (THF) and cooled to 0.degree. C. Lithium aluminum
hydride (1 M solution in THF, 3.05 mL) was added and the mixture
was stirred at 0.degree. C. for 25 minutes. Potassium bisulfate
(465 mg) in 20 mL water was added and the mixture was extracted
with EtOAc (3.times.80 mL). The combined organic layers were washed
with aqueous 5% HCl, saturated NaHCO.sub.3, and brine, then dried
over anhydrous MgSO.sub.4, filtered, and evaporated to afford the
product (430 mg) as a white solid.
EXAMPLE 2
Preparation of Z-L-Leucine-L-Leucine-L-Leucinal
[0123] a) Boc-L-Leucine-L-Leucine N,O-dimethylhydroxylamide
[0124] A mixture of N-Boc-L-leucine-L-leucine (1 g),
N,O-dimethylhydroxylamine hydrochloride (423 mg),
1-hydroxybenzotriazole monohydrate (509 mg), and N-methylmorpholine
(0.42 mL) was dissolved in DMF (20 mL).
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (610
mg) was added at 0.degree. C. for 2 h, then at room temperature for
40 h. The reaction was quenched with water (80 mL) and the mixture
was extracted with EtOAc (3.times.100 mL). The combined organic
layers were washed with aqueous 10% HCl, saturated NaHCO.sub.3, and
brine then dried over anhydrous MgSO.sub.4, filtered, and
evaporated to afford the product (923 mg) as a white solid.
[0125] b) Z-L-leucine-L-leucine-L-leucine
N,O-dimethylhydroxylamide
[0126] A solution of N-Boc-L-leucine-L-leucine
N,O-dimethylhydroxylamide (923 mg) and trifluoroacetic acid (10 mL)
in methylene chloride (20 mL) was stirred at 0.degree. C. for 3
hours. The solvent was evaporated under reduced pressure and the
residue was dried under vacuum. A portion of this product (488 mg)
was transferred to another flask and was combined with Z-L-leucine
(451 mg), 1-hydroxybenzotriazole monohydrate (276 mg),
N-methylmorpholine (0.22 mL), and DMF (15 mL).
1-Ethyl-3-(3-dimethylamino- propyl)carbodiimide hydrochloride (357
mg) was then added at 0.degree. C. The mixture was stirred at
0.degree. C. for 2 h, then at room temperature for 42 h. The
reaction was quenched with water (50 mL) and the mixture was
extracted with EtOAc (3.times.60 mL). The combined organic layers
were washed with aqueous 10% HCl, saturated NaHCO.sub.3, and brine,
then dried over anhydrous MgSO.sub.4, filtered, and evaporated to
afford the product as a white solid. This was further purified by
silica gel chromatography (hexane/acetone 80:20, 70:30) to give the
title compound (546 mg) as a white solid.
[0127] c) Z-L-leucine-L-leucine-L-leucinal
[0128] A solution of Z-L-leucine-L-leucine-L-leucine
N,O-dimethylhydroxylamide (546 mg) was dissolved in 15 mL dry
tetrahydrofuran (THF) and cooled to 0.degree. C. Lithium aluminum
hydride (1 M solution in THF, 4.1 mL) was added and the mixture was
stirred at 0.degree. C. for 30 minutes. Potassium bisulfate (1.39
g) in 30 mL water was added and the mixture was extracted with
EtOAc (3.times.50 mL). The combined organic layers were washed with
aqueous 5% HCl, saturated NaHCO.sub.3, and brine, then dried over
anhydrous MgSO.sub.4, filtered, and evaporated to afford the
product (446 mg) as a white solid. This was further purified by
reverse phase HPLC (water/acetonitrile).
EXAMPLE 3
Preparation of
Z-L-(2-naphthyl)-Alanine-L-(1-naphthyl)-Alanine-L-Leucinal
[0129] a) Boc-L-Leucine N,O-dimethylhydroxylamide
[0130] A mixture of N-Boc-L-leucine (2.47 g),
N,O-dimethylhydroxylamine hydrochloride (1.09 g),
1-hydroxybenzotriazole monohydrate (1.51 g), and N-methylmorpholine
(1.21 mL) was dissolved in DMF (40 mL),
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.14
g) was added at 0.degree. C. and the mixture was stirred at
0.degree. C. for 2 h, then at room temperature for 22 h. The
reaction was quenched with water (100 mL) and the mixture was
extracted with EtOAc (3.times.100 mL). The combined organic layers
were washed with aqueous 10% HCl, saturated NaHCO.sub.3, and brine,
then dried over anhydrous MgSO.sub.4, filtered, and evaporated to
afford the product (2.57 g) as an oil.
[0131] b) Boc-L-(1-naphthyl)-Alanine-L-Leucine
N,O-dimethylhydroxylamide
[0132] A solution of N-Boc-L-leucine N,O-dimethylhydroxylamide (983
mg) and trifluoroacetic acid (8 mL) in methylene chloride (20 mL)
was stirred at 0.degree. C. for 3 hours. The solvent was evaporated
under reduced pressure and the residue was dried under vacuum. A
portion of this product (208 mg) was transferred to another flask
and was combined with Boc-L-(1-naphthyl)-alanine (378 mg)
1-hydroxybenzotriazole monohydrate (178 mg) N-methylmorpholine
(0.15 mL), and DMF (10 mL). 1-Ethyl-3(3-dimethylaminopropyl)
carbodiimide hydrochloride (241 mg) was then added at 0.degree. C.
The mixture was stirred at 0.degree. C. for 2 h, then at room
temperature for 17 hours. The reaction was quenched with water (20
mL) and the mixture was extracted with EtOAc (3.times.50 mL). The
combined organic layers were washed with aqueous 10% HCl, saturated
NaHCO.sub.3, and brine, then dried over anhydrous MgSO.sub.4,
filtered, and evaporated to afford the product as a white solid
(459 mg).
[0133] c) Z-L-(2-naphthyl)-Alanine-L-(1-naphthyl)-Alanine-L-Leucine
N,O-dimethylhydroxylamide
[0134] A solution of Boc-L-(1-naphthyl)-alanine-L-leucine
N,O-dimethylhydroxylamide (459 mg), trifluoroacetic acid (5 mL),
and thioanisole (2 mL) was stirred at 0.degree. C. for 2.5 hours.
The solvent was evaporated and the residue was dried under vacuum.
A portion of this product (182 mg) was transferred to another flask
and was combined with Z-L-(2-naphthyl)-alanine (171 mg),
1-hydroxybenzotriazole monohydrate (99 mg), N-methylmorpholine
(0.08 mL), and DMF (10 mL). 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (112 mg) was then added at 0.degree. C.
The mixture was stirred at 0.degree. C. for 2 h, then at room
temperature for 41 hours. The reaction was quenched with water (20
mL) and the mixture was extracted with EtOAc (3.times.50 mL). The
combined organic layers were washed with aqueous 10% HCl, saturated
NaHCO.sub.3, and brine, then dried over anhydrous MgSO.sub.4,
filtered, and evaporated to afford the product as a white solid.
This was then purified by silica gel chromatography (hexane/acetone
80:20, 70:30) to give the title compound (321 mg).
[0135] d)
Z-L-(2-naphthyl)-Alanine-L(1-naphthyl)-Alanine-L-Leucinal
[0136]
Z-L-(2-naphthyl)-alanine-L-(1-naphthyl)-alanine-L-leucine-N,O-dimet-
hylhydroxylamide (321 mg) was dissolved in 15 mL dry
tetrahydrofuran (THF) and cooled to 0.degree. C. Lithium aluminum
hydride (1 M, solution in THF, 1.7 mL) was added and the mixture
was stirred at 0.degree. C. for 30 minutes. Potassium bisulfate
(0.59 g) in 30 mL water was added and the mixture was extracted
with EtOAc (3.times.40 mL). The combined organic layers were washed
with aqueous 5% HCl, saturated NaHCO.sub.3, and brine, then dried
over anhydrous MgSO.sub.4, filtered, and evaporated to afford the
product (274 mg) as a white solid.
EXAMPLE 4
Proteolytic Processing of the p60Tth Precursor to p50 In Vitro
Requires ATP
[0137] The p60Tth precursor (or p105) was translated in wheat germ
extract. The substrate protein was mixed with HeLa cell cytoplasmic
extract (S100) in a processing buffer containing 12 mM Tris, pH
7.5, 60 mM KCl, 20 mM creatine phosphate, 3.5 mM MgCl.sub.2 and 1
mM ATP. After incubation at 30.degree. C for one hour, the reaction
mixtures were subjected to immunoprecipitation with anti-p50 Ab and
the proteins were resolved by SDS-PAGE. In the Apyrase treated
sample, the p60 was incubated with 10 U of enzyme at 37.degree. C.
for 30 minutes to inactivate residual ATP in the wheat germ extract
before adding the HeLa cell S100. The control sample did not
receive the enzyme or ATP. The results are shown in FIG. 1.
EXAMPLE 5
Processing of p105/p60Tth in Proteasome-Depleted and
Proteasome-Enriched Extracts
[0138] HeLa cell S 100 was centrifuged for six hours at
100,000.times.g to remove proteasomes. Loss of proteasome activity
was verified using a fluorogenic peptide assay specific for the
proteasome. The results from two different depleted extracts
[Pr.sup.-(I & II)] are shown. The pellet contains most of the
proteasome activity. The processing reactions were carried out as
described in Example 4 and the reactions were immunoprecipitated
with either anti-p50 Ab or anti-myc peptide mAb. The anti-myc mAb
will recognize the N-terminal myc-peptide on a tagged p60 precursor
protein. The results are shown in FIG. 3.
EXAMPLE 6
[0139] Immunodepletion of the proteasome inhibits the processing of
NF-.kappa.B.sub.1
[0140] Monoclonal antibodies against specific components of the
proteasome (MCP20, 29K) and a control McAb against hemagglutinin
(HA 12CA5) were incubated with Pr(II) extract reconstituted with
proteasome activity from the pellet. The immune complexes were
removed and the depleted extracts were used in p60 processing
reactions as described in Example 4. The results are shown in FIG.
4.
EXAMPLE 7
Purified Proteasomes Stimulate the Processing of p60Tth
[0141] Increasing amounts of purified 20/26S proteasomes or a
proteasome-enriched fraction from reticulocyte lysate, fraction II,
were added alone or were combined with Pr.sup.-(II) extract in a
processing reaction (see Example 4). In addition, processing was
inhibited by ATP.gamma.S, a non-hydrolyzable analogue of ATP that
allows ubiquitination but inhibits proteasome function (lanes 3-5).
See FIG. 5.
EXAMPLE 8
The p60Tth Precursor Protein is Ubiquitinated
[0142] In this Example, and in those described above, there are
ladder-like bands that appear when the substrate is incubated with
extracts lacking proteasome activity (Pr.sup.-). The ubiquitination
of p60 is more pronounced when 7.5 .mu.g of purified ubiquitin (ub)
is added to the Pr.sup.-(II) extract in a processing reaction (lane
5). See FIG. 6.
EXAMPLE 9
Ubiquitin is Required for the Processing of NF-.kappa.B.sub.1
[0143] A. Different amounts of reticulocyte fraction II (+/-7.5
.mu.g of ub) were used in a processing reaction (see Example 4)
with p60 as substrate. Fraction II has proteasome activity, but has
very little ubiquitin.
[0144] B. HeLa cell S100 or fraction II was supplemented with E.
coli recombinant wild-type ub or mutant ub(L>R48) protein, which
inhibits ub chain formation. Processing reactions were as described
above.
[0145] The results are shown in FIG. 7.
EXAMPLE 10
Processing of p105 in Saccharomyces Cerevisiae Requires the
Proteasome
[0146] Both wild-type (PRE 1) and proteasome mutant (pre1-1) yeast
were transformed with human p105. The transformants were pulsed
with 35S-methionine/cysteine for 20 minutes and chased with cold
methionine/cysteine for various periods of time. Extracts were
prepared, the lysates were immunoprecipitated with anti-p50 Ab and
the proteins were resolved by SDS-PAGE. The results are shown in
FIG. 8.
EXAMPLE 11
Specific Inhibitors of the Proteasome Block the Processing of p105
In Vivo
[0147] COS cells were transfected with human p105. Inhibitors
(Calpain inhibitor, MG101, and MG115) in 50 .mu.M portions were
added to the cells one hour prior to the addition of
.sup.35S-methionine/cysteine. A typical 20 minute pulse-2 hour
chase experiment, immunoprecipitated with anti-p50 Ab followed by
SDS-PAGE, is shown in FIG. 9. Only proteasome-specific inhibitors
block p105 processing; non-specific protease inhibitors do not have
an effect. These results were verified in vitro.
EXAMPLE 12
Specific Inhibitors of the Proteasome Block Activation of
NF-.kappa.B
[0148] HeLa or MG63 cells were pretreated with inhibitors (50
.mu.M) for one hour. Cells were then treated with TNF-.alpha. (1000
U/ml) or IFN-.gamma. (1000 U/ml) for 30 and 60 minutes,
respectively. Whole-cell extracts were prepared and analyzed by an
electrophoretic mobility shift assay. The NF-.kappa.B site from the
interferon-.beta. gene was used to examine NF-.kappa.B binding
activity and the pIRE site from the IRF-1 gene was used to measure
gamma-activated factor (GAF) activity. The inhibitors only block
NF-.kappa.B activation and have no effect on GAF induction. The
results are shown in FIG. 10.
[0149] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those ordinarily skilled in the
art that various modifications may be made to the disclosed
embodiments and that such modifications are intended to be within
the scope of the present invention.
* * * * *