U.S. patent application number 12/291236 was filed with the patent office on 2009-04-16 for enzyme inhibition.
This patent application is currently assigned to Yale University. Invention is credited to Craig M. Crews, Mikael Elofsson, Kyung Bo Kim, Ute Splittgerber.
Application Number | 20090099097 12/291236 |
Document ID | / |
Family ID | 33492650 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090099097 |
Kind Code |
A1 |
Crews; Craig M. ; et
al. |
April 16, 2009 |
Enzyme inhibition
Abstract
Peptide-based compounds including heteroatom-containing,
three-membered rings efficiently and selectively inhibit specific
activities of N-terminal nucleophile (Ntn) hydrolases. The
activities of those Ntn having multiple activities can be
differentially inhibited by the compounds described. For example,
the chymotrypsin-like and PGPH activities of the 20S proteasome can
be selectively inhibited with the inventive compounds. The
peptide-based compounds include an electron withdrawing group
adjacent to the ring functionality, and the peptide include at
least three peptide units. Among other therapeutic utilities, the
peptide-based compounds exhibit anti-inflammatory and inhibition of
cell proliferation, involving therapeutic applications for these
compounds.
Inventors: |
Crews; Craig M.; (New Haven,
CT) ; Elofsson; Mikael; (Umea, SE) ;
Splittgerber; Ute; (Le Mesa, CA) ; Kim; Kyung Bo;
(Hamden, CT) |
Correspondence
Address: |
ROPES & GRAY LLP
PATENT DOCKETING 39/41, ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Yale University
New Haven
CT
|
Family ID: |
33492650 |
Appl. No.: |
12/291236 |
Filed: |
November 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10871752 |
Jun 17, 2004 |
7476650 |
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12291236 |
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09569748 |
May 11, 2000 |
6831099 |
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10871752 |
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60133847 |
May 12, 1999 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C07K 5/1016 20130101;
C07K 5/101 20130101; C07K 7/06 20130101; C07K 5/1008 20130101 |
Class at
Publication: |
514/18 |
International
Class: |
A61K 38/08 20060101
A61K038/08 |
Goverment Interests
FUNDING
[0002] This invention was made with support from the Government,
pursuant to a grant from the National Institutes of Health, Grant
CA74967-01. The Government has certain rights in this invention.
Claims
1. A method for inhibiting or reducing HIV infection or decreasing
the level of viral gene expression in a subject comprising
administering a compound of formula I: ##STR00018## X is a
heteroatom; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are,
independently, hydrogen or a branched or unbranched: C.sub.1-6
alkyl group, C.sub.1-6 hydroxy alkyl group, C.sub.1-6 alkoxy, or
aryl or aryl-substituted C.sub.1-6 alkyl group, any of which can be
substituted with amide linkages, amines, carboxylic acids and salts
thereof, carboxyl esters, thiols, or thioethers; and R.sub.5 is a
chain of amino acids, hydrogen, an acetyl group, C.sub.1-6
alkanoyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, or a protecting group; with the proviso
that, simultaneously, R.sub.1 is not iso-butyl, R.sub.2 is not
1-hydroxy ethyl, R.sub.3 is not sec-butyl, R.sub.4 is not
sec-butyl, and R.sub.5 is not an acetyl group.
2. The method of claim 1, wherein R.sub.1, R.sub.2, R.sub.3, and
R.sub.4 are, independently, branched or unbranched C.sub.1-6 alkyl,
C.sub.1-6 hydroxy alkyl, C.sub.1-6 alkoxy alkyl, aryl, or
aryl-substituted C.sub.1-6 alkyl.
3. The method of claim 2, wherein R.sub.1, R.sub.2, R.sub.3, or
R.sub.4 are, independently, isobutyl, 1-naphthyl, phenylmethyl, or
2-phenylethyl.
4. The method of claim 3, wherein R.sub.1 is isobutyl.
5. The method of claim 1, wherein X is O; R.sub.1, R.sub.3, and
R.sub.4 are iso-butyl; R.sub.2 is selected from methyl, iso-propyl,
phenyl, and 1-naphthyl; and R.sub.5 is acetyl.
6. The method of claim 1, wherein X is O; R.sub.1, R.sub.3, and
R.sub.4 are iso-butyl; R.sub.3 is selected from methyl, iso-propyl,
phenyl, and 1-naphthyl; and R.sub.5 is acetyl.
7. The method of claim 1, wherein X is O; R.sub.1, R.sub.2, and
R.sub.3 are iso-butyl; R.sub.4 is selected from methyl, iso-propyl,
phenyl, phenylmethyl, 1-naphthyl, and p-benzoylphenyl; and R.sub.5
is acetyl.
8. The method of claim 1, wherein R.sub.2 is a C.sub.1-6 hydroxy
alkyl group.
9. The method of claim 1, wherein R.sub.4 is a C.sub.1-6 hydroxy
alkyl group.
10. The method of claim 8, wherein R.sub.4 is a C.sub.1-6 hydroxy
alkyl group.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/871,752, filed on Jun. 17, 2004 which is a continuation of
U.S. application Ser. No. 09/569,748, filed on May 11, 2000, now
granted as U.S. Pat. No. 6,831,099,which claims the benefit of
priority from U.S. Provisional Application Ser. No. 60/133,847,
filed on May 12, 1999, the specifications of each of which are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0003] This invention relates to compounds and methods for enzyme
inhibition. In particular, the invention relates to therapeutic
methods deriving from enzyme inhibition.
BACKGROUND
[0004] In eukaryotes, protein degradation is predominately mediated
through the ubiquitin pathway in which proteins targeted for
destruction are ligated to the 76 amino acid polypeptide ubiquitin.
Once targeted, ubiquitinated proteins then serve as substrates for
the 26S proteasome, a multicatalytic protease, which cleaves
proteins into short peptides through the action of its three major
proteolytic activities. While having a general function in
intracellular protein turnover, proteasome-mediated degradation
also plays a key role in many processes such as major
histocompatibility complex (MHC) class I presentation, apoptosis,
cell division, and NF-.kappa.B activation.
[0005] The 20S proteasome is a 700 kDa cylindrical-shaped
multicatalytic protease complex comprised of 28 subunits organized
into four rings. In yeast and other eukaryotes, 7 different a
subunits form the outer rings and 7 different .beta. subunits
comprise the inner rings. The .alpha. subunits serve as binding
sites for the 19S (PA700) and 11S (PA28) regulatory complexes, as
well as a physical barrier for the inner proteolytic chamber formed
by the two .beta. subunit rings. Thus, in vivo, the proteasome is
believed to exist as a 26S particle ("the 26S proteasome"). In vivo
experiments have shown that inhibition of the 20S form of the
proteasome can be readily correlated to inhibition of 26S
proteasome. Cleavage of amino-terminal prosequences of .beta.
subunits during particle formation expose amino-terminal threonine
residues, which serve as the catalytic nucleophiles. The subunits
responsible for catalytic activity in proteaseome thus possess an
amino terminal nucleophilic residue, and these subunits belong to
the family of N-terminal nucleophile (Ntn) hydrolases (where the
nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and
other nucleophilic moieties). This family includes, for example,
penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine
PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. In
addition to the ubiquitously expressed .beta. subunits, higher
vertebrates also possess three .gamma.-interferon-inducible .beta.
subunits (LMP7, LMP2 and MECL1), which replace their normal
counterparts, X, Y and Z respectively, thus altering the catalytic
activities of the proteasome. Through the use of different peptide
substrates, three major proteolytic activities have been defined
for the eukaryote 20S proteasome: chymotrypsin-like activity
(CT-L), which cleaves after large hydrophobic residues;
trypsin-like activity (T-L), which cleaves after basic residues;
and peptidylglutamyl peptide hydrolyzing activity (PGPH), which
cleaves after acidic residues. Two additional less characterized
activities have also been ascribed to the proteasome: BrAAP
activity, which cleaves after branched-chain amino acids; and SNAAP
activity, which cleaves after small neutral amino acids. The major
proteasome proteolytic activities appear to be contributed by
different catalytic sites, since inhibitors, point mutations in
.beta. subunits and the exchange of .gamma. interferon-inducing
.beta. subunits alter these activities to various degrees.
[0006] The 20S proteasome plays important roles in cell growth
regulation, major histocompatibility complex class I presentation,
apoptosis, antigen processing, NF-.kappa.B activation, and
transduction of pro-inflammatory signals.
[0007] Small molecules which have been used to inhibit proteasome
activity include lactacystin, and short peptides including
aldehyde, vinyl sulfone, boronic acid and glyoxal functional
groups. These compounds generally lack the specificity, stability,
or potency necessary to explore the roles of the proteasome at the
cellular and molecular level. For example, peptide aldehydes also
inhibit lysosomal and Ca.sup.+2-activated proteases, thus
complicating a precise dissection of their effects on cells. Vinyl
sulfone-based inhibitors have been reported to bind and inhibit
intracellular cysteine proteases (for example, cathepsin S), in
addition to their actions against the proteasome. Lactacystin has a
rate of proteasome inactivation which is significantly slower than
that of vinyl sulfone peptide inhibitors. Lactacystin is also
non-specific for 20S proteasome, as it has been found to
significantly decrease the hydrolysis rate of human platelet
lysosomal cathepsin A-like enzyme at pH 5.5.
SUMMARY
[0008] Enzyme inhibitors are valuable tools that enable the
elucidation of details in cellular events that are regulated by
these enzymes. Additionally, enzyme inhibitors have therapeutic
applications and can be used to carry out mechanistic studies of
the machinery of enzymatic processes. The invention relates to the
discovery that classes of molecules known as peptide
.alpha.',.beta.'-epoxides and peptide .alpha.',.beta.'-aziridines
can bind efficiently, irreversibly and selectively to N-terminal
nucleophile (Ntn) hydrolases, and can specifically inhibit
particular activities of enzymes having multiple catalytic
activity.
[0009] Once thought merely to dispose of denatured and misfolded
proteins, the proteasome is now recognized as constituting
proteolytic machinery that regulates the levels of diverse
intracellular proteins through their degradation in a
signal-dependent manner. Hence, there is great interest in
identifying reagents that can specifically perturb the activities
of the proteasome and other Ntn hydrolases and thereby be used as
probes to study the role of these enzymes in biological processes.
Compounds that target the Ntn hydrolases are herein described,
synthesized and investigated. Peptide epoxides and peptide
aziridines that can potently, selectively, and irreversibly inhibit
particular proteasome activities are disclosed and claimed.
[0010] Particular peptide epoxides and peptide aziridines modify
three catalytic subunits of the 20S proteasome resulting in
inhibition primarily of the chymotrypsin-like activity; the
trypsin-like and PGPH activities were also inhibited at
approximately 100-fold and 1000-fold slower rates, respectively.
Furthermore, in comparison with other potent irreversible
proteasome inhibitors, peptide epoxides and peptide aziridines
inhibit the chymotrypsin-like activity at least about 80-fold
faster than lactacystin and at least about four-fold faster than
clasto-lactacystin .beta.-lactone. Even higher rates are
obtainable.
[0011] Other particular peptide epoxides and peptide aziridines
primarily inhibit PGPH activity, while having far less inhibitory
effect on chymotrypsin-like activity, and virtually no effect on
trypsin-like activity. In contrast to the enzyme inhibitors
described above, which are highly specific for chymotrypsin-like
activity of the proteasome, these other particular PGPH-specific
peptide epoxides and peptide aziridines inhibit a catalytic step
which is believe to be a rate-limiting step in protein degradation.
Their use in elucidating the role(s) of other proteasomal subunits
is thus limited. The PGPH-specific inhibitors allow separation of
contributions of this particular catalytic activity in biological
processes mediated by the proteasome.
[0012] Unlike several other peptide-based inhibitors, the peptide
epoxides and peptide aziridines described herein do not
substantially inhibit non-proteasomal proteases such trypsin,
chymonypsin, cathepsin B, papain, and calpain at concentrations up
to 50 .mu.M. At higher concentrations, inhibition is observed, but
is competitive and not irreversible, since the inhibitor merely
competes with the substrate. The novel peptide epoxides and peptide
aziridines are also shown to inhibit NF-.kappa.B activation and to
stabilize p53 levels in cell culture. Moreover, we have
demonstrated the potent anti-inflammatory activity of peptide
epoxides and peptide aziridines in a mouse model of cutaneous
inflammation. Thus, these compounds can be unique molecular probes,
which have the versatility to explore Ntn enzyme function in normal
biological and pathological processes.
[0013] In one aspect, the invention provides N-terminal nucleophile
hydrolase inhibitors comprising a heteroatom-containing,
three-membered ring, where the ring is bonded to an
electron-withdrawing group, and the electron-withdrawing group is
bonded to a peptide moiety. These inhibitors can inhibit catalytic
activity of N-terminal nucleophile hydrolase enzymes (for example,
the 20S proteasome, or the 26S proteasome) when said inhibitor is
present at concentrations below about 50 .mu.M, and do not inhibit
catalytic activity of non-proteasomal proteases when the inhibitor
is present at concentrations below about 50 .mu.M. Regarding the
20S proteasome, particular hydrolase inhibitors inhibit
chymotrypsin-like activity of the 20S proteasome when the inhibitor
is present at concentrations below about 5 .mu.M, and does not
inhibit trypsin-like activity or PGPH activity of the 20S
proteasome when is present at concentrations below about 5 .mu.M.
Other particular hydrolase inhibitors inhibit PGPH activity of the
20S proteasome when the inhibitor is present at concentrations
below about 50 .mu.M, and does not inhibit chymotrypsin-like or
trypsin-like activity of the 20S proteasome when the inhibitor is
present at concentrations below about 50 .mu.M. The hydrolase
inhibitor can be, for example, a peptide .alpha.',.beta.'-epoxy
ketone or .alpha.',.beta.'-aziridine ketone, and the peptide can be
a tetrapeptide. The tetrapeptide can include branched or unbranched
side chains such as hydrogen, C.sub.1-6 alkyl, C.sub.1-6 hydroxy
alkyl, C.sub.1-6 alkoxy alkyl, aryl, and aryl-substituted C.sub.1-6
alkyl, C.sub.1-6 amide, C.sub.1-6 amine, C.sub.1-6 carboxylic acid,
C.sub.1-6 carboxyl ester, C.sub.1-6 thiol, or C.sub.1-6 thioether,
for example isobutyl, 1-naphthyl, methylphenyl, and 2-ethylphenyl.
The .alpha.'-carbon of the .alpha.',.beta.'-epoxy ketone or
.alpha.',.beta.'-aziridine ketone can be a chiral carbon atom, such
as an (R) or .beta. configured carbon, as these are defined
herein.
[0014] In another aspect, the invention includes a method of making
a peptide .alpha.',.beta.'-epoxy ketone or
.alpha.',.beta.'-aziridine ketone. The method involves synthesizing
a first molecule by providing a tripeptide; acetylating the amino
terminal of the tripeptide to make an acetylated tripeptide; and
catalytically hydrogenating the acetylated tripeptide to make the
first molecule. The method further involves synthesizing a second
molecule by alkenylating a Weinreb amide of an amino acid having an
amino terminal protection group to form an unsaturated ketone;
forming a three-membered, heteroatom-containing ring at the
.alpha.',.beta.'-unsaturation side to form an
.alpha.',.beta.'-epoxy ketone or an .alpha.',.beta.'-azridine
ketone; removing the amino terminal protection group to form the
second molecule. The method also involves coupling the first and
second molecules to make a peptide .alpha.',.beta.'-epoxy ketone or
.alpha.',.beta.'-aziridine ketone. If the tripeptide has hydroxy
side chains, protecting the hydroxy side chain to make a protected
hydroxy side chain-containing tripeptide; and deprotecting the
hydroxy side chain after said coupling are also desirable.
[0015] In another aspect, the invention provides pharmaceutical
compositions, including a pharmaceutically acceptable carrier, and
a pharmaceutically effective amount of the hydrolase inhibitor,
which ameliorates the effects of Alzheimer's disease,
muscle-wasting diseases, cancer, chronic infectious diseases,
fever, muscle disuse, denervation, nerve injury, and wasting, among
others.
[0016] In another aspect, the invention provides anti-inflammatory
compositions.
[0017] In another aspect, the invention provides methods for the
following: inhibiting or reducing HIV infection in a subject;
affecting the level of viral gene expression in a subject; altering
the variety of antigenic peptides produced by the proteasome in an
organism; determining whether a cellular, developmental, or
physiological process or output in an organism is regulated by the
proteolytic activity of a particular Ntn hydrolase; treating
Alzheimer's disease in a subject; reducing the rate of muscle
protein degradation in a cell; reducing the rate of intracellular
protein degradation in a cell; reducing the rate of p53 protein in
a cell; inhibiting the growth of p53-related cancers in a subject;
inhibiting antigen presentation in a cell; suppressing the immune
system of a subject; inhibiting I.kappa.B-.alpha. degradation in an
organism; reducing the content of NF-.kappa.B in a cell, muscle,
organ or subject; affecting cyclin-dependent eukaryotic cell
cycles; treating proliferative disease in a subject; affecting
proteasome-dependent regulation of oncoproteins in a cell; treating
cancer growth in a subject; treating p53-related apoptosis in a
subject; and screening proteins processed by N-terminal nucleophile
hydrolases in a cell. Each of these methods involves administering
or contacting an effective amount of a composition comprising the
hydrolase inhibitors disclosed herein, to a subject, a cell, a
tissue, an organ or an organism.
[0018] In a further aspect, the invention provides a method of
making an .alpha.,.beta.-aziridine ketone, the method including
reacting an .alpha.-halo ketone with a) a boron-containing reagent,
and with b) an imine for a time and under conditions sufficient to
form an .alpha.,.beta.-aziridine ketone.
[0019] As used herein, the term "inhibitor" is meant to describe a
compound that blocks or reduces an activity of an enzyme (for
example, inhibition of proteolytic cleavage of standard fluorogenic
peptide substrates such as suc-LLVY-AMC (SEQ ID NO:1), Box-LLR-AMC
and Z-LLE-AMC, inhibition of various catalytic activities of the
20S proteasome). An inhibitor can act with competitive,
uncompetitive, or noncompetitive inhibition. An inhibitor can bind
reversibly or irreversibly, and therefore the term includes
compounds that are suicide substrates of an enzyme. An inhibitor
can modify one or more sites on or near the active site of the
enzyme, or it can cause a conformational change elsewhere on the
enzyme.
[0020] As used herein, "enzyme" can be any partially or wholly
proteinaceous molecule which carries out a chemical reaction in a
catalytic manner. Such enzymes can be native enzymes, fusion
enzymes, proenzymes, apoenzymes, denatured enzymes, farnesylated
enzymes, ubiquitinated enzymes, fatty acylated enzymes,
gerangeranylated enzymes, GPI-linked enzymes, lipid-linked enzymes,
prenylated enzymes, naturally-occurring or artificially-generated
mutant enzymes, enzymes with side chain or backbone modifications,
enzymes having leader sequences, and enzymes complexed with
non-proteinaceous material, such as proteoglycans, proteoliposomes.
Enzymes can be made by any means, including natural expression,
promoted expression, cloning, various solution-based and
solid-based peptide syntheses, and similar methods known to those
of skill in the art.
[0021] As used herein, the term "peptide" includes not only
standard amide linkage with standard .alpha.-substituents, but
commonly utilized peptidomimics, other modified linkages,
non-naturally occurring side chains, and side chain modifications,
as detailed below.
[0022] As used herein, the term "heteroatom-containing,
three-membered ring" includes moieties with two carbon atoms and a
single heteroatom, such as oxygen or nitrogen.
[0023] As used herein, the term "treating" includes reversing,
reducing, or arresting the symptoms, clinical signs, and underlying
pathology of a condition in manner to improve or stabilize a
subject's condition.
[0024] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0025] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DESCRIPTION OF DRAWINGS
[0026] FIG. 1 is a generalized molecular structure of epoxomicin
and epoxomicin-biotin.
[0027] FIG. 2 is a photocopy of an electrophoretic gel showing a)
separation of bovine brain proteasome catalytic subunits without
(lane 1) and with (lane 2) 50 .mu.M epoxomicin pretreatment, and b)
human B cell lymphoma cells LCL 721.45 (lane 3) and LCL 721.174
(lane 4), both labeled with 5 .mu.M epoxomicin-biotin.
[0028] FIG. 3 is a reaction scheme of the synthesis of
[.sup.3H]-epoxomicin.
[0029] FIG. 4 is an electrophoretic gel of the concentration
dependence of biotin-epoxomicin on protein binding.
[0030] FIG. 5A is an .alpha.-p53 immunoblot analysis showing
accumulation of p53 in epoxomicin-treated cells.
[0031] FIG. 5B is an .alpha.-ubiquitin immunoblot analysis showing
accumulation of ubiquitinated proteins in epoxomicin-treated
cells.
[0032] FIG. 6 is a plot of log k.sub.obs/[I] for inhibition of the
chymotrypsin-like activity of 20S proteasome versus log IC.sub.50
for inhibition of bovine aortic endothelial cell proliferation for
particular enzyme inhibitors.
[0033] FIG. 7 is an electrophoretic gel Western blot analysis
showing epoxomicin inhibition of activation of NF-.alpha.B.
[0034] FIG. 8 is a plot of dose-dependence of epoxomicin on
fold-change in radioactivity of gel-retarded bands, according to
EMSA analysis.
[0035] FIG. 9 is a plot of ear-swelling responses for contact and
irritant sensitivity according to picrylchloride challenges.
[0036] FIG. 10 is a plot of irritant sensitivity response (change
in mouse ear thickness) for various dosages of epoxomicin and a
particular anti-inflammatory compound.
DETAILED DESCRIPTION
[0037] The invention involves compounds useful as enzyme
inhibitors. These compounds include peptide and epoxide or
aziridine moieties. These compounds are generally useful as
inhibitors of enzymes having a nucleophilic group at the
N-terminus. For example, activities of enzymes or enzyme subunits
having N-terminal amino acids with nucleophiles in their side
chains, such as threonine, serine, or cysteine can be successfully
inhibited by the enzyme inhibitors described herein. For example,
activities of enzymes or enzyme subunits having non-amino acid
nucleophilic groups at their N-termini, such as for example,
protecting groups or carbohydrates, can also be successfully
inhibited by the enzyme inhibitors described herein.
[0038] While not bound by any particular theory of operation, it is
believed that such N-terminal nucleophiles of Ntn are able to form
covalent adducts with the epoxide or aziridine functional group of
the enzyme inhibitors described herein. For example, in the
.beta.5/Pre2 subunit of 20S proteasome, the N-terminal threonine is
believed to irreversibly form a morpholino or piperazino adduct
upon reaction with a peptide epoxide or peptide aziridine,
respectively, such as those described below. Such adduct formation
would involve a ring-opening reaction of the heteroatom-containing,
three-membered ring.
[0039] The epoxide- or aziridine-containing compounds preferably
contain groups proximate the heteroatom-containing, three-membered
rings, such that a ring-opening reaction of the
heteroatom-containing three-membered ring is facilitated. Such
groups include electron-withdrawing groups (E.W.G.) adjacent to
(for example, at a carbon vicinal to a carbon atom of the
three-membered, heteroatom-containing ring), or in electronic
communication with (for example, via a carbon atom, or an alkenyl
or alkynyl linkage), epoxide or aziridine functionalities. The
E.W.G. can be bonded to one of the carbon atoms of the
heteroatom-containing, three-membered ring. E.W.G. include, for
example, cyano, isocyano, nitro, amide, sulfonyl, .beta.-carboxy
vinyl, sulfinyl, .beta.,.beta.-dicyano vinyl, formyl, carboxyl,
alkyloxy- and aryloxy-carbonyl, 1-tetrazolyl, carbamoyl, sulfamoyl,
carbonyl, sulfoxide groups, and halogenated or dihalogenated carbon
atoms such as --CHX--, --CXX'--, --CRX-- (where X and X' are
independently selected halogens, and R is a carbon-containing
substituent such as alkyl, aryl alkenyl, alkynyl and the like). In
some preferred embodiments, E.W.G. is a carbonyl group.
[0040] In some embodiments, it may be desirable to utilize E.W.G.
that are of size, charge, and polarity sufficient to interact
electronically with particular polar or charged moieties within an
Ntn hydrolase. For example, an ionized aspartate or glutamate side
chain can be present in the Ntn, and interact with, and stabilize,
an electron-withdrawing group present in a peptide epoxide. Such
groups act as an "anion hole," with which E.W.G. can interact when
enzyme inhibitors are bound to Ntn, resulting in increased
electrophilicity of E.W.G.
[0041] Some peptide epoxide or peptide aziridine compounds have
ketone functionality as the electron-withdrawing group, along with
epoxide or aziridine functional groups. Particular examples are
peptide .alpha.',.beta.'-epoxy ketones or peptide
.alpha.',.beta.'-aziridine ketones, in which the carbon atoms
forming two of the three members of the epoxide or aziridine ring
are one (.alpha.') and two (.beta.') carbons from the ketone, and
the ketone carbon is bonded to one of the carbon atoms of the
heteroatom-containing, three-membered ring. Further groups can be
bonded to .alpha.' or .beta.' carbons such as hydrogen, branched or
unbranched C.sub.1-4 alkyl groups, including methyl, ethyl, propyl
and butyl groups. Groups bonded to .alpha.' or .beta.' carbons can
be further substituted with hydroxy, halogen, amino, carboxy,
carbonyl, thio, sulfide, ester, amide or ether functionality.
[0042] For example, a carboxylic acid group can be bonded directly
to the .alpha.' carbon, or via a linker. The linker can be
C.sub.1-4 alkylene, C.sub.2-5 alkenylene, C.sub.2-5 alkynylene,
aryl, oxygen, sulfur, amine. This carboxylic acid can be part of a
peptide moiety extending from the .alpha.' carbon of the
heteroatom-containing, three-membered ring. In this way, peptides
containing side chains can be constructed. Such side chains can be
labeled as P1', P2', and so forth, with P1' being the first side
chain adjacent to the .alpha.' carbon, P2' being the second, and so
forth. Optimization of side chains for P1', P2' and other positions
can result in enzyme inhibitors with desirable specificity, or
desirable inhibition rates. Side chains for P1', P2' and so forth
can be any of those side chains discussed herein.
[0043] In embodiments including such groups bonded to .alpha.'
carbons, the stereochemistry of the .alpha.'-carbon (that carbon
forming a part of the epoxide or aziridine ring) can be (R) or
(S).
[0044] The invention is based, in part, on the structure-function
information disclosed herein, which suggests the following
preferred stereochemical relationships. Note that a preferred
compound may have a number of stereocenters having the indicated
up-down (or .beta.-.alpha., where .beta. as drawn herein is above
the plane of the page) or (R)-(S) relationship (that is, it is not
required that every stereocenter in the compound conform to the
preferences stated). In some preferred embodiments, the
stereochemistry of the .alpha.' carbon is (R), that is, the X atom
is .beta., or above the plane of the molecule, when drawn as below.
For example, the following general structural formula I
demonstrates a preferred stereochemistry for some embodiments:
##STR00001##
where X is oxygen or an NH or N-alkyl group, E.W.G. is an electron
withdrawing group as described above, "peptide" is a peptide as
describe below, and R is a hydrogen atom, a branched or unbranched
C.sub.1-4 alkyl group, which can be further substituted with
hydroxy, halogen, amino, carboxy, carbonyl, thio, sulfide, ester,
amide or ether functionality. For some embodiments, the X atom
should be configured as above in order to facilitate interaction
with an N-terminal nucleophilic group in an Ntn hydrolase. For
example, irreversible interactions of enzyme inhibitors with the
.beta.5/Pre2 subunit of 20S proteasome which lead to inhibition
appear to be facilitated by the configuration illustrated above. In
the case of other Ntn hydrolases, the opposite stereochemistry of
the .alpha.-carbon of the peptide epoxides or peptide aziridines
may be preferred.
[0045] In the case illustrated above, the .beta.' carbon is
substituted with two hydrogen atoms. Regarding the stereochemistry,
the chiral .alpha.' carbon is indicated with a star, and the
Cahn-Ingold-Prelog rules for determining absolute stereochemistry
are followed. These rules are described, for example, in Organic
Chemistry, Fox and Whitesell; Jones and Bartlett Publishers,
Boston, Mass.(1994); Section 5-6, pp 177-178, which section is
hereby incorporated by reference. The stereochemistry of the
.alpha.' carbon is (R) when the oxygen or nitrogen has the highest
priority, the peptide-E.W.G. group has second highest priority, and
the --CH.sub.2--X-- group has third highest priority. If the
relative priorities of the peptide-E.W.G., --CH.sub.2--X--, and R
groups change, the nominal stereochemistry can change, but the
essential configuration of the groups can remain the same, for some
preferred embodiments. That is, referring to the general structure
immediately above, peptide-E.W.G. is joined to the chiral .alpha.'
carbon from the left, R is joined to the chiral .alpha.' carbon
from the right, and the X atom(s) project(s) from the plane of the
page. The nitrogen atom of an aziridine ring can also, in
principle, be chiral, as discussed in March, Advanced Organic
Chemistry, 4.sup.th Ed. (1992) Wiley-Interscience, New York, pp.
98-100, which pages are incorporated herein by reference.
[0046] The peptide epoxides or peptide aziridines also include a
peptide moiety. The peptide moiety is bonded to the
electron-withdrawing group. The bond is made between the
electron-withdrawing group and any portion of the peptide. For
example, in some preferred embodiments, the E.W.G. is bonded to the
terminal backbone unit, such as for example, to the carboxy
terminus of the peptide. Alternatively, the E.W.G. can be bonded to
the amino terminus of the peptide. In other embodiments, the E.W.G.
can be bonded to a side chain if the peptide moiety.
[0047] Peptides can have a repeating backbone structure with side
chains extending from the backbone units. Generally, each backbone
unit has a side chain associated with it, although in some cases,
the side chain is a hydrogen atom. In other embodiments, not every
backbone unit has an associated sidechain. Peptides useful in
peptide epoxides or peptide aziridines have two or more backbone
units. In some embodiments useful for inhibiting chymotrypsin-like
(CT-L) activity of the proteasome, between four and eight backbone
units are present, and in some preferred embodiments for CT-L
inhibition, between four and six backbone units are present. In
other embodiments useful for inhibiting the PGPH activity of the
proteasome, between two and eight backbone units are present, and
in some preferred embodiments for PGPH inhibition, between three
and six backbone units are present.
[0048] The side chains extending from the backbone units can
include natural aliphatic or aromatic amino acid side chains, such
as hydrogen (glycine), methyl (alanine), iso-propyl (valine),
sec-butyl (isoleucine), iso-butyl (leucine), methylphenyl
(phenylalanine), and the side chain constituting the amino acid
proline. The side chains can also be other branched or unbranched
aliphatic or aromatic groups such as ethyl, n-propyl, n-butyl,
t-butyl, and aryl substituted derivatives such as 1-phenylethyl,
2-phenylethyl, (1-naphthyl)-methyl, (2-naphthyl)-methyl,
1-(1-naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl,
2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can be
further substituted with branched or unbranched C.sub.1-6 alkyl
groups, or substituted alkyl groups, such as acetyl and the like,
or further aryl groups, or substituted aryl groups, such as benzoyl
and the like. Heteroaryl groups can also be used as side chain
substituents. Heteroaryl groups include nitrogen-, oxygen-, and
sulfur-containing aryl groups such as thienyl, benzothienyl,
naphthothienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl,
chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,
indolyl, purinyl, quinolyl, and the like.
[0049] In some embodiments, polar or charged residues can be
introduced into the peptide epoxides or peptide aziridines. For
example, naturally occurring amino acids such as hydroxy-containing
(Thr, Tyr, Ser) or sulfur-containing (Met, Cys) can be introduced,
as well as non-essential amino acids, for example taurine,
carnitine, citrulline, cystine, ornithine, norleucine and others.
Non-naturally occurring side chain substituents with charged or
polar moieties can also be included, such as, for example,
C.sub.1-C.sub.6 alkyl chains or C.sub.6-C.sub.12 aryl groups with
one or more hydroxy, short chain alkoxy, sulfide, thio, carboxyl,
ester, phospho, amido or amino groups, or such substituents
substituted with one or more halogen atoms. In some preferred
embodiments, there is at least one aryl group present in a side
chain of the peptide moiety.
[0050] In some embodiments, the backbone units are amide units
[--NH--CHR--C(.dbd.O)--], in which R is the side chain. Such a
designation does not exclude the naturally occurring amino acid
proline, or other non-naturally occurring cyclic secondary amino
acids, which will be recognized by those of skill in the art.
[0051] In other embodiments, the backbone units are N-alkylated
amide units (for example, N-methyl and the like), olefinic analogs
(in which one or more amide bonds are replaced by olefinic bonds),
tetrazole analogs (in which a tetrazole ring imposes a
cis-configuration on the backbone), or combinations of such
backbone linkages. In still other embodiments, the amino acid
.alpha.-carbon is modified by .alpha.-alkyl substitution, for
example, aminoisobutyric acid. In some further embodiments, side
chains are locally modified, for example, by .DELTA..sup.E or
.DELTA..sup.Z dehydro modification, in which a double bond is
present between the .alpha. and .beta. atoms of the side chain, or
for example by .DELTA..sup.E or .DELTA..sup.Z cyclopropyl
modification, in which a cyclopropyl group is present between the
.alpha. and .beta. atoms of the side chain. In still further
embodiments employing amino acid groups, D-amino acids can be used.
Further embodiments can include side chain-to-backbone cyclization,
disulfide bond formation, lactam formation, azo linkage, and other
modifications discussed in "Peptides and Mimics, Design of
Conformationally Constrained" by Hruby and Boteju, in "Molecular
Biology and Biotechnology: A Comprehensive Desk Reference", ed.
Robert A. Meyers, VCH Publishers (1995), pp. 658-664, which is
hereby incorporated by reference.
[0052] The enzyme inhibitors for inhibition of chymotrypsin-like
(CT-L) activity of Ntn include at least four backbone units. In
some particularly preferred CT-L inhibitor embodiments, at least
four amide units and an .alpha.',.beta.'-epoxy ketone or
.alpha.',.beta.'-aziridine ketone moiety are present (tetrapeptide
epoxy ketones or tetrapeptide aziridine ketones). In particular
CT-L inhibitor embodiments with at least four amide units, the
peptide moiety, and the ketone and epoxide or aziridine
functionalities of the enzyme inhibitors form compounds with the
general structure I:
##STR00002##
where X is oxygen, NH, or N-alkyl, R.sub.1, R.sub.2, R.sub.3 and
R.sub.4 are independently selected from the group consisting of
branched or unbranched C.sub.1-6 alkyl or branched or unbranched
C.sub.1-6 hydroxy alkyl or branched or unbranched C.sub.1-6 alkoxy
alkyl, aryl, and aryl-substituted branched or unbranched C.sub.1-6
alkyl, wherein such groups can further include: amide linkages;
amines; carboxylic acids and salts thereof; carboxyl esters,
including C.sub.1-5 alkyl esters and aryl esters; thiols and
thioethers; and R.sub.5 is a further chain of amino acids,
hydrogen, acetyl, or a protecting group, such as N-terminal
protecting groups known in the art of peptide syntheses, including
t-butoxy carbonyl (BOC), benzoyl (Bz), fluoren-9-ylmethoxycarbonyl
(Fmoc), triphenylmethyl(trityl) and trichloroethoxycarbonxyl (Troc)
and the like. The use of various N-protecting groups, e.g., the
benzyloxy carbonyl group or the t-butyloxycarbonyl group (BOC),
various coupling reagents, e.g., dicyclohexylcarbodiimide,
1,3-diisopropylcarbodiimide (DIC),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),
N-hydroxyazabenzotriazole (HATU), carbonyldiimidazole, or
1-hydroxybenzotriazole monohydrate (HBT), and various cleavage
reagents: for example, trifluoracetic acid; HCL in dioxane;
hydrogenation on Pd--C in organic solvents, such as methanol or
ethyl acetate; boron tris(trifluoroacetate); and cyanogen bromide,
and reaction in solution with isolation and purification of
intermediates is well-known classical peptide methodology.
[0053] In some embodiments of chymotrypsin-like activity
inhibitors, R.sub.1 is branched or unbranched C.sub.1-6 alkyl. In
some embodiments of chymotrypsin-like activity inhibitors, R.sub.1
is isobutyl. In some embodiments of chymotrypsin-like activity
inhibitors, R.sub.2 is branched or unbranched C.sub.1-6 alkyl or
aryl. In some embodiments of chymotrypsin-like activity inhibitors,
R.sub.2 is phenyl, methylphenyl, or 1-naphthyl. In some embodiments
of chymotrypsin-like activity inhibitors, R.sub.3 is branched or
unbranched C.sub.1-6 alkyl or aryl. In some embodiments of
chymotrypsin-like activity inhibitors, R.sub.3 is isobutyl, phenyl
or 1-naphthyl. In some embodiments of chymotrypsin-like activity
inhibitors, R.sub.4 is branched or unbranched C.sub.1-6 alkyl,
aryl, and aryl-substituted branched or unbranched C.sub.1-6 alkyl.
In some embodiments of chymotrypsin-like activity inhibitors,
R.sub.4 is isobutyl, phenyl, 1-naphthyl, methylphenyl, or
2-ethylphenyl. In some embodiments of chymotrypsin-like activity
inhibitors, R.sub.5 is hydrogen, C.sub.1-6 alkanoyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, where
substituents include halogen, carbonyl, nitro, hydroxy, aryl, and
C.sub.1-5 alkyl. In some embodiments of chymotrypsin-like activity
inhibitors, R.sub.5 is hydrogen, acetyl, substituted or
unsubstituted aryl.
[0054] In some preferred embodiments of chymotrypsin-like activity
inhibitors, simultaneously, R.sub.1 is isobutyl, R.sub.2 is
methylphenyl, R.sub.3 is isobutyl, and R.sub.4 is 2-ethylphenyl,
and R.sub.5 is acetyl.
[0055] The above definitions of R groups further include a proviso
that, simultaneously, R.sub.1 is not iso-butyl, R.sub.2 is not
1-hydroxyethyl, R.sub.3 is not sec-butyl, R.sub.4 is not sec-butyl,
and R.sub.5 is not acetyl.
[0056] In some embodiments of PGPH activity inhibitors, R.sub.1 is
hydrogen, branched or unbranched C.sub.1-6 alkyl. In some
embodiments of PGPH activity inhibitors, R.sub.1 is isobutyl. In
some embodiments of PGPH activity inhibitors, R.sub.2 is hydrogen,
branched or unbranched C.sub.1-6 alkyl or aryl. In some embodiments
of PGPH activity inhibitors, R.sub.2 is phenyl, methylphenyl, or
1-naphthyl. In some embodiments of PGPH activity inhibitors,
R.sub.3 is hydrogen, branched or unbranched C.sub.1-6 cyclic
alkylene bonded to the R.sub.3 backbone unit. In some embodiments
of PGPH activity inhibitors, R.sub.3 is ethylene bonded to the
amine of the R.sub.3 amino acid backbone, such as would be the case
for the amino acid proline. In some optional embodiments of PGPH
activity inhibitors, R.sub.4 is hydrogen, branched or unbranched
C.sub.1-6 alkyl, aryl, and aryl-substituted branched or unbranched
C.sub.1-6 alkyl. In some other optional embodiments of PGPH
activity inhibitors, R.sub.4 is hydrogen, or isopropyl. In some
optional embodiments of PGPH activity inhibitors, R.sub.5 is
hydrogen, C.sub.1-6 alkanoyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, where substituents include
halogen, carbonyl, monosubstituted-, disubstituted- or
unsubstituted-amino, nitro, hydroxy, aryl, and C.sub.1-5 alkyl. In
some optional embodiments of PGPH activity inhibitors, R.sub.5 is
acetyl, N-acetyl-piperidinecarbonyl, N-dimethylaminobenzyl,
isooctanoic, or benzoylbenzoic.
[0057] In some preferred embodiments of PGPH activity inhibitors,
simultaneously, R.sub.1 is isobutyl, R.sub.2 is phenyl, R.sub.3 is
ethylene bonded to the R.sub.3 amine of the amino acid backbone,
and R.sub.4 is hydrogen, and R.sub.5 is acetyl.
Selectivity for 20S Proteasome
[0058] The enzyme inhibitors disclosed herein are useful in part
because they inhibit the action of the 20S proteasome.
Additionally, unlike other 20S proteasome inhibitors, the compounds
disclosed herein are highly selective toward the 20S proteasome,
with respect to other protease enzymes. That is, the instant
compounds show selectivities for the 20S proteasome over other
proteases such as cathepsins, calpains, papain, chymotrypsin,
trypsin, tripeptidyl pepsidase II. The selectivities of the enzyme
inhibitors for 20S proteasome are such that at concentrations below
about 50 .mu.M, the enzyme inhibitors show inhibition of the
catalytic activity of the 20S proteasome, while not showing
inhibition of the catalytic activity of other proteases such as
cathepsins, calpains, papain, chymotrypsin, trypsin, tripeptidyl
pepsidase II. In preferred embodiments, the enzyme inhibitors show
inhibition of the catalytic activity of the 20S proteasome at
concentrations below about 10 .mu.M, while not showing inhibition
of the catalytic activity of other proteases at these
concentrations. In even more preferred embodiments, the enzyme
inhibitors show inhibition of the catalytic activity of the 20S
proteasome at concentrations below about 1 .mu.M, while not showing
inhibition of the catalytic activity of other proteases at these
concentrations. The enzyme kinetic assays are carried out as
described below in Example 2.
Selectivity for Chymotrypsin-Like Activity
[0059] Particular embodiments of the enzyme inhibiting compounds
described herein are further useful because they can efficiently
and selectively inhibit the chymotrypsin-like activity of the 20S
proteasome, as compared to the trypsin-like, and PGPH activities.
The chymotrypsin-like activity of 20S proteasome is characterized
by cleavage of peptides in the immediate vicinity of large
hydrophobic residues. In particular, the chymotrypsin-like activity
of Ntn hydrolases can be determined by cleavage of a standard
substrate. Examples of such substrates are known in the art. For
example, a leucylleucylvalinyltyrosine derivative can be used.
Particular enzyme inhibitors described herein can inhibit the
chymotrypsin-like activity of 20S proteasome on standard substrates
at least about 80-fold faster than lactacystin and at least about
four-fold faster than clasto-lactacystin .beta.-lactone. Particular
peptide epoxides and peptide aziridines primarily inhibit
chymotrypsin-like activity of 20S proteasome; the trypsin-like and
PGPH activities were also inhibited at approximately 100-fold and
1000-fold slower rates, respectively. The enzyme kinetic assays are
carried out as described below in Example 2.
Selectivity for PGPH Activity
[0060] Particular embodiments of the enzyme inhibiting compounds
described herein are further useful because they can efficiently
and selectively inhibit the PGPH activity of the 20S proteasome, as
compared to the chymotrypsin-like, and trypsin-like activities. The
PGPH activity of 20S proteasome is characterized by cleavage of
peptides in the immediate vicinity of acidic residues. In
particular, the PGPH activity of Ntn hydrolases can be determined
by cleavage of a standard substrate. Examples of such substrates
are known in the art. For example, a leucylleucylglutamate
derivative can be used. Particular peptide epoxides and peptide
aziridines primarily inhibit PGPH activity of 20S proteasome; the
chymotrypsin-like activity was inhibited at approximately a 40-fold
slower rate. The trypsin-like activity is generally very poorly
inhibited by the inhibitors optimized for PGPH inhibition. The
enzyme kinetic assays are carried out as described below in Example
2.
The Synthesis of Peptide Epoxides and Peptide Aziridines
[0061] Peptide epoxides and peptide aziridines, [3H]-peptide
epoxides, and [3H]-peptide aziridines, and biotinylated peptide
epoxides and biotinylated peptide aziridines can be synthesized as
described herein.
[0062] A general strategy for the synthesis of peptide epoxides and
peptide aziridines involves the synthesis of a first molecular
fragment (also referred to as a "left hand" fragment), the parallel
synthesis of a second molecular fragment (also referred to as a
"right hand" fragment), and the coupling of these fragments. The
use of amino terminal-, carboxy terminal-, and side
chain-protecting groups is involved in the synthetic procedure. Any
generally useful protecting groups can be used for the purposes of
this synthesis. A number of amino terminal-, carboxy terminal-, and
side chain-protecting groups are known to those of skill in the
art. A key step is the removal of protecting groups while keeping
the epoxide or aziridine ring intact. Additionally, the epoxide or
aziridine ring can be introduced when a carbonyl group is
present.
[0063] The synthesis of the first molecular fragment can begin with
the reaction of an amino terminal protected amino acid with a
carboxy terminal-protected amino acid to make a dipeptide with
amino terminal and carboxy terminal protected. Any natural or
non-natural amino acid can be used. For example, the synthesis of
epoxomicin would involve reaction of amino terminal-protected
isoleucine and carboxy terminal-protected threonine. Amino
terminal- and carboxy terminal-protecting groups, as well as
conditions and reagents for carrying out the reaction to make a
dipeptide, are known to those in the art. For example,
fluoren-9-ylmethoxycarbonylisoleucine (Fmoc-Ile-OH) can be coupled
to threonine benzyl ester (H-Thr-OBn) with
O-benzotriazo-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU) and 1-hydroxybenzotriazole (HOBt) in the presence of a
relatively hindered base (for example, i-Pr.sub.2NEt) in a
non-polar, non-protic solvent (for example, CH.sub.2Cl.sub.2), at
moderate temperature (for example, room temperature) for at least
about 4 hours (for example 24 hours) to give dipeptide
Fmoc-11e-Thr-OBn in good yield (for example 79%).
[0064] In some embodiments, this step is followed by protection of
a side chain hydroxyl group. For example, the synthesis of
epoxomicin would involve protection of the side chain of threonine
at this point. Hydroxyl side chain protecting groups are known in
the art. For example, reaction of dipeptide Fmoc-Ile-Thr-OBn with
t-butyldiphenylsilylchloride (TBDPSCl) in the presence of a base
(for example, imidazole) in a non-protic solvent (for example,
tetrahydrofuran) at a moderate temperature (for example, room
temperature), for at least about 10 hours (for example, for 48
hours) yields a TBDPSCl-protected threonine side chain in
Fmoc-Ile-Thr-OBn in moderate yield (for example, 64%).
[0065] If a hydroxyl-containing side chain is not involved, the
above step can be omitted, or postponed until a later point. In any
event, at this point, the amino terminal-protecting group is
removed, by methods known in the art. For example, in an epoxomicin
synthesis, Fmoc can be removed by reaction in the presence of a
base (for example, piperidine) in a polar, non-protic solvent (for
example, dimethylformamide), at a moderate temperature (for
example, room temperature), for at least about five minutes (for
example 20 minutes) to give the amino terminal deprotected
H-Ile-Thr-OBn (with Thr side chain protected, as detailed above) in
near-quantitative yield.
[0066] At this point, a third amino acid is coupled to the amino
terminus of the dipeptide to make an amino terminal- and carboxy
terminal-protected tripeptide. As above, conditions and reagents
for this coupling reaction are known in the art. For example, for
an epoxomicin synthesis, reaction of Fmoc-N-methyl-isoleucine
(Fmoc-MeIle-OH) with H-Ile-Thr-OBn (with Thr side chain protected)
in the presence of coupling reagents
O-benzotriazo-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU) and 1-hydroxybenzotriazole (HOBt) in the presence of a
relatively hindered base (for example, i-Pr.sub.2NEt) in a
non-polar, non-protic solvent (for example, CH.sub.2Cl.sub.2), at
moderate temperature (for example, room temperature) for at least
about 4 hours (for example 18 hours) to give tripeptide
Fmoc-MeIle-Ile-Thr-OBn in near-quantitative yield (for example
99%).
[0067] Acetylation of the amino terminus is then carried out, by
reaction of exemplary tripeptide
Fmoc-X.sub.1--X.sub.2--X.sub.3--OBn with an acetylating agent,
where X.sub.1, X.sub.2, and X.sub.3 are amino acids, to give
exemplary acetylated tripeptide Ac--X.sub.1--X.sub.2--X.sub.3--OBn.
For example, for an epoxomicin synthesis, acetylation of
Fmoc-MeIle-Ile-Thr-OBn is carried out with an acetylating agent
(for example, acetic anhydride) in the presence of a relatively
hindered base (for example, i-Pr.sub.2NEt) in a non-polar,
non-protic solvent (for example, CH.sub.2Cl.sub.2), at moderate
temperature (for example, room temperature) for at least about 1
hour (for example 3.5 hours) to give tripeptide
Ac-MeIle-Ile-Thr-OBn in near-quantitative yield (for example
99%).
[0068] Catalytic hydrogenation of exemplary acylated tripeptide
Ac--X.sub.1--X.sub.2--X.sub.3--OBn removes the carboxyl terminus
protecting group to give exemplary tripeptide
Ac--X.sub.1--X.sub.2--X.sub.3--OH. For example, for an epoxomicin
synthesis, hydrogenation of Ac-MeIle-Ile-Thr-OBn by a typical
catalytic hydrogenation agent (for example, 10% Pd--C) in the
presence of hydrogen gas removes the benzyl ester to yield the free
carboxyl terminus in Ac-MeIle-Ile-Thr-OH in essentially
quantitative yield. At this stage the first ("left hand") molecular
fragment is completed.
[0069] The second ("right hand") molecular fragment can be made in
parallel or in sequence. This procedure involves alkenylating a
Weinreb amide of an amino terminal protected amino acid. For
example, an amino terminal protected amino acid can be reacted with
a reagent (for example, MeNHOMe.times.HCl, EDCI, NMM, HoBt, DMF,
0.degree., 12 h, 80%) to make a Weinreb amide of the carboxyl
terminus of the amino acid. Alkenylation is carried out by exposing
the Weinreb amide to an alkenylating agent (for example,
propen-2-yl lithium) to give an .alpha.',.beta.'-unsaturated
ketone. For example, for an epoxomicin synthesis, Boc-Leu Weinreb
amide can be reacted with propen-2-yl lithium to give the
corresponding .alpha.',.beta.'-unsaturated ketone. Propen-2-yl
lithium can be generated by reaction of 2-bromopropene with t-butyl
lithium in a non-protic solvent (for example, diethyl ether) at low
temperature (for example -78.degree. C.), for at least 30 minutes
(for example 2.5 hours) to give the .alpha.',.beta.'-unsaturated
ketone in excellent yield (for example, 92%).
[0070] At this point, the .alpha.',.beta.'-unsaturated ketone can
be epoxidized to form an .alpha.',.beta.'-epoxy ketone;
alternatively, nitrogen can be added to the
.alpha.',.beta.'-unsaturated ketone to form an
.alpha.',.beta.'-aziridine ketone. Nitrogen can be added by thermal
or photochemical reaction with an azide (RN.sub.3 where R is aryl,
cyano, EtOOC and alkyl-SO.sub.2, as well as other groups). For
example, for an epoxomicin synthesis, a typical epoxidizing reagent
(for example, alkaline hydrogen peroxide) can be reacted with the
.alpha.',.beta.'-unsaturated ketone described above, in polar
solvent (for example, aqueous solution or methanol in water) in the
presence of a compound which gives a reactive intermediate, which
reacts with the unsaturated ketone to give an epoxide product (for
example, benzonitrile), and a moderately hindered base (for
example, i-Pr.sub.2Net) at moderately low temperature (for example,
0 to 4.degree. C.), for at least about 10 hours (for example, 43
hours) to give the .alpha.',.beta.'-epoxy ketone in good yield (for
example, 76%).
[0071] In another embodiment, an .alpha.',.beta.'-aziridine ring
can be introduced by forming an epoxide ring substantially as
disclosed above, opening the epoxide ring to give a tertiary
alcohol, using acid catalysis, base catalysis, specific catalysis,
or any other technique known to those of skill in the art.
Thereafter, substituting the alcohol with a suitable leaving group,
such as tosylate, for example, and addition of an azide, such as an
alkali or alkali metal salt of an azide, can result in an aziridine
ring.
[0072] In yet another embodiment, an .alpha.',.beta.'-aziridine
ring can be introduced by way of an enolate reaction, as follows. A
peptide, protected at the carboxy terminus with an ester group,
such as a branched or unbranched C.sub.1-6 alkyl ester, for example
a methyl ester, is reacted with a halogenated alkylating agent.
Such halogenated alkylating agents include, for example
1-halo-1-metalloalkanes, including 1-bromo-1-lithio-ethane. These
reagents can be generated according to methods known in the art,
such as by treating a dihalogenated alkane with an alkylating
reagent such as n-butyllithium in a nonpolar, nonprotic solvent
such as diethyl ether, at low temperature, for example -78.degree.
C. The reaction between the esterified peptide and the halogenated
alkylating agent results in an .alpha.-haloketone, which is
substantially racemic at the .alpha. carbon.
[0073] The .alpha.-haloketone is treated, in the presence of an
base (such as a nitrogenous base, for example, triethylamine), with
a boron-containing reagent such as XBL*.sub.2 where X is halogen, B
is boron, and L* is an H.C. Brown chiral auxiliary ligand, (for
example, diisopinocanpheyl; IPC) further examples of which are well
known to those of skill in the art. The selection of chiral
auxiliary ligand can be used to produce high stereoselectivity in
aziridine formation. Such stereoselectivity can simplify
purification procedures. It is believed that particular aziridine
and epoxide stereoisomers can possess superior utility for some
applications.
[0074] The .alpha.-haloketone can alternatively be treated, in the
presence of an base (such as a nitrogenous base, for example,
triethylamine), with a dialkyl boron reagent having a leaving group
bonded to boron. This reagent can take the form YBR.sub.2''', where
Y is a leaving group, for example, triflate, B is boron, and R'''
can be aryl, heteroaryl, or branched or unbranched C.sub.1-6 alkyl,
any of which can be further substituted with halogen, hydroxy,
amine, aryl, thiol, sulfide, ether, ester, amide or nitro groups. B
can alternatively be bonded to a single group which forms a cyclic
ligand, such as, for example, the alkylene --(CH.sub.2).sub.n--
where n is from 2 to 7 for example. Such groups can be further
substituted as described immediately above.
[0075] The product of the reaction of the .alpha.-haloketone with
either of these types of reagents is then treated with an imine in
a nonpolar, nonprotic solvent such as diethyl ether, at a low
temperature, for example, -78.degree. C. and raised to a more
moderate temperature, for example, room temperature, over a time
period of at least about 4 hours, for example, from about 4 hours
to about 18 hours. The imine can be of the form H.sub.2C.dbd.N-Z,
where Z can be a silyl, for example, trialkylsilyl, triarylsilyl,
or any combination of three alkyl or aryl silicon substituents, for
example, trimethylsilyl, or triisopropylsilyl; Z can also be a
sulfonyl, for example, alkylsulfonyl or arylsulfonyl, for example
benzenesulfonyl. As the reaction mixture temperature approaches a
moderate temperature, the reaction mixture is treated with a basic
solution of excess hydrogen peroxide, sodium perborate, or similar
workup reagent to yield the aziridine. A general scheme is given
below.
##STR00003##
where Z is as described above, X, X.sub.1 and X.sub.2 are
independently halogens, R can be branched or unbranched alkyl,
aryl, heteroaryl, including such groups substituted with hydroxy,
halogen, amide, ester, amine, ether, sulfide, and thiols. In
particular embodiments, R is a branched or unbranched aliphatic or
aromatic group such as ethyl, n-propyl, n-butyl, t-butyl, and aryl
substituted derivative such as 1-phenylethyl, 2-phenylethyl,
(1-naphthyl)-methyl, (2-naphthyl)-methyl, 1-(1-naphthyl)ethyl,
1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl, 2-(2-naphthyl)ethyl, and
similar compounds. The aryl groups can be further substituted with
branched or unbranched C.sub.1-6 alkyl groups, or substituted alkyl
groups, such as acetyl and the like, or further aryl groups, or
substituted aryl groups, such as benzoyl and the like. Heteroaryl
groups can also be used as R. Heteroaryl groups include nitrogen-,
oxygen-, and sulfur-containing aryl groups such as thienyl,
benzothienyl, naphthothienyl, thianthrenyl, furyl, pyranyl,
isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrazinyl, indolyl, purinyl, quinolyl, and the like.
[0076] In some embodiments, polar or charged residues can be used
as R. For example, groups corresponding to the side chains of
naturally occurring amino acids such as hydroxy-containing (Thr,
Tyr, Ser) or sulfur-containing (Met, Cys) can be used, as well as
those corresponding to the side chains of non-essential amino
acids, for example taurine, carnitine, citrulline, cystine,
ornithine, norleucine and others. Non-naturally occurring
substituents with charged or polar moieties can also be included,
such as, for example, C.sub.1-C.sub.6 alkyl chains or
C.sub.6-C.sub.12 aryl groups with one or more hydroxy, short chain
alkoxy, sulfide, thio, carboxyl, ester, phospho, amido or amino
groups, or such substituents substituted with one or more halogen
atoms.
[0077] R''' can be any group as described above with respect to the
boron-containing reagent. R'' can be branched or unbranched alkyl,
aryl, heteroaryl, including such groups substituted with hydroxy,
halogen, amide, ester, amine, ether, sulfide, and thiols. In
particular embodiments, R''' can be a peptide such as a dipeptide,
tripeptide, tetrapeptide and so forth, having side chains as
described above with respect to enzyme inhibitors, and R' is the
group derived from the imine, for example a silyl or sulfonyl group
such as trimethylsilyl or benzenesulfonyl. The R' group can be
converted to hydrogen by treatment with a reagent such as TBAF
(tetrabutylammonium fluoride) in a solvent suitable for such
conversion. R' can be converted to alkyl by treatment with an
S.sub.N2 substrate such as an alkyl halide, in solvents suitable
for such conversion.
[0078] The introduction of an epoxide or aziridine ring at this
stage makes the .alpha.' carbon chiral. Generally, some significant
amount of each stereoisomer is formed at this stage. For example,
in the above synthesis of epoxomicin using Boc-protected Leu, the
ratio of compound having the (R) .alpha.' carbon to the compound
having the (S) .alpha.' carbon was 1.7:1. The same synthesis using
Z-protected Leu gave a 4.0:1 (R):(S) ratio. These compounds were
readily separated, for example by flash chromatography, in common
solvent systems, for example hexanes:EtOAc 10:1). In some
embodiments, the R stereoisomer (as described above, with
peptide-E.W.G. attached from the left, and R attached from the
right, the X atom projects above the plane of the page) is a
preferred isomer, and further reactions were carried out with that
isomer.
[0079] At this stage, the amino terminal protection group of the
.alpha.',.beta.'-epoxy ketone or aziridine ketone is removed, but
without disturbing the epoxide or aziridine ring. For example, if
the amino terminal protecting group is Boc, brief treatment of the
.alpha.',.beta.'-epoxy ketone or the .alpha.',.beta.'-aziridine
ketone with a deprotection agent (for example, trifluoroacetic
acid), at a moderate temperature (for example, room temperature)
for a short time (for example, from about 2 to about 20 minutes,
preferably from about 3 to about 18 minutes, more preferably from
about 5 to about 15 minutes, and even more preferably, about 10
minutes) gives the second molecular fragment in excellent yield.
Under these conditions of trifluoroacetic acid treatment, there are
no very good nucleophiles available to attack the epoxide or
aziridine. The deprotected amine is immediately able to form a
trifluoroacetate salt, which is a good leaving group. Even if
trifluoroacetate were to open the three-membered,
heteroatom-containing ring, the reverse reaction is generally more
favorable.
[0080] At this stage, the second molecular fragment is completed
and can be coupled to the first molecular fragment. For example,
reaction of the first and second molecular fragments with
O-benzotriazo-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU) and 1-hydroxybenzotriazole (HOBt) in the presence of a
relatively hindered base (for example, i-Pr.sub.2NEt) in a
non-polar, non-protic solvent (for example, CH.sub.2Cl.sub.2), at
moderate temperature (for example, room temperature) for at least
about 4 hours (for example 18 hours) to give a tetrapeptide epoxy
ketone or tetrapeptide aziridine ketone in moderate yield (for
example 48%). If a hydroxyl side chain has been introduced, its
protecting group will be removed at this stage.
[0081] For example, for an epoxomicin synthesis, reaction of the
first molecular fragment, Ac-MeIle-Ile-Thr-OH (with Thr side chain
protected, as described above), with the second molecular fragment
derived from H-Leu-.alpha.',.beta.'-epoxy ketone and a fluorinating
agent (for example, tetrabutylammonium fluoride; TBAF), in a
non-protic solvent (for example, tetrahydrofuran; THF), at a
moderate temperature (for example, room temperature), for at least
about 10 minutes (for example, 1 hour), gives epoxomicin in
excellent yield (for example, 96%).
[0082] Peptide epoxides and peptide aziridines are also readily
amenable to synthesis and derivativization via solid phase
syntheses. Commercially available solid state synthetic supports,
such as for example, chlorinated resins, including 2-chlorotrityl
chloride resin (available from Advanced Chemtech) can be treated
with N-terminal protected amino acids. For the case of hydroxy-side
chain-containing amino acids, the hydroxy-side chain can also be
desirably protected. Standard peptide synthesis conditions can be
utilized to elongate the peptide chain. The peptide can be readily
detached from the solid support by known methods, including for
example, acetic acid/trifluoromethanol/dichloromethane in a ratio
of approximately 1:1:3 ratio, at a moderate temperature, for
example room temperature, for at least about 20 minutes, for
example for 2 hours. The detached peptide can then be coupled to a
"right hand" molecular fragment, and further reacted with reagents
to give peptide epoxides and peptide aziridines, as described
above.
[0083] In order to synthesize peptide epoxides and peptide
aziridines with tritiated acetylene at the N-terminal position, an
N-protected tetrapeptide-epoxide or -aziridine, as described above
is reacted with a base, such as piperidine in a polar solvent, such
as dimethylformamide, at a moderate temperature, for example, room
temperature, for at least about 2 minutes, for example, 20 minutes.
The resulting amine is reacted with a tritiating agent which can
react with an amino terminus, for example, [3H]-acetic anhydride in
a non-polar solvent, for example, methylene chloride, in the
presence of a base, for example diisopropylamine, at a moderate
temperature, for example, room temperature, for at least one hour,
for example, 8 hours. Concentration and chromatography yields a
protected tritiated peptide epoxide or peptide aziridine, which can
be deprotected according to methods known in the art.
[0084] Biotinylated peptide epoxides and aziridines can be prepared
by providing an N-terminal protected, C-terminal protected, and if
necessary, hydroxy-side chain protected left hand molecular
fragment (described above), reacting this fragment with a base such
as a nitrogenous base, for example, piperidine, in a non-protic
solvent, for example, tetrahydrofuran, at a moderate temperature,
for example, room temperature, for at least about 6 hours, for
example, 26 hours, to give an intermediate amine product in good
yield, for example about 91%. The intermediate amine product is
reacted with an amino-protected aminoalkanoic acid, such as
N-Fmoc-6-aminohexanioc acid in the presence of an activating agent
such as benzotriazolyl N-oxytrisdimethylamino-phosphonium
hexafluorophosphate (BOP),
bromo-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBrop),
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate
(PyBOP), O-benzotriazole-N,N,NO,NO-tetramethyluronium
hexafluoro-phosphate (HBTU),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU), or
O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluorophosphate (HATU). All of the previously named reagents
require activating bases, such as for example, hindered nitrogenous
bases such as diisopropylethylamine. The reaction is carried out in
a non-polar solvent such as methylene chloride, at a moderate
temperature, such as room temperature, for at least about 10
minutes, for example, 2 hours, to give a spacer derivative in
excellent yield, for example 98%. The C-terminal protecting group,
for example, a benzyl ester, is removed, for example, by treatment
with 10% Pd--C and H.sub.2.
[0085] Coupling of the C-terminal deprotected left hand fragment
with a right hand molecular fragment (as described above), with for
example, HATU/HOAt, in the presence of a nitrogenous base, such as
diisopropylethylamine, in a nonpolar solvent, for example,
methylene chloride, at a moderate temperature, such as room
temperature, for at least about 4 hours, for example 22 hours,
yields an amino-protected peptide epoxide or amino-protected
peptide aziridine in acceptable yield, for example, 60%.
Simultaneous removal of the amino-terminal and, if present, hydroxy
side chain protecting groups, for example by treatment with TBAF in
THF at room temperature for at least about 10 minutes, for example
1 hour, is followed by N-acylation with commercially available
N--(N-biotinyl-6-aminohexanoyl)-6-aminohexanoic acid succinimidyl
ester (Biotin-X--X--NHS) in a polar, non-protic solvent, for
example, DMSO, at a moderate temperature, for example, room
temperature, for at least about 2 hours, for example, for 24 hours,
gives a biotinylated peptide epoxide or biotinylated peptide
aziridine in good yield, for example 87%.
Uses of Enzyme Inhibitors
[0086] Using epoxomicin and its biotinylated affinity derivative,
we show that epoxomicin covalently binds the LMP7, X, Z, and MECL1
catalytic .beta. subunits of the 20S proteasome and selectively
inhibits the three major 20S proteasome proteolytic activities at
different rates. Evidence is presented in vitro and in vivo that
epoxomicin effectively inhibits NF-.kappa.B-mediated
pro-inflammatory signaling. Given its unique specificity and
potency, these antitumor, anti-inflammatory natural products
represent novel classes of irreversible inhibitors distinct from
those reagents currently in use and, thus, might prove useful in in
vivo and in vitro analyses of proteasome function.
[0087] Peptide epoxides and peptide aziridines can inhibit
NF-.kappa.B activation, and stabilize p53 levels in cell culture.
Since NF-.kappa.B is a key regulator of inflammation, it is an
attractive target for anti-inflammatory therapeutic intervention.
While several peptide aldehyde proteasomal inhibitors have been
shown to target NF-.kappa.B activation, their potential
cross-reactivity with other cellular proteases limits their
usefulness in studying the biological processes of proteasome
function and also raises concerns for their use in studying
pathological disease processes. In comparison to these other
proteasome inhibitors, peptide epoxides and peptide aziridines are
unique, demonstrating potent and specific inhibition of the
proteasome and its functions.
[0088] The biological consequences of proteasome inhibition are
numerous. At the cellular level, the accumulation of
polyubiquitinated proteins, cell morphological changes, and
apoptosis have been reported upon treatment of cells with various
proteasome inhibitors. In our analyses, we have observed that p53
levels are stabilized over 50-fold by epoxomicin treatment. In
addition, our results showing an accumulation of polyubiquitinated
proteins in epoxomicin-treated cells provide evidence that the
proteasome is the target of epoxomicin. Proteasome inhibition has
also been suggested as a possible antitumor therapeutic strategy.
The fact that epoxomicin was initially identified in a screen for
antitumor compounds validates the proteasome as an antitumor
chemotherapeutic target. Moreover, in parallel with this study, we
have found that another antitumor natural product, eponemycin,
targets the proteasome, however, less potently.
[0089] The disclosed compounds are used to treat conditions
mediated directly by the proteolytic function of the proteasome
such as muscle wasting, or mediated indirectly via proteins which
are processed by the proteasome such as NF-.kappa.B. The proteasome
participates in the rapid elimination and post-translational
processing of proteins (e.g., enzymes) involved in cellular
regulation (e.g., cell cycle, gene transcription, and metabolic
pathways), intercellular communication, and the immune response
(e.g., antigen presentation). Specific examples discussed below
include .beta.-amyloid protein and regulatory proteins such as
cyclins and transcription factor NF-.kappa.B.
[0090] Alzheimer's disease is characterized by extracellular
deposits of .beta.-amyloid protein (.beta.-AP) in senile plaques
and cerebral vessels. .beta.-AP is a peptide fragment of 39 to 42
amino acids derived from an amyloid protein precursor (APP). At
least three isoforms of APP are known (695, 751, and 770 amino
acids). Alternative splicing of mRNA generates the isoforms; normal
processing affects a portion of the .beta.-AP sequence, thereby
preventing the generation of .beta.-AP. It is believed that
abnormal protein processing by the proteasome contributes to the
abundance of .beta.-AP in the Alzheimer brain. The APP-processing
enzyme in rats contains about ten different subunits (22 kDa-32
kDa). The 25 kDa subunit has an N-terminal sequence of
X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser (SEQ ID NO:2), which is
identical to the .beta.-subunit of human macropain (Kojima, S. et
al., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing
enzyme cleaves at the Gln.sup.15-Lys.sup.16 bond; in the presence
of calcium ion, the enzyme also cleaves at the Met-.sup.1-Asp.sup.1
bond, and the Asp.sup.1-Ala.sup.2 bonds to release the
extracellular domain of .beta.-AP.
[0091] One embodiment, therefore, is a method of treating
Alzheimer's disease, including administering to a subject an
effective amount of a compound (e.g., pharmaceutical composition)
disclosed herein. Such treatment includes reducing the rate of
.beta.-AP processing, reducing the rate of .beta.-AP plaque
formation, and reducing the rate of .beta.-AP generation, and
reducing the clinical signs of Alzheimer's disease.
[0092] Other embodiments of the invention relate to cachexia and
muscle-wasting diseases. The proteasome degrades many proteins in
maturing reticulocytes and growing fibroblasts. In cells deprived
of insulin or serum, the rate of proteolysis nearly doubles.
Inhibiting the proteasome reduces proteolysis, thereby reducing
both muscle protein loss and the nitrogenous load on kidneys or
liver. Proteasome inhibitors are useful for treating conditions
such as cancer, chronic infectious diseases, fever, muscle disuse
(atrophy) and denervation, nerve injury, fasting, renal failure
associated with acidosis, and hepatic failure. See, e.g., Goldberg,
U.S. Pat. No. 5,340,736. Embodiments of the invention therefore
encompass methods for: reducing the rate of muscle protein
degradation in a cell; reducing the rate of intracellular protein
degradation; reducing the rate of degradation of p53 protein in a
cell; and inhibiting the growth of p53-related cancers. Each of
these methods includes the step of contacting a cell (in vivo or in
vitro, e.g., a muscle in a subject) with an effective amount of a
compound (e.g., pharmaceutical composition) disclosed herein.
[0093] Another protein processed by the proteasome is NF-.kappa.B,
a member of the Rel protein family. The Rel family of
transcriptional activator proteins can be divided into two groups.
The first group requires proteolytic processing, and includes p50
(NF-.kappa.B1, 105 kDa) and p52 (NF-.kappa.2, 100 kDa). The second
group does not require proteolytic processing, and includes p65
(RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can be
formed by Rel family members; NF-.kappa.B, for example, is a
p50-p65 heterodimer. After phosphorylation and ubiquitination of
I.kappa.B and p105, the two proteins are degraded and processed,
respectively, to produce active NF-.kappa.B which translocates from
the cytoplasm to the nucleus. Ubiquitinated p105 is also processed
by purified proteasomes (Palombella et al., Cell (1994)
78:773-785). Active NF-.kappa.B forms a stereospecific enhancer
complex with other transcriptional activators and, e.g., HMG I(Y),
inducing selective expression of a particular gene.
[0094] NF-.kappa.B regulates genes involved in the immune and
inflammatory response, and mitotic events. For example, NF-.kappa.B
is required for the expression of the immunoglobulin light chain
.kappa. gene, the IL-2 receptor .alpha.-chain gene, the class I
major histocompatibility complex gene, and a number of cytokine
genes encoding, for example, IL-2, IL-6, granulocyte
colony-stimulating factor, and IFN-.beta. (Palombella et al., Cell
(1994) 78:773-785). Some embodiments of the invention include
methods of affecting the level of expression of IL-2, MHC-I, IL-6,
IFN-.beta. or any of the other previously-mentioned proteins, each
method including administering to a subject an effective amount of
a compound disclosed herein. Complexes including p50 are rapid
mediators of acute inflammatory and immune responses (Thanos, D.
and Maniatis, T., Cell (1995) 80:529-532).
[0095] NF-.kappa.B also participates in the expression of the cell
adhesion genes that encode E-selectin, P-selectin, ICAm, and VCAM-1
(Collins, T., Lab. Invest. (1993) 68:499-508). One embodiment of
the invention is a method for inhibiting cell adhesion (e.g., cell
adhesion mediated by E-selectin, P-selectin, ICAm, or VCAM-1),
including contacting a cell with (or administering to a subject) an
effective amount of a compound (e.g., pharmaceutical composition)
disclosed herein.
[0096] NF-.kappa.B also binds specifically to the
HIV-enhancer/promoter. When compared to the Nef of mac239, the HIV
regulatory protein Nef of pbj14 differs by two amino acids in the
region which controls protein kinase binding. It is believed that
the protein kinase signals the phosphorylation of I-.kappa.B,
triggering I.kappa.B degradation through the ubiquitin-proteasome
pathway. After degradation, NF-.kappa.B is released into the
nucleus, thus enhancing the transcription of HIV (Cohen, J.,
Science, (1995) 267:960). Two embodiments of the invention are a
method for inhibiting or reducing HIV infection in a subject, and a
method for decreasing the level of viral gene expression, each
method including administering to the subject an effective amount
of a compound disclosed herein.
[0097] Intracellular proteolysis generates small peptides for
presentation to T-lymphocytes to induce MHC class I-mediated immune
responses. The immune system screens for autologous cells that are
virally infected or have undergone oncogenic transformation. One
embodiment is a method for inhibiting antigen presentation in a
cell, including exposing the cell to a compound described herein. A
further embodiment is a method for suppressing the immune system of
a subject (e.g., inhibiting transplant rejection), including
administering to the subject an effective amount of a compound
described herein.
[0098] Another further embodiment is a method for altering the
repertoire of antigenic peptides produced by the proteasome or
other Ntn with multicatalytic activity. For example, if the PGPH
activity of 20S proteasome is selectively inhibited, a different
set of antigenic peptides will be produced by the proteasome and
presented in MHC molecules on the surfaces of cells than would be
produced and presented either without any enzyme inhibition, or
with, for example, selective inhibition of chymotrypsin-like
activity of the proteasome.
[0099] Certain proteasome inhibitors block both degradation and
processing of ubiquitinated NF-.kappa.B in vitro and in vivo.
Proteasome inhibitors also block I.kappa.B-A degradation and
NF-.kappa.B activation (Palombella, et al. Cell (1994) 78:773-785;
and Traenckner, et al., EMBO J. (1994) 13:5433-5441). One
embodiment of the invention is a method for inhibiting
I.kappa.B-.alpha. degradation, including contacting the cell with a
compound described herein. A further embodiment is a method for
reducing the cellular content of NF-.kappa.B in a cell, muscle,
organ, or subject, including contacting the cell, muscle, organ, or
subject with a compound described herein.
[0100] Other eukaryotic transcription factors that require
proteolytic processing include the general transcription factor
TFIIA, herpes simplex virus VP16 accessory protein (host cell
factor), virus-inducible IFN regulatory factor 2 protein, and the
membrane-bound sterol regulatory element-binding protein 1.
[0101] Other embodiments of the invention are methods for affecting
cyclin-dependent eukaryotic cell cycles, including exposing a cell
(in vitro or in vivo) to a compound disclosed herein. Cyclins are
proteins involved in cell cycle control. The proteasome
participates in the degradation of cyclins. Examples of cyclins
include mitotic cyclins, G1 cyclins, (cyclin B). Degradation of
cyclins enables a cell to exit one cell cycle stage (e.g., mitosis)
and enter another (e.g., division). It is believed all cyclins are
associated with p34.sup.cdc2 protein kinase or related kinases. The
proteolysis targeting signal is localized to amino acids
42-RAALGNISEN-50 (destruction box; SEQ ID NO:19)). There is
evidence that cyclin is converted to a form vulnerable to a
ubiquitin ligase or that a cyclin-specific ligase is activated
during mitosis (Ciechanover, A., Cell, (1994) 79:13-21). Inhibition
of the proteasome inhibits cyclin degradation, and therefore
inhibits cell proliferation, for example, in cyclin-related cancers
(Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075).
One embodiment of the invention is a method for treating a
proliferative disease in a subject (e.g., cancer, psoriasis, or
restenosis), including administering to the subject an effective
amount of a compound disclosed herein. The invention also
encompasses a method for treating cyclin-related inflammation in a
subject, including administering to a subject an effective amount
of a compound described herein.
[0102] Additional embodiments are methods for affecting the
proteasome-dependent regulation of oncoproteins and methods of
treating or inhibiting cancer growth, each method including
exposing a cell (in vivo, e.g., in a subject or in vitro) to a
compound disclosed herein. HPV-16 and HPV-18-derived E6 proteins
stimulate ATP- and ubiquitin-dependent conjugation and degradation
of p53 in crude reticulocyte lysates. The recessive oncogene p53
has been shown to accumulate at the nonpermissive temperature in a
cell line with a mutated thermolabile E1. Elevated levels of p53
may lead to apoptosis. Examples of proto-oncoproteins degraded by
the ubiquitin system include c-Mos, c-Fos, and c-Jun. One
embodiment is a method for treating p53-related apoptosis,
including administering to a subject an effective amount of a
compound disclosed herein.
[0103] Finally, the disclosed compounds are also useful as
diagnostic agents (e.g., in diagnostic kits or for use in clinical
laboratories) for screening for proteins (e.g., enzymes,
transcription factors) processed by Ntn hydrolases, including the
proteasome. The disclosed compounds are also useful as research
reagents for specifically binding the X/MB1 subunit or
.alpha.-chain and inhibiting the proteolytic activities associated
with it. For example, the activity of (and specific inhibitors of)
other subunits of the proteasome can be determined.
[0104] Most cellular proteins are subject to proteolytic processing
during maturation or activation. Enzyme inhibitors disclosed herein
can be used to determine whether a cellular, developmental, or
physiological process or output is regulated by the proteolytic
activity of a particular Ntn hydrolase. One such method includes
obtaining an organism, an intact cell preparation, or a cell
extract; exposing the organism, cell preparation, or cell extract
to a compound disclosed herein; exposing the compound-exposed
organism, cell preparation, or cell extract to a signal, and
monitoring the process or output. The high selectivity of the
compounds disclosed herein permits rapid and accurate elimination
or implication of the Ntn (for example, the 20S proteasome) in a
given cellular, developmental, or physiological process.
Administration
[0105] The methods of the invention contemplate treatment of animal
subjects, such as mammals (e.g., higher primates, and especially
humans). The invention encompasses pharmaceutical compositions
which include novel compounds described herein, and pharmaceutical
compositions which include compounds described and first recognized
herein as Ntn inhibitors, such as the peptide epoxides and peptide
aziridines described herein.
[0106] Pharmaceutically acceptable salts may be formed, for
example, with 1, 2, 3, or more equivalents of hydrogen chloride,
hydrogen bromide, trifluoroacetic acid, and others known to those
in the art of drug formulation. Compounds of the invention can be
formulated into pharmaceutical compositions by admixture with
pharmaceutically acceptable non-toxic excipients and carriers. A
pharmaceutical composition of the invention may contain more than
one compound of the invention, and/or may also contain other
therapeutic compounds not encompassed by the invention, such as
anti-inflammatory, anti-cancer, or other agents. A compound of the
invention may be administered in unit dosage form, and may be
prepared by any of the methods well known in the pharmaceutical
art, for example, as described in Remington's Pharmaceutical
Sciences (Mack Pub. Co., Easton, Pa., 1980). The invention also
encompasses a packaged drug, containing a pharmaceutical
composition formulated into individual dosages and printed
instructions for self-administration.
[0107] Compounds disclosed herein as Ntn hydrolase inhibitors may
be prepared for use in parenteral administration, particularly in
the form of solutions or liquid suspensions; for oral
administrations, particularly in the form of tablets or capsules;
or intranasally, particularly in the form of powders, gels, oily
solutions, nasal drops, aerosols, or mists. Formulations for
parenteral administration may contain as common excipients sterile
water or sterile saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Controlled release of a compound of the invention may be
obtained, in part, by use of biocompatible, biodegradable polymers
of lactide, and copolymers of lactide/glycolide or
polyoxyethylene/polyoxypropylene. Additional parental delivery
systems include ethylene-vinyl acetate copolymer particles, osmotic
pumps, implantable infusion systems, and liposomes. Formulations
for inhalation administration contain lactose,
polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholate.
Formulations for buccal administration may include glycocholate;
formulations for vaginal administration may include citric
acid.
[0108] The concentration of a disclosed compound in a
pharmaceutically acceptable mixture will vary depending on several
factors, including the dosage of the compound to be administered,
the pharmacokinetic characteristics of the compound(s) employed,
and the route of administration. In general, the compounds of this
invention may be provided in an aqueous physiological buffer
solution containing about 0.1-10% w/v of compound for parenteral
administration. Typical dose ranges are from about 0.01 to about 50
mg/kg of body weight per day, given in 1-4 divided doses. Each
divided dose may contain the same or different compounds of the
invention. The dosage will be an effective amount depending on
several factors including the overall health of a patient, and the
formulation and route of administration of the selected
compound(s).
[0109] The effective amount of the active compound used to practice
the present invention for treatment of conditions directly or
indirectly mediate by Ntn hydrolases varies depending upon the
manner of administration, the age and the body weight of the
subject and the condition of the subject to be treated, and
ultimately will be decided by the attending physician or
veterinarian. Such amount of the active compound as determined by
the attending physician or veterinarian is referred to herein as
"effective amount".
[0110] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
[0111] The following examples illustrate some advantages and
properties of particular embodiments of the invention.
[0112] Materials and methods are generally described as follows.
Streptavidin-I-IRP, and streptavidin agarose were purchased from
Sigma. Suc-LLVY-AMC (SEQ ID NO:1), Boc-LRR-AMC were purchased from
Bachem. Z-LLE-AMC, clasto-lactacystin .beta.-lactone, NLVS, calpain
substrate I were obtained from CalBiochem. NeutrAvidin beads were
from Pierce. Picrylchloride was a gift from Dr. Philip Asekanse,
Yale University School of Medicine.
[0113] Cell culture and treatments are generally described as
follows. I-IUVE cells (generously provided by J. Pober, Yale
University) were cultured in Dulbecco's minimal essential medium
(DMEM, Gibco-BRL), containing 10% fetal bovine serum (FBS) and 50
.mu.g/ml endothelial cell growth factor (Sigma). The transformed
human kidney epithelial cell line (HEK-293) and Hela cell line were
cultured in DMEM containing 10% FBS. The mouse thymoma cell line
EL4 was cultured in RPMI (Gibco-BRL) plus 10% FBS. All cell culture
media were supplemented with 50 .mu.g/L penicillin and 50 .mu.g/L
streptomycin.
[0114] For .alpha.I.kappa.B western-blot analysis, 60% confluent
monolayers were treated in duplicate with 10 .mu.M epoxomicin for 2
hour in conjunction with 50 .mu.g/ml of cycloheximide. Cells were
then treated with or without TNF-.alpha. (10 ng/ml) and harvested
after 15 minutes of stimulation. For EMSAs, epoxomicin (doses
between 100 nM to 10 .mu.M) were added to Hela cells in duplicate
and subsequently treated with and without PMA (100 nM) for 1 hour
of PMA stimulation before nuclear lysates were prepared as
described in Mohan et al.; J. Biol. Chem. 273, (1998)
25903-25914.
Example 1
Purification of Epoxomicin-Binding Proteins
[0115] We synthesized a biotinylated epoxomicin analog to serve as
an affinity chromatography reagent. FIG. 1 shows the molecular
structure of epoxomicin and epoxomicin-biotin. Epoxomicin-biotin
was incubated with the murine thymoma cell line EIA for four hours,
cell lysates were analyzed by SDS-PAGE and subsequently the
membrane-immobilized cellular lysates were probed with
streptavidin-HRP. A detailed procedure is given below.
[0116] 10 L of EL4 cells (10.sup.6 cells/ml) were harvested and
resuspended in 50 ml of RPMI medium containing 10% PBS.
Epoxomicin-biotin was added to final concentration of 8 .mu.M and
cells were incubated for 4 hours at 37.degree. C. in 5% CO.sub.2.
After collection by centrifugation, cells were homogenized using a
Powergen homogenizer in lysis buffer (25 mM HEPES, 5 mM EGTA, 50 mM
NaF) plus protease inhibitors (10 .mu.g/ml of leupeptin, pepstatin
and soybean trypsin inhibitors and 1 mM PMSF). The high-speed
(100,000.times.g) supernatant was loaded into a 1 ml streptavidin
agarose to remove endogenous biotinylated proteins. The
flow-through fraction was then incubated for 10 minutes with 50 ml
of DE52 beads pre-equilibrated with lysis buffer, washed twice with
50 ml lysis buffer containing 0.1 M NaCl, and eluted with 50 ml
lysis buffer containing 0.3 mM NaCl. SDS was added to the eluant at
a final concentration of 0.5%, boiled for 10 minutes, and diluted
2.5.times. fold using lysis buffer. The diluted solution was loaded
onto a 0.4 ml NeutrAvidin agarose column. The flow-through fraction
was collected and reloaded onto the same column three times. After
extensive washes, epoxomicin-biotin binding proteins were eluted by
boiling the NeutrAvidin agarose in 0.4 ml 1.times.SDS sample
buffer. The purified protein complexes were separated by SDS-PAGE.
Excised protein bands were identified by the W. M. Keck Foundation
Biotechnology Resource Laboratory (Yale University) using liquid
chromatography quadrupole mass spectrometry/mass spectrometry (LCQ
(MS/MS)) and automated Edman degradation of internal tryptic
peptides.
[0117] Two major (23 and 30 kDa) and one minor (28 kDa) newly
biotinylated bands were detected (FIG. 2, lane 1). These proteins
were shown to specifically interact with epoxomicin-biotin since
pretreatment of cells with a five-fold molar excess of epoxomicin
prevents biotin binding to these proteins upon subsequent challenge
with epoxomicin-biotin (FIG. 2, lane 2).
[0118] After large-scale purification of these epoxomicin-binding
proteins using avidin affinity chromatography, the three
epoxomicin-binding proteins were digested with trypsin and the
resulting tryptic peptides subjected to Matrix assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-MS)
analysis. Comparison of these results with peptide masses generated
from theoretical tryptic digests of putative open reading frames in
GenBank revealed significant matches between two of the three
epoxomicin binding proteins (23 and 28 kDa) and two murine
proteasomal subunits.
[0119] The 23 kDa band was identified as LMP7, a
.gamma.-interferon-inducible catalytic .beta. subunit of the 20S
proteasome. The percentage of the LMP7 protein sequence covered was
31.9% for the tryptic peptides from the 23 kDa band. In some
experiments, an additional 24 kDa band was observed. MALDI-MS
analysis revealed that this band is also LMP-7. This minor LMP-7
band may represent a post-translational modification or possibly
allelic variation.
[0120] MALDI-MS analysis of tryptic peptides from the 28 kDa band
identified this epoxomicin-binding protein as the
.gamma.-interferon-inducible proteasome catalytic subunit, MECL1,
with 29% coverage between the tryptic peptides and the MECL1
protein sequence. Although the tryptic peptide masses of the 30 kDa
band matched 19.9% of the predicted protein sequence of the Z
catalytic subunit of the proteasome, this was less than the 20%
match criteria used to confirm protein identity. Therefore,
automated Edman degradation was performed on an internal tryptic
peptide from this 30 kDa protein. The resulting eleven amino acid
sequence was identical to residues 185-195 of the murine Z .beta.
catalytic proteasome subunit. Thus, epoxomicin was shown to bind
covalently the LMP7, MECL1, and Z proteasome catalytic
subunits.
[0121] Given that epoxomicin binds both Z and its
IFN-.gamma.-inducible counterpart MECL1, we tested whether
epoxomicin also binds in an analogous manner to the subunit X,
which is the housekeeping part of LMP7. Epoxomicin-biotin binding
to the human B cell line LCL 721.45 gave a similar pattern as
observed with the murine lymphoma line EL4 (FIG. 2, lane 3).
However, epoxomicin-biotin binding to a LCL 721.45 derivative line
which does not express LMP7 or LMP2, resulted in two
epoxomicin-biotin-binding proteins of 23 and 30 kDa (FIG. 2, lane
4). Immunoblot analysis confirmed that these bands are the X and Z
proteasome subunits, respectively.
Example 2
Enzyme Kinetic Assays
[0122] Peptide-AMC (AMC is 7-aminomethyl coumarin) substrates (5
.mu.M Suc-LLVY-AMC (SEQ ID NO:1), 5 .mu.M Z-LLE-AMC, 5-10 .mu.M
Boc-LRR-AMC) and inhibitors in DMSO were added to 50 .mu.L of assay
solutions (20 mM Tris-HCl; pH 8.0, 0.5 mM EDTA, plus 0.035% SDS for
Suc-LLVY-AMC (SEQ ID NO:1) and Z-LLE-AMC assays). Bovine red blood
cell proteasome was added to 50 .mu.L of the assay buffer
containing substrates and inhibitors for a final volume of 100
.mu.L at room temperature (22-26.degree.) in DynEx Microfluor
96-well plates and the fluorescence emission was immediately
measured at 460 nm (.lamda..sub.ex, 360 nm) using a Cytofluor
fluorescence plate reader for 50 minutes. The k.sub.obs/[I] values
were obtained using Kaleidagraph by non-linear least squares fit of
the data to the following equation for slow and tight binding
inhibition:
Fluorescence=v.sub.st+[(v.sub.O-v.sub.s)/k.sub.obs][1-e.sup.(-kobs.sup.t)-
], where v.sub.O and v.sub.s are the initial and final velocities,
respectively and k.sub.obs is the reaction rate constant. Values of
k.sub.obs represent an average of typically nine measurements
(generally three independent experiments with three different
inhibitor concentrations). Peptide epoxides exhibiting low or no
inhibition at 150 .mu.M were not tested at higher concentrations in
order to avoid problems with low solubility. It is assumed that
k.sub.obs/[I] values for these compounds are low compared with
inhibitors that allow curve-fit to data collected at concentrations
lower than 150 .mu.M. Dilutions of bovine erythrocyte 20S
proteasome (2.5 mg/ml) were as follows: 1:1200 dilution final for
Suc-LLVY-AMC (SEQ ID NO:1) activity, 1:3000 for Z-LLE-AMC, 1:800
for Boc-LRR-AMC. Inhibition reactions were performed as previously
described in Meng et al., Cancer Research, 59:2798-2801 (1999). For
calpain inhibition assays, the enzyme was used at 1 unit/mL, and
Suc-LLVY-AMC (SEQ ID NO:1) was used at a final concentration of 10
.mu.M in assay buffer containing 20 mM Tris, pH 8.0/1 mM
CaCl.sub.2/2 mM DTT. Cathepsin B was used at a concentration of
0.005 unit/mL in 100 mM sodium acetate/5 mM EDTA, pH 5.5, and
cathepsin substrate III was used as substrate at 40 .mu.M. Kinetic
assays were performed as described for the proteasome.
Example 3
Effect of Inhibitor Length on Proteasome Inhibition
[0123] We investigated the potency of inhibitors of various length.
Mono-, di-, tri- and tetraleucine .alpha.',.beta.'-epoxides were
ranked by determination of the k.sub.obs/[I] values for inhibition
of the chymotrypsin-like, trypsin-like, and PGPH activities of
purified bovine erythrocyte 20S proteasome. Enzyme kinetic assays
were performed as described in Example 2. The results are presented
in Table 1.
TABLE-US-00001 TABLE 1 Proteasomal Inhibition by Peptide Epoxides
of Varying Length k.sub.obs/[I] (M.sup.-1s.sup.-1)
Chymotrypsin-like trypsin-like activity activity PGPH activity
##STR00004## 14,000 (50-150 nm) .dagger. 9.2 (100-160 .mu.M)
##STR00005## 780 (1-2.5 .mu.M) 5.1 (100-150 .mu.M) 120 (8-12 .mu.M)
##STR00006## 3.1 (100-160 .mu.M) .dagger. .dagger. ##STR00007##
.dagger. .dagger. .dagger.
[0124] The dagger symbol indicates that inhibition at 150 .mu.M was
either absent or insufficient to allow curve-fit to the collected
data. The tetrapeptide showed strong inhibition of the
chymotrypsin-like activity and had essentially no effect on the
trypsin-like and PGPH activities. The tripeptide showed
significantly reduced inhibition of the chymotrypsin-like activity
with a small increase in the inhibition of PGPH activity. The di-
and monopeptides were found to be essentially inactive against all
three proteasomal activities.
Example 4
Proteasome/Protease Inhibition Studies
[0125] Purified bovine erythrocyte proteasome was enzymatically
assayed using a variety of different substrates and inhibitors. In
order to evaluate the rates of proteolytic inactivation by
inhibitors, k.sub.association (k.sub.obs/[I]) values were
determined using fluorogenic peptide substrates over a range of
inhibitor concentrations. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 Proteasomal Association Contants k.sub.assoc
= k.sub.obs/[I] (M.sup.-1 s.sup.-1) Chymotrypsin-like Trypsin-like
compound activity activity PGPH activity epoxomicin 35,400 .+-.
1400 287 .+-. 71 34 .+-. 4.8 (40-80 nM) (6-10 .mu.M) (25-75 .mu.M)
clasto-lactacystin 8530 .+-. 280 253 .+-. 41 37 .+-. 4.7 .beta.
lactone (200-400 nM) (6-10 .mu.M) (25-75 .mu.M) NLVS 6790 .+-. 919
5.3 .+-. 2.8 6.4 .+-. 2.3 (200-400 nM) (50-100 .mu.M) (50-100
.mu.M)
[0126] As shown in Table 2, epoxomicin most potently inhibited the
chymotrypsin-like activity of the 20S proteasome with a k.sub.assoc
value of 35,400 M.sup.-1 s.sup.-1. This rate of inactivation is
greater than four- and five-fold faster than clastolactacystin
.beta.-lactone and the vinyl sulfone NLVS, respectively.
Interestingly, epoxomicin and clasto-lactacystin .beta.-lactone
displayed near identical inhibitory activities against the
trypsin-like and the PGPH activities.
Example 5
Inhibition of PGPH Activity
[0127] As the first step toward developing a peptide inhibitor(s)
selective for PGPH activity of the proteasome (that is, cleavage of
peptide bonds after acidic residues), we synthesized a series of
peptidyl .alpha.',.beta.'-epoxyketones with the acidic residue
glutamate at a first amino acid position. Both isooctanoyl-Ser-Glu-
and isooctanoyl-Thr-Glu-.alpha.',.beta.'-epoxyketones proved to be
poor PGPH-selective inhibitors (data not shown). This was
surprising since eponemycin
(isooctanoyl-Ser-Leu-.alpha.',.beta.'-epoxyketone) shows a modest
selectivity toward the PGPH activity (Table 1). Similarly, other
compounds, such as Ac-Ile-Ile-Thr-Glu- (SEQ ID NO: 3),
Ac-Ala-Val-Glu-, Ac-Ile-Val-Glu-, Ac-Gly-Pro-Phe-Glu- (SEQ ID
NO:4), and Ac-Pro-Phe-Glu-.alpha.',.beta.'-epoxyketones, did not
display any significant selectivity for the PGPH activity (data not
shown).
TABLE-US-00003 TABLE 3 Enzyme Inhibition for 20S Proteasome
Peptidase Activities k.sub.obs/[I] (M.sup.-1s.sup.-1) Compounds
Chymotrypsin-like PGPH Trypsin-like Ac-GPFL-Ex 5 .+-. 1 (50-150
.mu.M) 190 .+-. 10 (4-215 .mu.M) NI (100-150 .mu.M) (SEQ ID NO: 5)
Ac-NorLPFL-Ex 1.9 .+-. 0.6 (100-150 .mu.M) 115 .+-. 15 (10-40
.mu.M) NI (100-150 .mu.M) (SEQ ID NO: 6) Ac-PFL-Ex 42 .+-. 2
(50-100 .mu.M) 32 .+-. 6 (25-75 .mu.M) NI (100-150 .mu.M)
N-dimethylaminobenzoyl- 220 .+-. 70 (1-2 .mu.M) 150 .+-. 40 (10-40
.mu.M) NI (100-150 .mu.M) PFL-EX Isooctanioc-PFL-Ex 190 .+-. 60
(1-2 .mu.M) 194 .+-. 4 (10-40 .mu.M) NI (100-150 .mu.M)
Benzoylbenzoic-PFL-Ex 8.7 .+-. 0.2 (80-100 .mu.M) 140 .+-. 30
(10-40 .mu.M) NI (100-150 .mu.M) Pyrazinecarbonyl-PFL-Ex 520 .+-.
40 (1-2 .mu.M) 23 .+-. 3 (40-80 .mu.M) 3.7 .+-. 1.1 (125-150 .mu.M)
Ac-GGPFL-Ex 170 .+-. 60 (20-60 .mu.M) 170 .+-. 30 (10-40 .mu.M) 1.3
.+-. 0.3 (125-150 .mu.M) (SEQ ID NO: 7) Ac-PPFL-Ex 6.4 .+-. 3.3
(20-60 .mu.M) 10.6 .+-. 0.3 (40-80 .mu.M) NI (125-150 .mu.M) (SEQ
ID NO: 8) N-acetyl- 31.2 .+-. 0.6 (60-80 .mu.M) 45.3 .+-. 2.2
(40-80 .mu.M) NI (125-150 .mu.M) piperidinecarbonyl-PFL-Ex
Ac-IVL-Ex 5 .+-. 0.1 (100-150 .mu.M) 14.6 .+-. 4.2 (50-125 .mu.M)
6.7 .+-. 0.1 (100-150 .mu.M) Ac-AVL-Ex 10.8 .+-. 0.2 (100-150
.mu.M) 12.7 .+-. 1.3 (50-100 .mu.M) 1.8 .+-. 0.1 (125-150 .mu.M)
Eponemycin 58 .+-. 14 (25-62.5 .mu.M) 175 .+-. 2 (12.5-50 .mu.M) 17
.+-. 3 (100-150 .mu.M)
[0128] The one-letter code for amino acids is used in Table 3. The
values in parentheses indicate the concentration range of inhibitor
used. The entry "NI" indicates no inhibition observed in the
concentration range indicated. The prefix "Ac" is acetyl, the
suffix "Ex" is .alpha.',.beta.'-epoxide having an .alpha.'-methyl
substituent, and "NorL" is nor-leucine. The stereochemistry of all
epoxides is such that the oxygen atom is above the plane of the
molecule when drawn as shown in formula I (that is, the .beta.
stereoisomer).
[0129] As seen in Table 3, the first two entries (Ac-GPFL-Ex (SEQ
ID NO:5), and Ac-NorLPFL-Ex (SEQ ID NO:6)) showed the highest
selectivity toward the PGPH activity with 35- and 60-fold higher
values of k.sub.obs/[I] for inhibition of the PGPH activity than
the chymotrypsin-like activity, respectively. In contrast, the
compounds N-dimethylaminobenzoyl-PFL-EX and isooctanioc-PFL-Ex
showed no selectivity by inhibiting both the chymotrypsin-like and
the PGPH activities. Despite their differences in selectivity
toward the PGPH activity, compounds Ac-GPFL-Ex (SEQ ID NO:5),
Ac-NorLPFL-Ex (SEQ ID NO:6), N-dimethylaminobenzoyl-PFL-EX and
isooctanioc-PFL-Ex showed similar values of k.sub.obs/[I] for
inhibition of the PGPH activity, whereas all showed no inhibition
of the trypsin-like activity of the proteasome.
[0130] Table 1 shows that .alpha.',.beta.'-epoxyketone inhibitors
with Pro-Phe-Leu at the P3-P1 positions displayed no significant,
if any, inhibition of the trypsin-like activity, regardless of the
nature of residues at the P4 position. These results suggest that
the presence of Pro-Phe-Leu as the backbone of the proteasome
inhibitor would prevent these inhibitors from binding the subunits
responsible for the trypsin-like activity. Secondly, the presence
of a protecting group consisting a bulky aromatic group in place of
an acetyl group at the amino terminus provided a stronger
inhibition (.about.5-12-fold) towards the chymotrypsin-like
activity, thus making them far less PGPH-selective. Similarly,
compounds with larger groups at the P4 position displayed a modest
increase in inhibition of the chymotrypsin-like activity.
Example 6
Specificity for 20S Proteasome
[0131] We also investigated whether epoxomicin shared protease
inhibitory specificities with two other classes of peptide-based
proteasome inhibitors (i.e., peptide aldehydes and peptide vinyl
sulfones). While several peptide aldehydes potently inhibit the
proteasome, they also inhibit other intracellular non-proteasomal
proteolytic activities such as calpain and lysosomal proteases
(cathepsins). Epoxomicin did not display any inhibitory activity
against the calcium-dependent protease calpain, papain,
chymotrypsin, trypsin and cathepsin B at concentrations up to 50
.mu.M, whereas, the vinyl sulfone NLVS and clasto-lactacystin
.beta.-lactone significantly inhibited cathepsin B enzymatic
activity at 10 .mu.M and 50 .mu.M, respectively.
Example 7
Effect of Biotin on Epoxomicin Binding Specificity
[0132] Since biotin-epoxomicin was used to identify epoxomicin
binding proteins, it was important to determine if the biotin
affinity linker spuriously affects the binding specificity of the
natural product. A possible change in subunit binding specificity
is plausible since it has been shown that the length of
peptide-based proteasome inhibitors influences their specificity
for different proteasome catalytic subunits. For example, the vinyl
sulfone peptide, NLVS, has been shown to bind to the X subunit and
its .gamma.-interferon-inducible counterpart LMP7, both of which
are responsible for the CT-L proteolytic activity. However, an
extension of the peptide backbone by an additional amino acid
allows the resulting tetrapeptide vinyl sulfone,
H-Leu-Leu-Leu-Leu-VS (SEQ ID NO:9) to bind LMP7/X subunits as well
as the Z subunit and its .gamma.-interferon-inducible counterpart,
MECL1. The latter two subunits are responsible for the trypsin-like
(T-L) catalytic activity of the proteasome. To exclude the
possibility that biotinylation alters the binding of epoxomicin to
its target proteins, we prepared [.sup.3H]-labeled epoxomicin, as
shown in FIG. 3. The benzyl ester of fully protected tripeptide was
removed by hydrogenolysis to furnish the acid, which was coupled to
the right-hand fragment. The resulting Fmoc-protected epoxomicin
was successively treated with piperidine, [.sup.3H]-acetic
anhydride, and tetrabutylammonium fluoride to give
[.sup.3H]-epoxomicin. Various concentrations of epoxomicin-biotin
were added to different sets of murine thymoma EL4 cells for 6
hours. Cell lysates were analyzed by denaturing sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by
protein immobilization on PVDF membrane and visualization of
biotinylated proteins via avidin-horseradish peroxidase enhanced
chemiluminescence. Since SDS denatures proteins, the presence of
biotinylated proteins after addition of biotinylated epoxomicin and
SDS-PAGE indicates the presence of covalent protein adduct
formation. As shown in FIG. 4, the addition of increasing
concentrations of biotinylated epoxomicin resulted in newly
biotinylated proteins of 23 kDa and 30 kDa in size. In addition to
these two major epoxomicin-binding proteins, a minor 28 kDa protein
band was also observed. MALDI-MS analyses of peptides generated by
trypsin digestion of 23, 30, and 28 kDa epoxomicin-binding proteins
identified them as LMP2, Z and MECLI catalytic subunits,
respectively. In addition, the use of different concentrations of
epoxomicin-biotin demonstrated that LMP7 is more rapidly modified
than the Z or MECL1 subunits. Adduct formation between
epoxomicin-biotin and these binding proteins most likely occurs via
nucleophilic attack on the epoxyketone of epoxomicin by the
proteasome subunits' catalytic amino-terminal threonine. This has
been previously shown for lactacystin binding. Incubation with a
ten equivalent excess of epoxomicin with cells for 30 minutes prior
to challenge with epoxomicin-biotin resulted in a significant
decrease in biotin incorporation into all three protein targets
(FIG. 4, lane 5). This competition for epoxomicin-biotin binding
confirms that the interaction is specific.
[0133] As shown in FIG. 4, lane 6, addition of [.sup.3H] epoxomicin
to purified murine spleen proteasome resulted in the same protein
binding pattern observed with epoxomicin-biotin (FIG. 4, lane 4).
This finding validates the use of epoxomicin-biotin as a probe of
epoxomicin's biological activity.
Example 8
Effect of Epoxomicin on p53 Cell Levels
[0134] Given that epoxomicin covalently binds proteasome subunits
in intact cells and inhibits the proteolytic activity of purified
proteasome in vitro, we investigated the ability of epoxomicin to
inhibit in vivo proteasome function in cultured cells. The tumor
suppressor p53 is a known target of the proteasome.
[0135] As shown in FIG. 5A, incubation of HUVECs with 100 nM
epoxomicin (Epx) for 6 hours resulted in a 30-fold increase in p53
protein levels. In contrast, incubation with a 50-fold higher
concentration (5 .mu.M) of lactacystin (Lac) resulted in only a
10-fold increase of p53 levels over that of untreated cells. The
control experiment (Crtl) was carried out with vehicle only. Since
inhibition of proteasome function should result in increased levels
of ubiquitinated proteins, we also analyzed the effect of
epoxomicin on total intracellular ubiquitinated protein
accumulation.
[0136] Incubation of Hela cells with 10 .mu.M epoxomicin for 2
hours (FIG. 5B, lane 2) resulted in the accumulation of multiple
higher molecular weight bands recognized by anti-ubiquitin
antibody. These upper molecular weight bands were also observed in
cellular lysates of cells treated with the peptide aldehyde
inhibitor, Z-LLL-H (FIG. 5B, lane 3). The control experiment (Crtl)
was carried out with vehicle only.
Example 9
Optimized Enzyme Inhibitors
[0137] Investigations of variation in the identities of amino acid
sidechains were carried out to determine the effect of the
sidechains on inhibition of proteasomal activities. The effect of
steric bulk was investigated at each of the various positions of a
tetrapeptide epoxide, with other side chains held constant as
leucine.
[0138] The effect of steric bulk at P.sub.2 was investigated with
the following series of compounds, represented by the general
structure below.
##STR00008##
[0139] When P.sub.2 was methyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
16,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was 4.1, and the k.sub.obs/[I] for PGPH activity using
Z-LLE-AMC was 20.
[0140] When P.sub.2 was isopropyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
14,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was 9.2.
[0141] When P.sub.2 was phenyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO: 1) was
54,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0142] When P.sub.2 was 1-naphthyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO: 1) was
29,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0143] The effect of steric bulk at P.sub.3 was investigated with
the following series of compounds, represented by the general
structure below.
##STR00009##
[0144] When P.sub.3 was methyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
1,300, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was 2.0, and the k.sub.obs/[I] for PGPH activity using
Z-LLE-AMC was 130.
[0145] When P.sub.3 was isopropyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
14,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was 9.2.
[0146] When P.sub.3 was phenyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
8,500, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0147] When P.sub.3 was 1-naphthyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
31,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0148] The effect of steric bulk at P.sub.4 was investigated with
the following series of compounds, represented by the general
structure below.
##STR00010##
[0149] When P.sub.4 was methyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
5,300, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was 3.8, and the k.sub.obs/[I] for PGPH activity using
Z-LLE-AMC was 220.
[0150] When P.sub.4 was isopropyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
14,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was 9.2.
[0151] When P.sub.4 was phenyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
37,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was 5.5, and the k.sub.obs/[I] for PGPH activity using
Z-LLE-AMC was 78.
[0152] When P.sub.4 was methylphenyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
63,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was 5.4, and the k.sub.obs/[I] for PGPH activity using
Z-LLE-AMC was 50.
[0153] When P.sub.4 was 1-naphthyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
29,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0154] When P.sub.4 was p-benzoylphenyl, the k.sub.obs/[I] for
chymotryptic-like activity using Suc-LLVY-AMC (SEQ ID NO:1) was
23,000, the k.sub.obs/[I] for tryptic-like activity using
Boc-LRR-AMC was not measurable, and the k.sub.obs/[I] for PGPH
activity using Z-LLE-AMC was not measurable.
[0155] Compounds with aromatic residues in positions P.sub.2 and
P.sub.3 increased the inhibition of chymotrypsin-like activity,
with phenylalanine (P.sub.2) and 3-(1-naphthyl)alanine (P.sub.3) as
useful substituents. Small, neutral side chains in positions
P.sub.2 and P.sub.3 can be used to make compounds showing
inhibition of trypsin-like and PGPH activities, with alanine
(P.sub.2, P.sub.3) as a useful substituent. Compounds with aromatic
residues in position P.sub.4 increased the inhibition of
chymotrypsin-like activity, with phenylalanine and
homophenylalanine as useful substituents. The same experiments
carried out with bovine brain 20S proteasome resulted in similar
rankings of substituents.
Example 10
A Particular Optimized Enzyme Inhibitor
[0156] A particular optimized inhibitor, referred to as "compound
16", was prepared and evaluated similarly. This compound had
leucine at positions P.sub.1 and P.sub.3, phenylalanine at position
P.sub.2, and homophenylalanine at position P.sub.4, as shown
below.
##STR00011##
[0157] The k.sub.obs/[I] for chymotryptic-like activity using
Suc-LLVY-AMC (SEQ ID NO:1) was 183,000. The k.sub.obs/[I] for
tryptic-like activity using Boc-LRR-AMC was 7.1, and the
k.sub.obs/[I] for PGPH activity using Z-LLE-AMC was 21.
[0158] The deacylated version of compound 16, is referred to as
"compound 18", and was less potent a chymotrypsin-like inhibitor
(k.sub.obs/[I] for Suc-LLVY-AMC (SEQ ID NO:1) of 5200), but also
exhibited a major increase in potency for inhibition of
trypsin-like activity (k.sub.obs/[I] for Boc-LRR-AMC of 580).
Compound 18 was prepared directly from compound 16 by treatment
with the N-terminal deprotecting agent, tetrabutylammoniumfluoride,
in tetrahydrofuran.
Example 11
Comparison of Various Inhibitors of Proteasomal Activity
[0159] Efforts to optimize an inhibitor of CT-L activity in the
proteasome lead to the development of peptide-based enzyme
inhibitors having particular substituents in the P1-P4 positions.
The results are presented in Table 4. In Table 4, compound 16 is
the particular optimized enzyme inhibitor of Example 10. Compound
18 is the free amino terminal derivative of compound 16, also of
Example 10.
TABLE-US-00004 TABLE 4 Proteasome Inhibition by Various Inhibitors
k.sub.assoc = k.sub.obs/[I] (M.sup.-1s.sup.-1) Chymotrypsin-like
Trypsin-like compound activity activity PGPH activity ##STR00012##
166,000(5-12 nM) 7.1(80-130 .mu.M) 21(80-150 .mu.M) ##STR00013##
5200(0.11 .mu.M) 580(0.5-5 .mu.M) 11(10-150 .mu.M) ##STR00014##
37,000(30-80 nM) 79(8-12 .mu.M) 37(50-100 .mu.M) ##STR00015##
53,000(30-100 nM) 150(5-35 .mu.M) 3200(0.3-1 .mu.M) ##STR00016##
5000(200-500 nM) 3.4(50-120 .mu.M) 4.0(50-100 .mu.M) ##STR00017##
7400(150-500 nM) 68(8-12 .mu.M) 47(30-80 .mu.M)
Example 12
Correlation of Inhibition of Chymotrypsin-Like Activity and Growth
Inhibition of Bovine Aortic Endothelial Cells
[0160] The in vitro data obtained with respect to inhibition of
enzymatic, specifically chymotrypsin-like activity of 20S
proteasome can be translated to in vivo applicability by
correlating the inhibition with the inhibition of cell
proliferation of bovine aortic endothelial (BAE) cells. BAE cells
were cultured at 7% CO.sub.2 and were grown in DMEM supplemented
with 10% FBS, 1% penicillin/streptomycin (GIBCO), and 1 mM sodium
pyruvate.
[0161] Approximately 1000 BAE cells were plated into each well of a
96-well culture plate. After an overnight incubation, different
concentrations of proteasome inhibitors were added and incubation
was continued for another five days. After five days, all
inhibitors consistently inhibited cell growth in a dose-dependent
manner. Subsequently, 74 kBq of [methyl-3H]-thymidine was added to
each well of the plate and the plate was incubated for another
additional 4 hours. Cells were harvested using a Skatron Cell
Harvester and incorporated radioactivity was quantified using
liquid scintillation. Proliferation of BAE cells was assayed in
triplicate in the presence of different concentrations of peptide
epoxides. The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Chymotrypsin-Like Activity Inhibition and
BAE Cell Proliferation Inhibitor k.sub.assoc = k.sub.obs/[I]
(M.sup.-1 s.sup.-1) IC.sub.50 (nM) Ac-LLAL-epoxide 16,000 34 (SEQ
ID NO: 13) Ac-LLFL-epoxide 54,000 7 (SEQ ID NO: 14) Ac-LALL-epoxide
1300 700 (SEQ ID NO: 15) Ac-LFLL-epoxide 8500 16 (SEQ ID NO: 16)
Ac-ALLL-epoxide 5300 83 (SEQ ID NO: 17) Ac-hFLFL-epoxide 166,000 20
(SEQ ID NO: 18)
[0162] The group "Ac" refers to an acetyl group as N-terminal
protecting group, L is leucine, A is alanine, F is phenylalanine,
and hF is homophenylalanine. "Epoxide" is the
.alpha.'-methyl-.alpha.',.beta.'-epoxide group as detailed
herein.
[0163] FIG. 6 shows the correlation of the log k.sub.obs/[I] values
for inhibition of the chymotrypsin-like activity of 20S proteasome
with the log IC.sub.50 values for inhibition of bovine aortic
endothelial cell proliferation by peptide epoxides. The trend
observed in FIG. 6 suggests that the proteasome is the biological
target for peptide epoxide compounds.
Example 13
Correlation of Inhibition of PGPH Activity and Growth Inhibition of
Bovine Aortic Endothelial Cells
[0164] Perturbation of the proteasome activity can result in
inhibition of cell growth, and can further lead to cell death at
elevated concentration of the proteasome inhibitors. This provides
an excellent cell-based assay to assess the importance of each
catalytic activity of the 20S proteasome in degradation of proteins
involved in cell growth. We measured inhibition of proliferation of
bovine aortic endothelial (BAE) cells with our
.alpha.',.beta.'-epoxyketone-based inhibitors with varying degree
of specificity for the PGPH activity. When BAE cells were treated
with Ac-GPFL-Ex (SEQ ID NO:5), Ac-NorLPFL-Ex (SEQ ID NO:6),
N-dimethylaminobenzoyl-PFL-EX and isooctanioc-PFL-Ex, the compounds
with the higher values of k.sub.obs/[I] for the inhibition of the
chymotrypsin-like activity proved to be more active as
antiproliferative agents (Table 6).
TABLE-US-00006 TABLE 6 PGPH Activity Inhibition and BAE Cell
Proliferation Inhibitor IC.sub.50 (.mu.M) Ac-GPFL-epoxide 58 (SEQ
ID NO: 5) Ac-NorLPFL-epoxide 53 (SEQ ID NO: 6)
N-dimethylaminobenzoyl- 0.4 PFL-epoxide Isooctanoic-PFL-epoxide 0.6
Pyrazinecarbonyl-PFL- 0.4 epoxide
[0165] While all compounds arrested cell growth, a greater than
100-fold higher concentration of Ac-GPFL-Ex (SEQ ID NO:5) and
Ac-NorLPFL-Ex (SEQ ID NO:6) in comparison to
N-dimethylaminobenzoyl-PFL-EX and isooctanioc-PFL-Ex was needed to
cause 50%-inhibition of proliferation. This result reflects that
despite all these compounds have similar potency for inhibition of
the PGPH activity, N-dimethylaminobenzoyl-PFL-EX and
isooctanioc-PFL-Ex inhibit the chymotrypsin-like activity
40-1.5-fold more strongly than Ac-GPFL-Ex (SEQ ID NO:5) and
Ac-NorLPFL-Ex (SEQ ID NO:6) (see Table 6 above). Therefore, the
greater than 100-fold difference in IC.sub.50 values suggests that
inhibition of PGPH activity of the proteasome in the cell is not
sufficient to cause inhibition of proliferation of BAE cells.
Example 14
Electrophoretic Mobility Shift Assays
[0166] EMSAs were performed as described in Mohan et al.; J. Biol.
Chem. 273, (1998) 25903-25914. In brief, consensus DNA binding
oligonucleotide sequences for transcription is factors NF-.kappa.B
(5'-AGTTGAGGGGACTTTCCCAGGC-3'; SEQ ID NO: 10) and AP-2
(5'-GATCGAACTGACCGCCCGCGGCCCGT-3'; SEQ ID NO:11) from Santa Cruz
Biotechnology were labeled with .sup.32P-.gamma.-ATP and incubated
with equal amounts of nuclear lysates. Protein-DNA complexes were
separated on 4% polyacrylamide gels under non-denaturing and
non-reducing conditions. The gels were dried and exposed to a
phosphoimaging screen (Molecular Dynamics) for quantitation of
radioactivity in retarded bands. Results were representative of
experiments performed at least twice.
[0167] We investigated whether epoxomicin blocks
TNF-.alpha.-induced activation of NF-.kappa.B by stabilizing
1.kappa.B.alpha.. Western blot analysis revealed that 10 ng/ml
TNF-.alpha. potently induced the degradation of 1-.alpha. in Hela
cells within 15 minutes of treatment (FIG. 7, lane 2). However,
pretreatment with 10 .mu.M epoxomicin for 2 hours in the presence
of cycloheximide inhibited I.kappa.B.alpha. degradation by 10 fold
(FIG. 7, lane 3), to a level similar to that produced by treating
TNF-.alpha.-stimulated cells with 10 .mu.M of the peptide aldehyde
inhibitor Z-LLLH (SEQ ID NO:12) (FIG. 7, lane 4). Control
experiments with performed with vehicle (lane 1) and 10 ng/mL
TNF.alpha..
[0168] Next, using electrophoretic mobility shift assays (EMSA), we
tested whether epoxomicin inhibits NF-.kappa.B DNA binding
activity. Hela cells were treated with increasing concentrations of
epoxomicin for 2 hours, and subsequently, 10 ng/mL TNF-.alpha. was
added to drug-treated cells or to untreated cultures and incubated
for one hour. Equal amounts of protein from nuclear extracts
prepared from untreated and treated cultures were incubated with a
radiolabeled NF-.kappa.B oligonucleotide or an control AP-2
oligonucleotide and fractionated on 4% polyacrylamide gels. Dried
gels were exposed to a PhosphorImaging screen. The amount of
radioactivity in the transcription factor-retarded bands was
quantitated and represented as fold-change over that of untreated
samples.
[0169] EMSA of nuclear lysates derived from Hela cell cultures
treated with TNF-.alpha. alone (FIG. 8, lanes 1 and 5) showed
greater than 2-fold induction of NF-.kappa.B DNA binding activity
over that of untreated cells. Epoxomicin pretreatment for 1 hour
produced a significant dose-dependent reduction in
TNF-.alpha.-stimulated NF-.kappa.B DNA binding activity (FIG. 8:
100 nM epoxomicin, lanes 2 and 6; 1 .mu.M epoxomicin, lanes 3 and
7; 10 .mu.M epoxomicin, lanes 4 and 8). These effects were
selective, since we determined that the DNA binding activity of
AP-2 was unaltered. Additionally, incubation of HEK 293 cells with
100 nM epoxomicin for up to 5 hours did not inhibit the
PMA-stimulated DNA binding of transcription factor AP-2.
[0170] Epoxomicin targets NF-.kappa.B-mediated signaling, as shown
by the fact that the natural product stabilized levels of
I.kappa.B.alpha. resulting in inhibition of the DNS binding
activity of NF-.kappa.B in the nucleus. This transcriptional
inhibition is specific since DNA binding activity of activator
protein-2 (AP-2), which was also stimulated by TNF-.alpha. and PMA,
was not affected by epoxomicin up to 10 .mu.M.
Example 15
Assay for Contact Sensitivity
[0171] In order to address the role of the proteasome in
pathological inflammatory processes, we explored epoxomicin's
potential as an in vivo anti-inflammatory agent. Contact
sensitivity (CS) is a cutaneous immune inflammatory response that
is mediated by CD4.sup.+ T-cells in the classical delayed-type
hypersensitivity (DTH) reaction. The DTH response in mice immunized
with such haptens as picrylchloride (2,4,6-trinitro chlorobenzene),
2,4-dinitrofluorobenzene (DNFB), or 2,4,6-trinitrobenzene sulfone
has been well characterized, being mediated by the proinflammatory
cytokines .gamma.-IFN and TNF-.alpha.. The DTH response, which is
initiated by antigen-specific and nonspecific factors leads to
local increase in vascular permeability, in part, by serotonin
release that enables circulating CD4.sup.+ effector T-cells to
migrate into local sites of antigen challenge. Using the
picrylchloride model of CS, we demonstrated that epoxomicin
administration at non-toxic doses (as judged by absence of weight
loss) was found to reduce CS significantly. Since picrylchloride
also exerts a degree of non-specific irritation and nonspecific
inflammation, we tested the efficacy of epoxomicin in the skin
irritation assay using nonimmunized mice. A single bolus of
epoxomicin abrogated 95% of the inflammatory response.
[0172] Contact sensitivity (CS) and irritant response assays to
picrylchloride challenge was performed essentially as described in
Tsuji et al. J. Immunol., 156, (1996) 4444-50, with slight
modifications. In brief, mice were injected intraperiteoneally
(i.p.) daily for six days with vehicle or epoxomicin (0.58 mg/kg
body weight) solubilized in 10% DMSO-phosphate buffered saline. Six
days after immunization with picrylchloride, ear thickness
measurements (0 hour) of both ears were made in triplicate with an
engineer's micrometer (Peacock dial thickness gauge, Ozaki
Manufacturing Co., LTD., Japan). Mice were subsequently challenged
on both ear lobes by application of 10-15 microliters of a 0.2-0.8%
solution of picrylchloride or DNFB (solubilized in
acetone/high-grade extra virgin olive oil). Ear swelling
measurements were made again 24 hours post-ear challenge. In a
second assay, elicitation of inflammatory response to the
nonspecific vascular activation and permeability effects of
irritant response were determined using two groups of four
non-immunized mice. The 0-hour ear thickness measurements were
made, a single high-dose injection of epoxomicin (2.9 mg/kg) was
delivered intraperiteoneally to one group and the control group was
treated with vehicle. After one hour, the ears of all mice were
challenged with irritant as in CS assay and ear thickness
measurements taken 24 hours post-ear challenge.
[0173] Contact sensitivity is an inflammatory response to certain
classes of chemical compounds and haptens. Based on inhibition of
NF-.kappa.B activation in cell culture, described in Example 14, we
hypothesized that epoxomicin would have anti-inflammatory activity
in vivo. To test this hypothesis, epoxomicin was evaluated in the
picrylchloride mouse model of contact sensitivity. Mice were
immunized with picrylchloride and 6 days post-immunization were
challenged by application of picrylchloride on their ears. Ear
thickness measurements were made at 0 and 24 hours after
picrylchloride ear-challenge. As shown in FIG. 9A, daily treatment
with epoxomicin at a non-toxic dose of 0.58 mg/kg/day reduced the
contact sensitivity response by 44% relative to the control group
of mice treated with vehicle alone.
[0174] Since the hapten can elicit a non-specific
irritation-related inflammatory response, we explored the effects
of epoxomicin on skin irritation-mediated inflammation using
non-immunized mice. In a second experiment, mice were pretreated
with epoxomicin at a dose 5 times higher than that used previously
to test the idea that a single injection of the drug could reduce
inflammation in response to picrylchloride ear challenge.
Epoxomicin administered at 2.9 mg/kg potently inhibited the
irritant-associated inflammatory response by 95% when ear edema
measurements were made 24-hour post challenge (FIG. 9B).
[0175] Mice were injected with either a low dose (0.32 mg/kg) or a
high dose (2.0 mg/kg) of epoxomicin, the inhibitor compound 16 (see
Example 10) or vehicle (10% DMSO) alone, one hour before applying
DNFB to the ear. The low dose corresponds to 0.58 .mu.mol/kg and
the high dose to 3.6 .mu.mol/kg for both epoxomicin and compound
16. Ear thickness was measured at 0 and 24 hours after DNFB
application. As shown in FIG. 10, a single dose of epoxomicin at
2.0 mg/kg inhibited ear inflammation by nine-fold over that of
vehicle-treated controls. Interestingly, a six-fold lower amount of
epoxomicin decreased inflammation by this same margin. A low dose
of optimized inhibitor (compound 16) also exerted potent
anti-inflammatory activity, reducing ear swelling by the same
amount as epoxomicin. At the higher dose, compound 16 decreased ear
swelling by 12-fold, demonstrating a small increase, but an overall
higher level of potency compared to epoxomicin.
[0176] For this class of inhibitors, strong in vitro enzyme
inhibition correlates with strong in vivo activity, as illustrated
by the cell anti-proliferative data of Example 12, and the
anti-inflammatory data of Example 15.
OTHER EMBODIMENTS
[0177] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
forgoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
1914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Leu Leu Val Tyr1210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Xaa
Gln Asn Pro Met Xaa Thr Gly Thr Ser1 5 1034PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Ile
Ile Thr Glu144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Gly Pro Phe Glu154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Pro Phe Leu164PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 6Xaa Pro Phe Leu175PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Gly
Gly Pro Phe Leu1 584PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 8Pro Pro Phe Leu194PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Leu
Leu Leu Leu11022DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 10agttgagggg actttcccag gc
221126DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 11gatcgaactg accgcccgcg gcccgt
26124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Leu Leu Leu His1134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Leu
Leu Ala Leu1144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Leu Leu Phe Leu1154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Leu
Ala Leu Leu1164PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 16Leu Phe Leu Leu1174PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Ala
Leu Leu Leu1184PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 18Xaa Leu Phe Leu11910PRTHomo sapiens
19Arg Ala Ala Leu Gly Asn Ile Ser Glu Asn1 5 10
* * * * *