U.S. patent application number 15/050502 was filed with the patent office on 2016-06-16 for proteasome inhibitors and uses thereof.
This patent application is currently assigned to Yeda Research and Development Co. Ltd.. The applicant listed for this patent is Yeda Research and Development Co. Ltd.. Invention is credited to Benjamin GEIGER, Zvi KAM, Irina LAVELIN, Ami NAVON, Moshe OREN, Varda ROTTER.
Application Number | 20160166582 15/050502 |
Document ID | / |
Family ID | 42536328 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160166582 |
Kind Code |
A1 |
LAVELIN; Irina ; et
al. |
June 16, 2016 |
PROTEASOME INHIBITORS AND USES THEREOF
Abstract
A method of treating a disease in which inhibiting of a
proteasome is advantageous is provided. The method comprises
administering to the subject a therapeutically effective amount of
a compound which binds to a proteasome of a cell, the compound
comprising a copper bound to a ligand, the ligand being configured
such that upon binding to the proteasome, the copper interacts with
cysteine 31 of a Beta2 subunit of the proteasome and further
interacts with cysteine 118 of a Beta3 subunit of the proteasome,
thereby treating the disease. Additional novel proteasome
inhibitors are also provided as well as methods of identifying
proteasome inhibitors.
Inventors: |
LAVELIN; Irina; (Rehovot,
IL) ; ROTTER; Varda; (Rehovot, IL) ; OREN;
Moshe; (Rehovot, IL) ; NAVON; Ami; (Yavne,
IL) ; KAM; Zvi; (Rehovot, IL) ; GEIGER;
Benjamin; (Rechovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yeda Research and Development Co. Ltd. |
Rehovot |
|
IL |
|
|
Assignee: |
Yeda Research and Development Co.
Ltd.
Rehovot
IL
|
Family ID: |
42536328 |
Appl. No.: |
15/050502 |
Filed: |
February 23, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13872247 |
Apr 29, 2013 |
9289433 |
|
|
15050502 |
|
|
|
|
13322453 |
Nov 24, 2011 |
|
|
|
PCT/IL2010/000417 |
May 26, 2010 |
|
|
|
13872247 |
|
|
|
|
61213299 |
May 27, 2009 |
|
|
|
Current U.S.
Class: |
514/185 ;
435/366; 435/7.23; 514/263.2; 514/311; 530/350; 536/23.5 |
Current CPC
Class: |
G01N 33/5035 20130101;
A61K 31/60 20130101; C12N 9/6421 20130101; C12Y 304/25001 20130101;
A61K 33/34 20130101; C07K 2319/40 20130101; A61P 25/28 20180101;
A61P 35/00 20180101; C07K 2319/60 20130101; A61K 31/47 20130101;
A61P 29/00 20180101; A61K 31/522 20130101; A61K 31/555 20130101;
A61K 31/52 20130101; C07K 14/4746 20130101 |
International
Class: |
A61K 31/555 20060101
A61K031/555; G01N 33/50 20060101 G01N033/50; C07K 14/47 20060101
C07K014/47; A61K 31/522 20060101 A61K031/522; A61K 31/47 20060101
A61K031/47 |
Claims
1. A method of treating a disease in which inhibiting of a
proteasome is advantageous, the method comprising administering to
the subject a therapeutically effective amount of a compound
selected from the group consisting of NSC321206, NSC310551,
NSC99671 and NSC3907, thereby treating the disease.
2. A method of treating a disease in which inhibiting of a
proteasome is advantageous, the method comprising administering to
the subject a therapeutically effective amount of a compound which
binds to a proteasome of a cell, said compound comprising a copper
bound to a ligand, said ligand being configured such that upon
binding to said proteasome, said copper interacts with cysteine 31
of a .beta.2 subunit of said proteasome and further interacts with
cysteine 118 of a .beta.3 subunit of said proteasome, thereby
treating the disease.
3. The method of claim 1, wherein said disease is selected from the
group consisting of cancer, an inflammatory disease and a
neurodegenerative disease.
4. The method of claim 2, wherein said disease is selected from the
group consisting of cancer, an inflammatory disease and a
neurodegenerative disease.
5. An isolated polypeptide comprising a p53 amino acid sequence,
having a different cellular location in a presence or absence or a
proteasome inhibitor, the polypeptide being linked to a detectable
moiety.
6. The isolated polypeptide of claim 5, comprising an amino acid
sequence as set forth by SEQ ID NO: 3.
7. The isolated polypeptide of claim 5, comprising an amino acid
sequence as set forth by SEQ ID NO: 6.
8. The isolated polypeptide of claim 5, having a nuclear location
in a presence of a proteasome inhibitor and a cytoplasmic location
in an absence of a proteasome inhibitor.
9. An isolated polynucleotide comprising a nucleic acid sequence
encoding the polypeptide of claim 5.
10. The isolated polynucleotide of claim 9, comprising a nucleic
acid sequence as set forth in SEQ ID NO: 4.
11. The isolated polynucleotide of claim 9, comprising a nucleic
acid sequence as set forth in SEQ ID NO: 5.
12. A cell population expressing the polypeptide of claim 5.
13. The cell population of claim 12 comprising H1299 non-small cell
lung carcinoma cells.
14. A method of identifying a proteasome inhibitor, the method
comprising: (a) contacting a candidate inhibitor with a population
of cells which express the isolated polypeptide of claim 5; and (b)
analyzing a cellular location of said polypeptide in said
population of cells, wherein a change in localization of said
polypeptide is indicative of said candidate inhibitor being a
proteasome inhibitor.
15. A pharmaceutical composition comprising as an active ingredient
a compound selected from the group consisting of NSC321206,
NSC310551, NSC99671 and NSC3907 and a pharmaceutically acceptable
carrier.
16. A pharmaceutical composition comprising as an active ingredient
a compound which binds to a proteasome of a cell, said compound
comprising a copper bound to a ligand, said ligand being configured
such that upon binding to said proteasome, said copper interacts
with cysteine 31 of a .beta.2 subunit of said proteasome and
further interacts with cysteine 118 of a .beta.3 subunit of said
proteasome.
17. A pharmaceutical composition comprising as an active ingredient
a compound identified according to the method of claim 14 and a
pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 13/872,247 filed on Apr. 29, 2013, which is a division of
U.S. patent application Ser. No. 13/322,453 filed on Nov. 24, 2011,
which is a National Phase of PCT Patent Application No.
PCT/IL2010/000417 filed on May 26, 2010, which claims the benefit
of priority of U.S. Provisional Patent Application No. 61/213,299
filed on May 27, 2009. The contents of the above applications are
all incorporated herein by reference.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 65176SequenceListing.txt, created
on Feb. 23, 2016, comprising 83,456 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to proteasome inhibitors and uses thereof.
[0004] The proteasome is the major proteolytic complex,
responsible, in eukaryotic cells, for the degradation of a
multitude of cellular proteins. This multi-protein complex, present
in both the cytoplasm and the nucleus, catalyzes the ATP-dependent
proteolysis of short-lived regulatory proteins, as well as the
rapid elimination of damaged and abnormal proteins. The 26S
proteasome is a large complex of .about.2.5 MDa. Based on
biochemical analyses, this complex can be dissociated into two
functionally distinct subcomplexes, the 20S core particle (CP)
which is the proteolytic component, and the 19S regulatory particle
(RP), that is responsible for recognizing, unfolding, and
translocating polyubiquitinated substrates into the 20S CP, where
they are degraded.
[0005] The 20S CP is a 670 kDa barrel-shaped protein complex made
up of four stacked, seven-membered rings (4.times.7 subunits), two
outer a rings and two inner .beta. rings
(.alpha..sub.1-7.beta..sub.1-7.beta..sub.1-7.alpha..sub.1-7). The
two matching a rings are situated in the outer rims of the barrel,
facing the 19S regulatory complex. The proteolytic active sites are
located on the two identical .beta.-rings, which are positioned in
the center of the 20S complex. In eukaryotes, the catalytic
activities of the proteasomes are confined to only three of the
.beta.-subunits. Although proteasomes can hydrolyze the amide bonds
between most amino acids, proteolytic activities measured using
fluorogenic substrates define three distinct (although not
conclusive) cleavage preferences [5]: .beta.2 possesses tryptic
activity (i.e., cleaving after basic residues); .beta.5 displays
chymotryptic activity (i.e., cleaving after hydrophobic residues);
and .beta.1 has "caspase-like" or "post-acidic" activity. In all
three active .beta.-subunits, proteolytic activity is associated
with their N-terminal threonine residue, which acts as a
nucleophile in peptide-bond hydrolysis.
[0006] The use of proteasome inhibitors as drug candidates emerged
from the observation that at specific concentrations, they can
induce apoptosis in certain leukemia- and lymphoma-derived cells
without similarly affecting their non-transformed counterparts.
Further development and clinical trials led to the approval of the
modified boronic dipeptide Pyz-Phe-boroLeu, known as Bortezomib as
a drug for the treatment of multiple myeloma. Most synthetic
proteasome inhibitors are short peptides that mimic protein
substrates. Typically, the pharmacophore that reacts with and
inhibits the threonine residue in the 20S proteasome's active site
is bound to the carboxyl residue of the peptide. Some of the
typical synthetic inhibitors are peptide aldehydes, peptide vinyl
sulfones, peptide boronates, and peptide epoxyketones. Most notable
among the natural, bacterially derived non-peptide inhibitors is
claso-lactacystin-.beta.-lactone (Omuralide). Related drugs such as
Salinosporamide A (NPI-0052) and Carfilzomib (PR-171) are currently
in advanced clinical trials. However, despite the extensive efforts
invested in proteasome inhibitor development, there is a growing
need for novel inhibitory molecules, due to the emergence of
drug-resistant cells and the variable effects of existing
inhibitors on different cells.
[0007] Most of the current assays for proteasome inhibition are
based on cell-free assays, which require purification of 26S or 20S
proteasomes from different sources. Such assays may, in principle,
be adapted to high-throughput screens, yet they may fail to predict
the inhibitory activity in live cells. To overcome this problem,
cell-based screens have been incorporated into the drug discovery
process. For example, a modified "classical" method for measurement
of the chymotrypsin-like, trypsin-like, or caspase-like proteasome
activities in cultured cells [Moravec R A et al., 2009, Anal
Biochem 387: 294-302] is currently available from Promega
Corporation. A number of fluorescent reporter molecules have been
also usefully employed to monitor the activity of the proteasome.
Dantuma et al constructed a fusion of GFP to Ubiquitin
(Ubi[G76V]-GFP) using a standard peptide bond at the N-terminus
[Nat Biotechnol 18: 538-543, 2000], Another proteasome sensor
construct, which is a GFP fusion to an artificial peptide, CL1,
identified in yeast has been designed by Bence et al (Science 292:
1552-1555, 2001). The Andreatta group and BD Biosciences Clontech
has introduced a sensor cell line expressing a GFP fusion protein
with a fragment of the mouse ornithine decarboxylase (MODC), which
is degraded by the proteasome without the requirement for
ubiquitination [Andreatta et al, 2001, Biotechniques 30: 656-660].
An additional reporter cell line, based on the stable expression of
a p27.sup.kip1-GFP fusion was recently employed for the discovery
of a novel proteasome inhibitor, argyrin A [Nickeleit I et al.,
2008, Cancer Cell 14: 23-35]. The common feature of most of these
GFP-fused reporters is that they are based on proteins rapidly
degraded by the proteasome under normal conditions, leading to very
low fluorescence of the cells, while following inhibition of
proteasome activity, the overall fluorescent signal of the cells
rapidly increases as a result of accumulation of the reporter
proteins.
SUMMARY OF THE INVENTION
[0008] According to an aspect of some embodiments of the present
invention there is provided a method of treating a disease in which
inhibiting of a proteasome is advantageous, the method comprising
administering to the subject a therapeutically effective amount of
a compound selected from the group consisting of NSC321206,
NSC310551, NSC99671 and NSC3907, thereby treating the disease.
[0009] According to an aspect of some embodiments of the present
invention there is provided a method of treating a disease in which
inhibiting of a proteasome is advantageous, the method comprising
administering to the subject a therapeutically effective amount of
a compound which binds to a proteasome of a cell, the compound
comprising a copper bound to a ligand, the ligand being configured
such that upon binding to the proteasome, the copper interacts with
cysteine 31 of a .beta.2 subunit of the proteasome and further
interacts with cysteine 118 of a .beta.3 subunit of the proteasome,
thereby treating the disease.
[0010] According to an aspect of some embodiments of the present
invention there is provided a method of identifying a proteasome
inhibitor, the method comprising:
[0011] (a) contacting a candidate inhibitor with a population of
cells which express the isolated polypeptide of the present
invention; and
[0012] (b) analyzing a cellular location of the polypeptide in the
population of cells, wherein a change in localization of the
polypeptide is indicative of the candidate inhibitor being a
proteasome inhibitor.
[0013] According to an aspect of some embodiments of the present
invention there is provided an isolated polypeptide comprising a
p53 amino acid sequence, having a different cellular location in a
presence or absence or a proteasome inhibitor, the polypeptide
being linked to a detectable moiety.
[0014] According to an aspect of some embodiments of the present
invention there is provided a cell population expressing the
polypeptide of the present invention.
[0015] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient a compound selected from the group
consisting of NSC321206, NSC310551, NSC99671 and NSC3907 and a
pharmaceutically acceptable carrier.
[0016] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient a compound which binds to a proteasome of a
cell, the compound comprising a copper bound to a ligand, the
ligand being configured such that upon binding to the proteasome,
the copper interacts with cysteine 31 of a .beta.2 subunit of the
proteasome and further interacts with cysteine 118 of a .beta.3
subunit of the proteasome.
[0017] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient a compound identified according to the
method of the present invention and a pharmaceutically acceptable
carrier.
[0018] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding the polypeptide of the present
invention.
[0019] According to some embodiments of the invention, the disease
is cancer.
[0020] According to some embodiments of the invention, the disease
is an inflammatory disease.
[0021] According to some embodiments of the invention, the disease
is a neurodegenerative disease.
[0022] According to some embodiments of the invention, the isolated
polypeptide comprises an amino acid sequence as set forth by SEQ ID
NO: 3.
[0023] According to some embodiments of the invention, the isolated
polypeptide comprises an amino acid sequence as set forth by SEQ ID
NO: 6.
[0024] According to some embodiments of the invention, the isolated
has a nuclear location in a presence of a proteasome inhibitor and
a cytoplasmic location in an absence of a proteasome inhibitor.
[0025] According to some embodiments of the invention, the isolated
polynucleotide comprises a nucleic acid sequence as set forth in
SEQ ID NO: 4.
[0026] According to some embodiments of the invention, the isolated
polynucleotide comprises a nucleic acid sequence as set forth in
SEQ ID NO: 5.
[0027] According to some embodiments of the invention, the cell
population comprises H1299 non-small cell lung carcinoma cells.
[0028] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0029] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0030] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings
and images. With specific reference now to the drawings in detail,
it is stressed that the particulars shown are by way of example and
for purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0031] In the drawings:
[0032] FIG. 1 is a schematic representation of the PIR protein. PIR
protein consists of the yellow fluorescent protein (YFP) fused to
the C-terminus of the human mutant (R175H) p53, carrying triple
mutation in the bipartite Nuclear Localization Signal (SEQ ID NO:
7) in which three consecutive lysine residues Lys-319, -320, and
-321 were replaced with alanines.
[0033] FIGS. 2A-2I are photographs illustrating nuclear
accumulation of the PIR protein upon treatment with proteasome
inhibitors. A-H: PIR cells were exposed to MG132, bortezomib and
ALLN at the indicated concentrations for 6 hours and monitored for
YFP-fluorescence for PIR reporter (upper panel) or stained with
anti-beta-catenin antibody (lower panel). I: PIR cells were
incubated for 6 h without proteasome inhibitor (control) or with 10
.mu.M MG132. Cell lysates were separated for cytoplasm and nuclear
fractions. A band corresponding to the PIR reporter was detected by
western blot with a polyclonal anti-p53 antibody.
[0034] FIGS. 3A-3H are photographs illustrating that murine double
minute 2 (MDM2) promotes PIR nuclear translocation. Overexpression
of MDM2 results in PIR nuclear localization in the absence of
additional stimuli. PIR cells were transfected with wild-type MDM2,
MDM2 mutant deficient on p53 binding (D 9-58), or MDM2 mutant with
abolished E3 ligase site (Ser 440). Cells expressing both p53 and
MDM2 were visualized by immunofuorescence staining with the
anti-MDM2 monoclonal antibodies. PIR has a nuclear localization in
the cells expressing wt MDM2 and MDM2 (Ser 440), and remains
cytoplasmic in the cells transfected with MDM2 (D 9-58).
[0035] FIGS. 4A-4H are photographs illustrating that Mdm2 siRNA
prevents bortezomide-induced translocation of PIR to the nucleus.
PIR cells were transiently transfected with 200 pmol control-siRNA
or Mdm2-siRNA. Forty-eight hours after transfection, bortezomide
(0.1 .mu.M) was added for an additional 6 hours, and
immunofuorescence staining for MDM2 was performed as described in
Materials and Methods.
[0036] FIG. 5 is a flow chart of the screen procedure. For the
screening assay H11299-PIR reporter cells were plated in 384-well
plates for 24 hours and treated with compounds of NCI Diversity Set
library at two concentrations (1 and 10 .mu.M), using a single
compound per well. Following 12 hours of incubation, the cells were
fixed by 3% paraformaldehyde and screened for localization of the
PIR protein by automated microscope system. Cell images were
analyzed for PIR nuclear translocation and selected hits were
confirmed by microscopic and biochemical methods, followed by test
for compound cytotoxicity.
[0037] FIG. 6 is a table illustrating the effect of the candidate
inhibitors identified in the screen on PIR cellular
localization.
[0038] FIGS. 7A-7B are photographs and graphs illustrating an
increase of ubiquitinated proteins of a whole-cell lysate upon
treatment with hit compounds. PIR cells were treated with hit
compounds for 6 hours in following concentrations: NSC3907-20
.mu.M, NSC99671-50 .mu.M, NSC310551-0.3 .mu.M, NSC321206-0.15
.mu.M. Known proteasome inhibitor MG132 (5 .mu.M) was used as a
positive control. Whole lysates of the PIR cells were immunoblotted
for ubiquitin (upper panel) and beta-catenin (middle panel). The
tubulin (lower panel) signal represents the internal loading
control.
[0039] FIG. 8 is a graph illustrating in vitro proteasomal
inhibition by the candidate inhibitors. Rabbit muscle purified 26S
proteasome was incubated for the indicated time in the presence of
30 .mu.M of the candidate inhibitors (100 .mu.M for NSC3907),
MG-132 at 5.mu.M concentration serves as a control.
[0040] FIG. 9 is a graph illustrating cytotoxic activity of the
candidate inhibitors. A: PIR-H1299 cells were treated with hit
compounds for 48 hours at 11 concentrations between 0.1 and 100
.mu.M. Cell viability assay was carried out as described (Materials
and Methods). Results are expressed as GI.sub.50, concentration
that reduced by 50% the growth of treated cells with respect to
untreated controls. All results are displayed as the mean and
standard deviation from six replicate wells.
[0041] FIG. 10 is a graph illustrating the effect of NSC321206 on
cancer cell viability.
[0042] FIG. 11 is a chart presenting the in vitro cytotoxicity of
the candidate inhibitors on NCI-60 panel of human tumor cell lines.
The results is based on the data from the anticancer drug screening
against the full panel of 60 human cancer cell lines conducted as a
part of the Developmental Therapeutics Program at the National
Cancer Institute. The panel is organized into nine subpanels
representing diverse histologies: leukemia, melanoma, and cancers
of lung, colon, kidney, ovary, breast, prostate, and central
nervous system. The results obtained with this test expressed as
the -log of the molar concentration that inhibited the cell growth
by 50% (-log GI.sub.50>4.00 for active compounds).
[0043] FIGS. 12A-12B are illustrations of the potential docking
site for NSC321206 pertaining to the Trypsin-like active site. FIG.
12A illustrates the deep hydrophobic pocket in the trypsin-like
active site and two Cys residues therein positioned in the ideal
conformation with regards to the Cu atom of the NSC321206. FIG. 12B
is a space filling cartoon of the Trypsin-like active site. The
catalytic threonine is depicted as an orange patch and the Velcade
backbone is outlined in red. The three most energetically favored
clusters of NSC321206 are presented.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0044] The present invention, in some embodiments thereof, relates
to proteasome inhibitors and uses thereof.
[0045] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0046] Proteasomes are enzymes with a complex structure and
function. They are found abundantly in all cells, both normal and
cancerous, and are responsible for the degradation of all
regulatory proteins. Since regulatory proteins are key to the
activation or repression of many cellular processes, including
cell-cycle progression, transcription, and apoptosis, the
proteasome has become a potential target for inhibition for the
treatment of a myriad of disorders.
[0047] Using a novel image-based screening approach, the present
inventors tested a battery of chemical compounds, and identified
four with pronounced proteasome inhibitory activity (FIGS. 7A-B and
FIG. 8). The screen was based on the use of H1299 reporter cell
line stably expressing a fluorescent Proteasome Inhibition Reporter
(PIR) protein. The rational of the screen builds on the finding
that upon inhibition of proteasomal activity this reporter
translocates to the nucleus, resulting in a distinct and detectable
nuclear fluorescent signal. The findings demonstrate that this
approach is highly sensitive and compatible with high-throughput
microscopy.
[0048] The present inventors further showed that at least one of
these inhibitors (NSC321206) could selectively kill cancerous cells
as opposed to non-cancerous cells (FIG. 10).
[0049] Computer analysis of the 3D structure of the proteasome and
the NSC321206 inhibitor, served to identify putative docking sites
on the proteasome for the inhibitor. The present inventors
postulate that the identified sites may be used to design
additional small molecules based on NSC321206 that would strongly
interact with these binding sites and thus would inhibit
proteasomal activity.
[0050] As is described in the Examples section that follows, it was
deduced from these studies that the proteasome comprise two
cysteines in, or close to, the trypsin-like active site of the
proteasome, which interact with a copper of the NSC321206 inhibitor
(FIGS. 12A-B). It was thus deduced that in order to achieve a
maximized number and strength of interactions with these binding
sites, small molecules may be designed such that they comprise a
copper in a suitable proximity and orientation to these binding
sites.
[0051] Thus, according to one aspect of the present invention there
is provided an isolated polypeptide comprising a p53 amino acid
sequence, having a different cellular location in a presence or
absence or a proteasome inhibitor, the polypeptide being linked to
a detectable moiety.
[0052] The term "p53" refers to the human p53 polypeptide that has
at least 70%, at least 80%, at least 90% or at least 95% sequence
homology to SEQ ID NO: 1.
[0053] The present inventors contemplate all p53 sequences having a
cellular location which is influenced by the presence of a
proteasome inhibitor e.g., soluble (i.e. cytoplasmic) or membrane
bound, a specific cellular organelle, or a specific biochemical
pathway, e.g., replication, transcription or translation, etc.
Exemplary cellular organelles include nuclei, mitochondrion,
chloroplast, ribosome, ER, Golgi apparatus, lysosome, proteasome,
secretory vesicle, vacuole and microsome.
[0054] The present inventors have shown that a mutation in the
nuclear localization signal of the p53 polypeptide serves to alter
the native nuclear localization of the polypeptide to a cytoplasmic
location. However, in the presence of a proteasome inhibitor, the
p53 polypeptide reverts to its nuclear location.
[0055] Thus, according to one embodiment, the p53 polypeptide has a
cytoplasmic location in an absence of a proteasome inhibitor and a
nuclear location in a presence of a proteasome inhibitor.
[0056] With reference to a particular location, the present
invention contemplates that at least 70%, more preferably at least
75%, more preferably at least 80%, more preferably at least 85%, at
least 90% and even more preferably at least 95% of the polypeptide
is situated at that particular location, wherein the remaining
polypeptide is situated at any other location in the cell.
[0057] Contemplated mutations include for example a mutation at a
position corresponding to lysine 319 of SEQ ID NO: 1, a mutation at
a position corresponding to lysine at 320 of SEQ ID NO: 1 and a
mutation at a position corresponding to lysine 321 of SEQ ID NO:
1.
[0058] As used herein, the term "mutation" refers to an alteration
in an amino acid sequence compared to the wild type sequence
(GenBank Accession No: NP_000537.3--SEQ ID NO: 1)
[0059] The mutation may comprise a deletion or a substitution.
Exemplary mutations include at least one of an alanine
corresponding to lysine at position 319, an alanine corresponding
to lysine at position 320 and an alanine corresponding to lysine at
position 321.
[0060] Thus, for example the present inventors contemplate the use
of a polypeptide having an amino acid sequence as set forth in SEQ
ID NO: 2.
[0061] The p53 polypeptide may further comprise mutations which act
to increase the half-life thereof.
[0062] Accordingly, the present inventors contemplate the use of a
p53 polypeptide having a mutation of an arginine corresponding to
histidine at position 175.
[0063] Thus, for example the present inventors contemplate the use
of a polypeptide having an amino acid sequence as set forth in SEQ
ID NO: 3.
[0064] Other mutations that may serve to increase the half life of
the p53 include those described in Joerger A C, Fersht A R.
Structural biology of the tumor suppressor p53 and
cancer-associated mutants. Adv Cancer Res. 2007; 97:1-23),
incorporated herein by reference.
[0065] In addition, the p53 polypeptide of the present invention
may comprise other conservative variations of SEQ ID NO: 1.
[0066] The phrase "conservative variation" as used herein refers to
the replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine, or methionine for another, or the substitution of one
solar residue for another, such as the substitution of arginine for
lysine, glutamic acid for aspartic acid, or glutamine for
asparagine, and the like. The term "conservative variation" also
includes the use of a substituted amino acid in place of an
unsubstituted parent amino acid provided that antibodies raised to
the substituted polypeptide also immunoreact with the unsubstituted
polypeptide.
[0067] Other mutations which impart stability or alter the cellular
localization of p53 to a cytoplsmic one can be uncovered using
computational biology. For example, various mutated P53 peptide
sequences can be computationally analyzed for an ability to impart
stability and cellular localization using a variety of three
dimensional computational tools. Software programs useful for
displaying three-dimensional structural models, such as RIBBONS
(Carson, M., 1997. Methods in Enzymology 277, 25), O (Jones, T A.
et al., 1991. Acta Crystallogr. A47, 110), DINO (DINO: Visualizing
Structural Biology (2001) www.dino3d.org); and QUANTA, INSIGHT,
SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP (reviewed in
Kraulis, J., 1991. Appl Crystallogr. 24, 946) can be utilized to
model prospective mutant peptide sequences to identify useful
mutations.
[0068] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0069] Table 1 below lists naturally occurring amino acids which
can be used with the present invention.
TABLE-US-00001 TABLE 1 Three-Letter Amino Acid Abbreviation
One-letter Symbol alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid
Glu E glycine Gly G Histidine His H isoleucine Iie I leucine Leu L
Lysine Lys K Methionine Met M phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T tryptophan Trp W tyrosine Tyr Y Valine
Val V Any amino acid as above Xaa X
[0070] As mentioned, the p53 polypeptide of this aspect of the
present invention is linked to a detectable moiety. An exemplary
p53 polypeptide linked to a detectable moiety has an amino acid
sequence as set forth in SEQ ID NO: 6.
[0071] The detectable moiety can be any one which is capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, a fluorescent
or chemiluminescent compound, or a tag (to which a labeled antibody
can bind).
[0072] Examples of suitable fluorescent detectable moieties
include, but are not limited to, phycoerythrin (PE), fluorescein
isothiocyanate (FITC), Cy-chrome, rhodamine, Texas red, PE-Cy5,
green fluorescent protein, the yellow fluorescent protein, the cyan
fluorescent protein and the red fluorescent protein as well as
their enhanced derivatives.
[0073] Table 2 below provides examples of sequences of identifiable
moieties.
TABLE-US-00002 TABLE 2 Amino Acid sequence Nucleic Acid sequence
(Genebank (Genebank TIdentifiable Moiety Accession No.) Accession
No.) Green Fluorescent protein AAL33912 AF435427 Alkaline
phosphatase AAK73766 AY042185 Peroxidase NP_568674 NM_124071
Histidine tag AAK09208 AF329457 Myc tag AF329457 AF329457 Biotin
lygase tag NP_561589 NC_003366 orange fluorescent protein AAL33917
AF435432 Beta galactosidase NM_125776 NM_125776 Fluorescein
isothiocyanate AAF22695 AF098239 Streptavidin S11540 S11540
[0074] For additional guidance regarding fluorophore selection,
methods of linking fluorophores to various types of molecules see
Richard P. Haugland, "Molecular Probes: Handbook of Fluorescent
Probes and Research Chemicals 1992-1994", 5th ed., Molecular
Probes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.;
Hermanson, "Bioconjugate Techniques", Academic Press New York, N.Y.
(1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al.,
1996. Biochemistry 35:937; Gakamsky D. et al., "Evaluating Receptor
Stoichiometry by Fluorescence Resonance Energy Transfer," in
"Receptors: A Practical Approach," 2nd ed., Stanford C. and Horton
R. (eds.), Oxford University Press, UK. (2001); U.S. Pat. No.
6,350,466 to Targesome, Inc.].
[0075] In order to express the polypeptides of the present
invention in cell populations, the encoding DNA sequence is
inserted into nucleic acid constructs and cells are transfected
using methods commonly known in the art as described further herein
below.
[0076] Thus, according to another aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence encoding a polypeptide which comprises a p53 amino
acid sequence, the polypeptide having a different cellular location
in a presence or absence or a proteasome inhibitor, the polypeptide
being linked to a detectable moiety.
[0077] The phrase "an isolated polynucleotide" refers to a single
or double stranded nucleic acid sequence which is isolated and
provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0078] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0079] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0080] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exon sequences required to encode the polypeptide
of the present invention, as well as some intronic sequences
interposing therebetween. The intronic sequences can be of any
source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0081] Exemplary polynucleotides of the present invention comprise
the sequences as set forth in SEQ ID NOs: 4 and 5.
[0082] As mentioned, the polynucleotides of this aspect of the
present invention are typically inserted into nucleic acid
constructs suitable for mammalian cell expression.
[0083] Such a nucleic acid construct typically includes a promoter
sequence for directing transcription of the polynucleotide sequence
in the cell in a constitutive or inducible manner.
[0084] Constitutive promoters suitable for use with the present
invention are promoter sequences which are active under most
environmental conditions and most types of cells such as the
cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible
promoters suitable for use with the present invention include for
example the tetracycline-inducible promoter (Zabala M, et al.,
Cancer Res. 2004, 64(8): 2799-804).
[0085] The nucleic acid construct may include additional sequences
which render this vector suitable for replication and integration
in prokaryotes, eukaryotes, or preferably both (e.g., shuttle
vectors). In addition, typical cloning vectors may also contain a
transcription and translation initiation sequence, transcription
and translation terminator and a polyadenylation signal.
[0086] Eukaryotic promoters typically contain two types of
recognition sequences, the TATA box and upstream promoter elements.
The TATA box, located 25-30 base pairs upstream of the
transcription initiation site, is thought to be involved in
directing RNA polymerase to begin RNA synthesis. The other upstream
promoter elements determine the rate at which transcription is
initiated.
[0087] Preferably, the promoter utilized by the nucleic acid
construct of the present invention is active in the specific cell
population transformed.
[0088] Enhancer elements can stimulate transcription up to 1,000
fold from linked homologous or heterologous promoters. Enhancers
are active when placed downstream or upstream from the
transcription initiation site. Many enhancer elements derived from
viruses have a broad host range and are active in a variety of
tissues. For example, the SV40 early gene enhancer is suitable for
many cell types. Other enhancer/promoter combinations that are
suitable for the present invention include those derived from
polyoma virus, human or murine cytomegalovirus (CMV), the long term
repeat from various retroviruses such as murine leukemia virus,
murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic
Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
1983, which is incorporated herein by reference.
[0089] In the construction of the expression vector, the promoter
is preferably positioned approximately the same distance from the
heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0090] Polyadenylation sequences can also be added to the
expression vector in order to increase RNA stability [Soreq et al.,
1974; J. Mol Biol. 88: 233-45).
[0091] Two distinct sequence elements are required for accurate and
efficient polyadenylation: GU or U rich sequences located
downstream from the polyadenylation site and a highly conserved
sequence of six nucleotides, AAUAAA, located 11-30 nucleotides
upstream. Termination and polyadenylation signals that are suitable
for the present invention include those derived from SV40.
[0092] In addition to the elements already described, the
expression vector of the present invention may typically contain
other specialized elements intended to increase the level of
expression of cloned nucleic acids or to facilitate the
identification of cells that carry the recombinant DNA. For
example, a number of animal viruses contain DNA sequences that
promote the extra chromosomal replication of the viral genome in
permissive cell types. Plasmids bearing these viral replicons are
replicated episomally as long as the appropriate factors are
provided by genes either carried on the plasmid or with the genome
of the host cell.
[0093] The vector may or may not include a eukaryotic replicon. If
a eukaryotic replicon is present, then the vector is amplifiable in
eukaryotic cells using the appropriate selectable marker. If the
vector does not comprise a eukaryotic replicon, no episomal
amplification is possible. Instead, the recombinant DNA integrates
into the genome of the engineered cell, where the promoter directs
expression of the desired nucleic acid.
[0094] Examples for mammalian expression vectors include, but are
not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-),
pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5,
DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from
Invitrogen, pCI which is available from Promega, pMbac, pPbac,
pBK-RSV and pBK-CMV which are available from Strategene, pTRES
which is available from Clontech, and their derivatives.
[0095] Nucleic acid constructs containing regulatory elements from
eukaryotic viruses such as retroviruses can be also used. SV40
vectors include pSVT7 and pMT2. Vectors derived from bovine
papilloma virus include pBV-1MTHA, and vectors derived from Epstein
Bar virus include pHEBO, and p205. Other exemplary vectors include
pMSG, pAV009/A.sup.+, pMT010/A.sup.+, pMAMneo-5, baculovirus pDSVE,
and any other vector allowing expression of proteins under the
direction of the SV-40 early promoter, SV-40 later promoter,
metallothionein promoter, murine mammary tumor virus promoter, Rous
sarcoma virus promoter, polyhedrin promoter, or other promoters
shown effective for expression in eukaryotic cells.
[0096] According to one embodiment, the nucleic acid construct is a
viral vector, such as adenovirus, lentivirus, Herpes simplex I
virus, or adeno-associated virus (AAV) and lipid-based systems.
[0097] As mentioned herein above, the present inventors have found
that cells expressing the polypeptide of this aspect of the present
invention can be used as a system for identifying proteasome
inhibitors.
[0098] Various methods can be used to introduce the expression
vector of the present invention into cell populations. Such methods
are generally described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989,
1992), in Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic
Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene
Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, Butterworths, Boston
Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986]
and include, for example, stable or transient transfection,
lipofection, electroporation and infection with recombinant viral
vectors. Useful lipids for lipid-mediated transfer of the gene are,
for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer
Investigation 14(1): 54-65 (1996)].
[0099] Contemplated cell populations that may be used for
identifying proteasome inhibitors include immortalized cell
populations (i.e. cell lines) or cells taken directly from a living
organism. According to one embodiment, the cell populations are
homogeneous (i.e. comprise only one cell type). According to
another embodiment the cells express MDM2 (E3 ubiquitin-protein
ligase Mdm2; NC_000012.11). According to one embodiment, the cell
population comprises H1299 non-small cell lung carcinoma cells.
Other exemplary cell populations include, but are not limited to
MCF7 cells, MCF10a (human breast cells), human foreskin fibroblasts
and U2OS (human osteosarcoma) cells.
[0100] Thus, according to another aspect of the present invention
there is provided a method of identifying a proteasome inhibitor,
the method comprising:
[0101] (a) contacting a candidate inhibitor with a population of
cells which express an isolated polypeptide comprising a p53 amino
acid sequence, having a different cellular location in a presence
or absence or a proteasome inhibitor when expressed in a cell, the
polypeptide being linked to a detectable moiety; and
[0102] (b) analyzing a cellular location of the polypeptide in the
population of cells, wherein a change in a localization of the
polypeptide is indicative of the candidate inhibitor being a
proteasome inhibitor.
[0103] The term "proteasome" as used herein refers to the
multi-protein complex responsible, in eukaryotic cells, for the
degradation of cellular proteins.
[0104] The 26S proteasome can be dissociated into two functionally
distinct subcomplexes, the 20S core particle (CP) which is the
proteolytic component, and the 19S regulatory particle (RP), that
is responsible for recognizing, unfolding, and translocating
polyubiquitinated substrates into the 20S CP, where they are
degraded.
[0105] The 20S CP is a 670 kDa barrel-shaped protein complex made
up of four stacked, seven-membered rings (4.times.7 subunits), two
outer a rings and two inner .beta. rings
(.alpha..sub.1-7.beta..sub.1-7.beta..sub.1-7.alpha..sub.1-7). The
two matching a rings are situated in the outer rims of the barrel,
facing the 19S regulatory complex. The proteolytic active sites are
located on the two identical .beta.-rings, which are positioned in
the center of the 20S complex.
[0106] As used herein, the phrase "proteasome inhibitor" refers to
a substance (e.g. compound) that inhibits at least one enzymatic
activity of the proteasome. Exemplary enzymatic activities of the
proteasome include tryptic activity (i.e., cleaving after basic
residues) present in the .beta.2 subunit; chymotryptic activity
(i.e., cleaving after hydrophobic residues) present in the .beta.5
subunit; and "caspase-like" or "post-acidic" activity present in
the .beta.1 subunit.
[0107] The phrase "change in location" refers to a change in the
cellular distribution of the polypeptide such that at least 70%, at
least 80%, at least 90% of the polypeptide is translocated from a
first location in the cell (e.g. cytoplasm) to a second location in
the cell (e.g. nucleus).
[0108] Since the polypeptides of the present invention comprise
detectable moieties, analyzing their location in the cell may be
performed by various techniques including for example fluorescent
microscopy, immunohistochemistry, Western blot analysis (by
generating nuclear and cytoplasmic extracts of the cells).
[0109] The candidate inhibitors of this aspect of the present
invention that may be tested as potential proteasome inhibitors
according to the method of the present invention include, but are
not limited to, nucleic acids, e.g., polynucleotides, ribozymes,
siRNA and antisense molecules (including without limitation RNA,
DNA, RNA/DNA hybrids, peptide nucleic acids, and polynucleotide
analogs having altered backbone and/or bass structures or other
chemical modifications); proteins, polypeptides (e.g. peptides),
carbohydrates, lipids and "small molecule" drug candidates. "Small
molecules" can be, for example, naturally occurring compounds
(e.g., compounds derived from plant extracts, microbial broths, and
the like) or synthetic organic or organometallic compounds having
molecular weights of less than about 10,000 daltons, preferably
less than about 5,000 daltons, and most preferably less than about
1,500 daltons.
[0110] Using the above screen, the present inventors tested 1,992
low molecular weight compounds comprising the NCI Diversity Set
chemical library and identified four compounds NSC321206,
NSC310551, NSC99671 and NSC3907 that were positive in the assay and
therefore may be considered as proteasome inhibitors.
[0111] As mentioned herein above, the present inventors
computer-analyzed the 3D structure of the proteasome and the
NSC321206 inhibitor, identifying putative docking sites on the
proteasome for the inhibitor. It was deduced from these studies
that the proteasome comprise two cysteines in, or close to, the
trypsin-like active site of the proteasome, which interact with a
copper atom of the NSC321206 inhibitor (FIGS. 12A-B of the Examples
section herein below). It was thus deduced that in order to achieve
a maximized number and strength of interactions with these binding
sites, small molecules may be designed such that they comprise a
metal ion in a suitable proximity and orientation to these binding
sites.
[0112] Thus, according to another aspect of the present invention
there is provided a method of treating a disease in which
inhibiting of a proteasome is advantageous, the method comprising
administering to the subject a therapeutically effective amount of
a compound which binds to a proteasome of a cell, the compound
comprising copper bound to a ligand, the ligand being configured
such that upon binding to the proteasome, the copper interacts with
cysteine 31 of a .beta.2 subunit of the proteasome and further
interacts with cysteine 118 of a .beta.3 subunit of the
proteasome.
[0113] The copper can be either a copper atom (having a 0 oxidation
state), or a copper ion (having I or II oxidation state).
[0114] In cases where the copper is a copper ion, it is bound to
one or two anions such as, for example, halides (e.g.,
bromides).
[0115] In some embodiments, the copper is a copper ion. In some
embodiments, the copper ion is Cu.sup.+1. In some embodiments, the
copper ion is bound to a bromide.
[0116] As used herein the term "ligand" describes a chemical
moiety, ion or atom that is associated with a central metal atom,
via covalent bonds, ionic bonds and/or coordinative bonds. The
ligand can be associated with copper atom or a copper ion.
[0117] According to embodiments of the invention, the ligand is
selected such that it includes one or more chemical groups that
interact with one or more amino acids in the trypsin-like active
site of the proteasome, whereby this interaction results in a
configuration of the ligand within this active site in which the
copper is present in a proximity and orientation of the
above-indicated cysteine residues that allows its interaction with
these cysteine residues.
[0118] In some embodiments, the ligand includes one or more
electron donating atoms for forming a complex with the
electron-poor copper atom or ion. Suitable electron donating atoms
include, but are not limited to, nitrogen, sulfur, and oxygen.
[0119] In some embodiments, the ligand includes at least two
nitrogen atoms that form coordinative bonds with the copper.
Optionally, the ligand includes one or more nitrogen atoms and one
or more sulfur atoms.
[0120] In some embodiments, the ligand is such that the electron
donating atoms (e.g., nitrogen and/or sulfur atoms) form stable
5-membered or 6-membered ring(s) upon coordinatively binding the
copper.
[0121] In some embodiments, the nitrogen and/or sulfur atoms in the
ligand are such that upon coordinatively binding the copper, a
rigid structure is formed.
[0122] By "rigid structure" it is meant that the number of
free-to-rotate bonds in the compound is minimal, namely, is no more
than 1 or 2.
[0123] The rigid structure assures that the ligand is configured in
the active site as desired (as indicated supra) selectively, such
that is cannot be subjected to changes in its three-dimensional
configuration that could reduce the interaction of the copper with
the above-indicated cysteine residues in the active site.
[0124] Thus, in some embodiments, the ligand includes electron
donating atoms as described herein, which form, upon coordinating
the copper, a complex that comprises fused rings.
[0125] In some embodiments, the ligand includes electron donating
atoms which form with copper a complex that comprises 2 fused
rings, optionally and preferably 3 fused rings, and further
optionally 4 and even 5 fused rings.
[0126] By "fused ring" it is meant that the two or more rings that
have two adjacent atoms and the bond therebetween in common.
[0127] Each ring in the part of the ligand that is directly
associated with the copper can independently be heteroalicyclic or
heteroaromatic ring.
[0128] In some embodiments, one or more rings, and optionally each
ring in this part of the ligand is a heteroaromatic ring.
Heteroaromatic rings form a more rigid structure as compared to
heteroalicyclic rings.
[0129] Exemplary ligands that are suitable for a compound as
described herein include, but are not limited to, ligands having a
core structure represented by the following general Formula:
##STR00001##
[0130] wherein:
[0131] the dashed line denotes a saturated or unsaturated bond;
[0132] X, Y and Z are each independently an electron donating atom
as described herein, preferably selected from the group consisting
of nitrogen and sulfur;
[0133] A.sub.1-A.sub.4 are each independently carbon or a
heteroatom (e.g., nitrogen, sulfur or oxygen); and
[0134] Rx, Rz and R.sub.1-R.sub.4 are each independently hydrogen,
alkyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl, alkoxy,
thioalkoxy, hydroxy, thiol, amine, amide, sulfonamide, carboxy,
thiocarboxy, carbamate, thiocarbamate, or absent, or,
alternatively, two or more of Rx, Rz and R.sub.1-R.sub.4 form a 5-
or 6-membered cyclic or heterocyclic ring.
[0135] Accordingly, a compound according to these embodiments of
the invention has a formula:
##STR00002##
[0136] wherein the broken line denotes a coordinative bond; and
"Cu" represents either Cu(0) or Cu(I) bound to an anion (e.g.,
bromide).
[0137] It will be appreciated by one of skills in the art that the
nature of each of the variables (X, Y, Z, A.sub.1-A.sub.4, Rx, Rz,
and R.sub.1-R.sub.4) depends on the valency and chemical
compatibility of the variable and its position with respect to
adjacent variables. Hence, the present invention is aimed at
encompassing all the feasible options for any variable.
[0138] In some embodiments, the chemical groups flanking the part
of the ligand that is directly associated with the copper (e.g.,
Rx, Rz and R.sub.1-R.sub.4) are selected so as to interact with
amino acids in the above-indicated active site in such a way that
results in the desired configuration in which the copper is in a
proximity and orientation suitable for binding the above-indicated
cysteine residues.
[0139] These groups can therefore include, for example, aromatic
groups for interacting with aromatic groups in corresponding amino
acid residues in the active sites (e.g., phenylalanine, tyrosine,
or tryptophan (e.g. the tryptophan may interact with a flanking
pyridine), also please indicate other optional interactions);
heteroatoms for forming hydrogen bonds with corresponding groups of
amino acid residues in the active sites (e.g., lysine, threonine,
methionine, serine, asparagine or glutamine)
[0140] In some embodiments, one or more of Rx, Rz and
R.sub.1-R.sub.4 comprises an aryl or heteroaryl. In an exemplary
embodiment, R.sub.2 is an aryl or heteroaryl.
[0141] In some embodiments, one or more of Rx, Rz and
R.sub.1-R.sub.4 is alkoxy or thioalkoxy. In an exemplary
embodiment, R.sub.4 is alkoxy or thioalkoxy.
[0142] In some embodiments, two or more of Rx, Rz and
R.sub.1-R.sub.4 form together a cyclic ring, so as to form a
compound that comprises at least 3 fused rings.
[0143] In some embodiments, Rz and R.sub.1 form a heterocyclic
ring. In some embodiments, the heterocyclic ring is a heteroaryl.
In some embodiments, X is nitrogen and the heteroaryl is
pyridine.
[0144] In some embodiments, X and Y are each nitrogen and Z is
sulfur. Alternatively, X and Z are each sulfur and Y is nitrogen.
Further alternatively, each of X, Y and X is nitrogen. Other
combinations are also contemplated.
[0145] In some embodiments, at least one of the bonds denoted by a
dashed line in the formulae hereinabove is an unsaturated bond. In
some embodiments, X, Y, Z, A.sub.1-A.sub.4 and the bonds
therebetween are selected so as to form an aromatic system when
complexed with copper.
[0146] In some embodiments, A.sub.1, A.sub.2 and A.sub.4 are each
carbon and A.sub.3 is a heteroatom (e.g., nitrogen).
[0147] In each of the above-described embodiments, the heteroatom
can be neutral, positively charged or negatively charged.
[0148] In some embodiments, the compound comprises two or more
ligands as described herein, which are coordinating with the
copper.
[0149] As used herein, the term "amine" describes both a --NR'R''
group and a --NR'-- group, wherein R' and R'' are each
independently hydrogen, alkyl, cycloalkyl, aryl, as these terms are
defined hereinbelow.
[0150] The amine group can therefore be a primary amine, where both
R' and R'' are hydrogen, a secondary amine, where R' is hydrogen
and R'' is alkyl, cycloalkyl or aryl, or a tertiary amine, where
each of R' and R'' is independently alkyl, cycloalkyl or aryl.
[0151] Alternatively, R' and R'' can each independently be
hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide,
phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, cyano, nitro, azo, sulfonamide, carbonyl,
C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate,
urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl,
guanidine and hydrazine.
[0152] The term "alkyl" describes a saturated aliphatic hydrocarbon
including straight chain and branched chain groups. Preferably, the
alkyl group has 1 to 20 carbon atoms. Whenever a numerical range;
e.g., "1-20", is stated herein, it implies that the group, in this
case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3
carbon atoms, etc., up to and including 20 carbon atoms. More
preferably, the alkyl is a medium size alkyl having 1 to 10 carbon
atoms. Most preferably, unless otherwise indicated, the alkyl is a
lower alkyl having 1 to 4 carbon atoms. The alkyl group may be
substituted or unsubstituted. Substituted alkyl may have one or
more substituents, whereby each substituent group can independently
be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide,
sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy,
thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo,
sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,
O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide,
N-amide, guanyl, guanidine and hydrazine.
[0153] The term "cycloalkyl" describes an all-carbon monocyclic or
fused ring (i.e., rings which share an adjacent pair of carbon
atoms) group where one or more of the rings does not have a
completely conjugated pi-electron system. The cycloalkyl group may
be substituted or unsubstituted. Substituted cycloalkyl may have
one or more substituents, whereby each substituent group can
independently be, for example, hydroxyalkyl, trihaloalkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,
amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy,
aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo,
sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,
O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide,
N-amide, guanyl, guanidine and hydrazine.
[0154] The term "heteroalicyclic" describes a monocyclic or fused
ring group having in the ring(s) one or more atoms such as
nitrogen, oxygen and sulfur. The rings may also have one or more
double bonds. However, the rings do not have a completely
conjugated pi-electron system. The heteroalicyclic may be
substituted or unsubstituted. Substituted heteroalicyclic may have
one or more substituents, whereby each substituent group can
independently be, for example, hydroxyalkyl, trihaloalkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,
amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy,
aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo,
sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,
O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide,
N-amide, guanyl, guanidine and hydrazine. Representative examples
are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane,
morpholino and the like.
[0155] The term "aryl" describes an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups having a completely conjugated pi-electron
system. The aryl group may be substituted or unsubstituted.
Substituted aryl may have one or more substituents, whereby each
substituent group can independently be, for example, hydroxyalkyl,
trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate,
hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,
cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate,
N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate,
O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.
[0156] The term "heteroaryl" describes a monocyclic or fused ring
(i.e., rings which share an adjacent pair of atoms) group having in
the ring(s) one or more atoms, such as, for example, nitrogen,
oxygen and sulfur and, in addition, having a completely conjugated
pi-electron system. Examples, without limitation, of heteroaryl
groups include pyrrole, furane, thiophene, imidazole, oxazole,
thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline
and purine. The heteroaryl group may be substituted or
unsubstituted. Substituted heteroaryl may have one or more
substituents, whereby each substituent group can independently be,
for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide,
sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy,
thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo,
sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,
O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide,
N-amide, guanyl, guanidine and hydrazine. Representative examples
are pyridine, pyrrole, oxazole, indole, purine and the like.
[0157] The term "hydroxyl" describes a --OH group.
[0158] The term "alkoxy" describes both an --O-alkyl and an
--O-cycloalkyl group, as defined herein.
[0159] The term "thiohydroxy" or "thiol" describes a --SH
group.
[0160] The term "thioalkoxy" describes both a --S-alkyl group, and
a --S-cycloalkyl group, as defined herein.
[0161] The term "thioaryloxy" describes both a --S-aryl and a
--S-heteroaryl group, as defined herein.
[0162] The term "carboxylate" encompasses "C-carboxylate", which
describes a --C(.dbd.O)--OR' group, where R' is as defined herein;
and "O-carboxylate", which describes a --OC(.dbd.O)R' group, where
R' is as defined herein.
[0163] The term "thiocarboxylate" encompasses "C-thiocarboxylate",
which describes a --C(.dbd.S)--OR' group, where R' is as defined
herein; and "O-thiocarboxylate", which describes a --OC(.dbd.S)R'
group, where R' is as defined herein.
[0164] The term "carbamate" encompasses "N-carbamate", which
describes an R''OC(.dbd.O)--NR'-- group, with R' and R'' as defined
herein; and "O-carbamate", which describes an --OC(.dbd.O)--NR'R''
group, with R' and R'' as defined herein.
[0165] The term "thiocarbamate" encompasses "O-thiocarbamate",
which describes a --OC(.dbd.S)--NR'R'' group, with R' and R'' as
defined herein; "N-thiocarbamate", which describes an
R''OC(.dbd.S)NR'-- group, with R' and R'' as defined herein;
"S-dithiocarbamate", which describes a --SC(.dbd.S)--NR'R'' group,
with R' and R'' as defined herein; and "N-dithiocarbamate", which
describes an R''SC(.dbd.S)NR'-- group, with R' and R'' as defined
herein.
[0166] The term "amide" encompasses "C-amide", which describes a
--C(.dbd.O)--NR'R'' group, where R' and R'' are as defined herein;
and "N-amide", which describes a R'C(.dbd.O)--NR''-- group, where
R' and R'' are as defined herein.
[0167] The term "S-sulfonamide" describes a
--S(.dbd.O).sub.2--NR'R'' group, with R' as defined herein and R''
is as defined herein for R'.
[0168] The term "N-sulfonamide" describes an
R'S(.dbd.O).sub.2--NR''-- group, where R' and R'' are as defined
herein.
[0169] Exemplary compounds include NSC321206 and NSC310551, as
described herein.
[0170] Thus, according to another aspect of the present invention
there is provided a method of treating a disease in which
inhibiting of a proteasome is advantageous, the method comprising
administering to the subject a therapeutically effective amount of
a compound selected from the group consisting of NSC321206,
NSC310551, NSC99671 and NSC3907, thereby treating the disease.
[0171] Because unregulated, proteasome-mediated degradation of
vital cell cycle regulatory proteins is an essential component of
tumor development, a possible way of arresting or limiting tumor
development is by inhibition of the proteasome. Proteasome
inhibition leads to the stabilization of these substrates, and, as
a result, cell-cycle arrest occurs and the cells ultimately undergo
apoptosis. Transformed cells seem to be particularly sensitive to
any disturbances of the cell cycle and/or the coordinated
production and degradation of all proteins involved in this
process, including proteasome inhibitor-induced growth retardation.
Consequently, proteasome inhibitors are being actively explored for
the treatment of a variety of hematologic malignant neoplasms and
solid tumors.
[0172] Thus, according to one embodiment, the disease in which
inhibiting a proteasome is advantageous is cancer.
[0173] Examples of cancers that may be treated using the
proteaseome inhibitors of this aspect of the present invention
include, but are not limited to adrenocortical carcinoma,
hereditary; bladder cancer; breast cancer; breast cancer, ductal;
breast cancer, invasive intraductal; breast cancer, sporadic;
breast cancer, susceptibility to; breast cancer, type 4; breast
cancer, type 4; breast cancer-1; breast cancer-3; breast-ovarian
cancer; Burkitt's lymphoma; cervical carcinoma; colorectal adenoma;
colorectal cancer; colorectal cancer, hereditary nonpolyposis, type
1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal
cancer, hereditary nonpolyposis, type 3; colorectal cancer,
hereditary nonpolyposis, type 6; colorectal cancer, hereditary
nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial
carcinoma; esophageal cancer; gastric cancer, fibrosarcoma,
glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma;
hepatocellular cancer; hepatocellular carcinoma; leukemia, acute
lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid,
with eosinophilia; leukemia, acute nonlymphocytic; leukemia,
chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer;
lung cancer, small cell; lymphoma, non-Hodgkin's; lynch cancer
family syndrome II; male germ cell tumor; mast cell leukemia;
medullary thyroid; medulloblastoma; melanoma, malignant melanoma,
meningioma; multiple endocrine neoplasia; multiple myeloma, myeloid
malignancy, predisposition to; myxosarcoma, neuroblastoma;
osteosarcoma; ovarian cancer; ovarian cancer, serous; ovarian
carcinoma; ovarian sex cord tumors; pancreatic cancer; pancreatic
endocrine tumors; paraganglioma, familial nonchromaffin;
pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma;
prostate cancer; renal cell carcinoma, papillary, familial and
sporadic; retinoblastoma; rhabdoid predisposition syndrome,
familial; rhabdoid tumors; rhabdomyosarcoma; small-cell cancer of
lung; soft tissue sarcoma, squamous cell carcinoma, basal cell
carcinoma, head and neck; T-cell acute lymphoblastic leukemia;
Turcot syndrome with glioblastoma; tylosis with esophageal cancer;
uterine cervix carcinoma, Wilms' tumor, type 2; and Wilms' tumor,
type 1, and the like.
[0174] According to a specific embodiment, the cancer is multiple
myeloma.
[0175] The formation of new blood vessels, angiogenesis, is
critical for the progression of many diseases, including cancer
metastases, diabetic retinopathy, and rheumatoid arthritis. Many
factors associated with angiogenesis, eg, cell adhesion molecules,
cytokines, and growth factors, are regulated through the
proteasome, and, hence, alteration of its activity will affect the
degree of vessel formation. Oikawa et al [Biochem Biophys Res
Commun. 1998; 246:243-248] demonstrated that a particular
proteasome inhibitor, lactacystin significantly reduced
angiogenesis, suggesting that it, or related compounds, could be
beneficial in disease states that rely on the formation of new
blood vessels.
[0176] Thus, according to another embodiment, the disease in which
inhibiting a proteasome is advantageous is an angiogenesis
associated disease.
[0177] The proteasome is intimately linked to the production of the
majority of the class I antigens. It is therefore conceivable that
excessive inhibition of the proteasome might also increase the
chance of viral infections such as HIV.
[0178] Through its regulation of NF-kappa B, the proteasome is
central to the processing of many pro-inflammatory signals. Once
released from its inhibitory complex through proteasome degradation
of I kappa B, NF-kappa B induces the activation of numerous
cytokines and cell adhesion molecules that orchestrate the
inflammatory response. Thus, the present invention contemplates use
of the proteasome inhibitors of the present invention for the
treatment of inflammatory diseases including but not limited to
asthma, ischemia and reperfusion injury, multiple sclerosis,
rheumatoid arthritis, psoriasis, autoimmune thyroid disease,
cachexia, Crohn disease, hepatitis B, inflammatory bowel disease,
sepsis, systemic lupus erythematosus, transplantation rejection and
related immunology and autoimmune encephalomyelitis.
[0179] In addition, it has been shown that blocking proteasome
activity reduces neuron and astrocyte degeneration and neutrophil
infiltration and therefore can be potential therapy for stroke and
neurodegenerative diseases including Parkinson's disease, Alzheimer
disease, and amyotrophic lateral sclerosis (ALS).
[0180] The proteaseome inhibitors of this aspect of the present
invention may be provided per se or as part of a pharmaceutical
composition, where it is mixed with suitable carriers or
excipients.
[0181] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0182] Herein the term "active ingredient" refers to the proteasome
inhibitors accountable for the biological effect.
[0183] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0184] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0185] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0186] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
inrtaperitoneal, intranasal, or intraocular injections.
[0187] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0188] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0189] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0190] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0191] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0192] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0193] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0194] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner
[0195] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0196] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuos infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0197] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0198] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0199] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0200] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients (proteasome inhibitor)
effective to prevent, alleviate or ameliorate symptoms of a
disorder (e.g., cancer) or prolong the survival of the subject
being treated.
[0201] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0202] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0203] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0204] Dosage amount and interval may be adjusted individually to
ensure blood or tissue levels of the active ingredient are
sufficient to induce or suppress the biological effect (minimal
effective concentration, MEC). The MEC will vary for each
preparation, but can be estimated from in vitro data. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. Detection assays can
be used to determine plasma concentrations.
[0205] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0206] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0207] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as is further detailed
above.
[0208] The term "treating" refers to inhibiting, preventing or
arresting the development of a pathology (disease, disorder or
condition) and/or causing the reduction, remission, or regression
of a pathology. Those of skill in the art will understand that
various methodologies and assays can be used to assess the
development of a pathology, and similarly, various methodologies
and assays may be used to assess the reduction, remission or
regression of a pathology.
[0209] As used herein, the term "preventing" refers to keeping a
disease, disorder or condition from occurring in a subject who may
be at risk for the disease, but has not yet been diagnosed as
having the disease.
[0210] As used herein, the term "subject" includes mammals,
preferably human beings at any age which suffer from the pathology.
Preferably, this term encompasses individuals who are at risk to
develop the pathology.
[0211] As used herein the term "about" refers to .+-.10%
[0212] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0213] The term "consisting of means "including and limited
to".
[0214] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0215] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0216] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0217] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0218] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0219] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Discovery of Novel Proteasome Inhibitors Using a High-Content
Cell-Based Screening System
[0220] Materials and Methods
[0221] DNA constructs, generation of a stable reporter cell line
and transient transfection: To construct a YFP-tagged Proteasome
Inhibitor Reporter (PIR) protein, cDNA encoding a full-length human
p53 R175H mutant was amplified by PCR from a cDNA clone, and three
consecutive lysine residues in the bipartite Nuclear Localization
Signal (NLS) were replaced with alanines by PCR-based,
site-directed mutagenesis [Higuchi R, et al., (1988) Nucleic Acids
Res 16: 7351-7367; Ho S N, et al (1989) Gene 77: 51-59]. The DNA
fragment was cloned into the BglII and NotI sites of pLPCX
retroviral vector (Clontech) in-frame to the N-terminus of the
yellow fluorescent protein (YFP), using the NotI/ClaI restriction
sites. The PIR protein was expressed in an H1299 non-small cell
lung carcinoma cell line, following retroviral infection, and the
cells were cultured in RPMI 1640 medium containing 10% fetal bovine
serum, 2 mM glutamine and 1% penicillin-streptomycin (all from
Sigma-Aldrich) in a humidified atmosphere of 5% CO.sub.2 at
37.degree. C. Fluorescent cells were isolated by flow cytometry,
and single-cell cloning was used to generate a morphologically
uniform cell population.
[0222] Transfections employing plasmid DNA were carried out using
Lipofectamine 2000.TM. reagent (Invitrogen) as per the
manufacturer's instructions. For RNA interference, PIR cells were
transfected with 50 pM control or MDM2 siRNA oligonucleotides
(Dharmacon, ON-TARGETplus SMARTpool), with Dharmafect 2 (Dharmacon)
according to the manufacturer's protocol
[0223] Immunofluorescence Microscopy: Cells were cultured on glass
coverslips, fixed, and permeabilized for 2 minutes in
phosphate-buffered saline (PBS) containing 0.5% Triton X-100 and 3%
formaldehyde, and post-fixed with 3% formaldehyde in PBS for 30
minutes. The cells were then rinsed and stained with polyclonal
anti-.beta.-catenin antibody (Sigma) or a mixture of anti-MDM2
monoclonal antibodies SMP14, 2A10, and 4B11 for 1 hour, washed, and
further incubated with Cy3-conjugated goat anti-mouse IgG (Enco).
Images were acquired using the DeltaVision system (Applied
Precision Inc.).
[0224] Compound Library: The chemical compound library screened for
proteasomal inhibitors consisted of the NCI Diversity Set,
containing 1,992 low molecular weight synthetic compounds selected
from and representing nearly 140,000 compounds available from the
NCI DTP chemical library
(www.dtp.nci.nih.gov/branches/dscb/diversity explanation.html).
[0225] The library compounds were dissolved in dimethyl sulfoxide
(DMSO) to a concentration of 10 mM, placed in 96-well plates, and
stored at -70.degree. C. for future use. Image-Based Screening
Assay for Proteasome Activity: PIR-expressing H1299 cells were
seeded at a density of 800 cells per well in 384-well assay plates
(F-bottom, pClear, black, tissue-culture-treated) (Greiner
Bio-One). Cells were cultured for 24 hours and treated with the
library compounds at two concentrations (1 and 10 .mu.M) in RPMI
1640. In each assay plate, cells in 24 wells treated with 0.2% DMSO
alone were used as controls. As a positive control, 1 .mu.M MG132
was added to a single column of the assay plate. Following 12 hours
of incubation, cells were fixed in 3% paraformaldehyde for 20
minutes, then washed with PBS and screened for localization of the
PIR protein.
[0226] Microscope automation and image processing: WiScan.TM. Cell
Imaging System (Idea Bio-Medical) was used for this screen [Paran Y
et al., 2006, Methods Enzymol 414: 228-247]. The system is based on
an IX71 microscope (Olympus), equipped with a fast laser AutoFocus
device and an automated stage. Thirty-six fields were acquired from
every well using a 60.times./0.9 air objective, stored, and tiled
into montages to detect consistent effects. Scoring of the nuclear
translocation of the fluorescent PIR protein was carried out
manually.
[0227] In vitro proteasome activity assay: For measuring proteasome
activity, purified 20S or 26S proteasomes prepared from rabbit
muscles were incubated at a final concentration of 16 nM, with 100
.mu.M fluorogenic 7-amido-4-methylcoumarin (AMC) tetrapeptide
substrate Suc-LLVY-AMC (Bachem) and the stated concentration of the
hit compounds in the presence of 100 .mu.l of assay buffer (50 mM
Hepes, 5 mM MgCl.sub.2, 2 mM ATP and 1 mM DTT). The well-documented
proteasomal inhibitor MG132 was used as a positive control, and an
equivalent volume of solvent as a negative control. The
time-dependent increase of hydrolyzed AMC groups was measured in a
96-well plate equilibrated to 37.degree. C., using a Varioscan
multi-well plate reader (Thermo Fisher Scientific, Inc.) in a
kinetic mode, in which the recording intervals were set to 1
minute. The excitation wavelength was 370 nm; fluorescence emission
was recorded at 465 nm
[0228] Immunobloting: H1299-PIR cells were lysed with radioimmune
precipitation assay buffer (1% NP-40, 1% sodium deoxycholate, 0.1%
SDS, 150 mM NaCl, 50 mM Tris, pH 8.0) containing a protease
inhibitor cocktail from Roche Applied Science. Protein extracts
were subjected to 8% SDS-PAGE, transferred to a nitrocellulose
membrane (Whatman), and probed with monoclonal anti-p53 (DO1, Santa
Cruz Biotechnology), anti-ubiquitin (Covance), anti-.beta.-catenin
(Sigma) and anti-.beta.-tubulin (Sigma) primary antibodies.
[0229] For sub-cellular fractionation, cells were harvested and
resuspended in ice-cold hypotonic lysis buffer [10 mM HEPES pH 7.9,
1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, supplemented with a complete
protease inhibitor cocktail (Roche)], incubated on ice for 15
minutes, then NP40 was added, to a final concentration of 0.6%. The
samples were vortexed for 10 seconds and immediately centrifuged at
12,000 g for 30 seconds. The supernatant (cytoplasmic fraction) was
transferred to a fresh tube. The nuclei pellet was washed once with
hypotonic lysis buffer, and lysed with SDS sample buffer (100 mM
Tris-HCl pH 6.8, 2% SDS, 100 mm DTT, and 10% glycerol). Cell
viability assay: The effect of each hit compound on cell viability
was tested at 11 different concentrations, ranging from 0.1-100
.mu.M. PIR cells were plated onto 384-well microplates for 24
hours, and then treated for 48 h with the library compounds. Cell
viability was determined using the colorimetric AlamarBlue.RTM.
(Invitrogen) viability assay, according to the manufacturer's
instructions. Results are expressed as GI.sub.50; namely, the
compound concentration that reduces the AlamarBlue.RTM. score by
50%, compared to untreated controls.
Results
Development H1299-PIR Reporter Cell Line and its Application for
Image-Based, High-Throughput Screening Assay for Proteasome
Inhibitors
[0230] As seen in FIG. 1, the PIR protein, used here for proteasome
inhibitor screening, was constructed of yellow fluorescent protein
(YFP) fused to the C-terminus of the human p53 R175H mutant. The
cytoplasm-to-nucleus transport of this p53 mutant was attenuated by
additional triple mutation in the bipartite NLS in which three
consecutive lysine residues were replaced with alanines (K319A,
K320A, and K321A), as described by O'Keefe et al [Mol Cell Biol 23:
6396-6405, 2003]. In agreement with previous reports, under normal
cell culture conditions, this point mutation leads to cytoplasmic
localization of the PIR protein in H1299 cells [O'Keefe et al., Mol
Cell Biol 23: 6396-6405, 2003], confirming that the three basic
residues at position 319-321 are indeed part of a nuclear
localization signal. However, upon treatment with known proteasome
inhibitors (e.g. MG132, Bortezomib and ALLN), PIR translocates into
the nucleus in a manner reminiscent of p53 mutated in its NLS
(FIGS. 2A-D). .beta.-catenin, whose cellular levels are primarily
regulated by the proteasome, underwent similar nuclear
translocation in response to proteasome inhibition (FIGS. 2E-H),
yet this was accompanied by significant stabilization and increase
in its quantity unlike PIR, whose overall levels did not markedly
change. To validate these results, the levels of PIR in the nuclear
and cytoplasmic fractions of treated and control H1299-PIR cells
were quantified using immunoblotting. This assay confirmed the
microscopy-based observation, and pointed to a .about.3-fold
increase in the nuclear/cytoplasmic ratio of PIR, in response to
MG132 treatment (FIG. 2I).
[0231] To assess the sensitivity of the PIR cell-based assay,
H1299-PIR cells were incubated for 8 hours with different
concentrations (0.01-10 .mu.M) of known proteasome inhibitors
(MG132 and Bortezomib). The cells were then fixed and scored for
nuclear translocation of PIR. The score (EC.sub.50) refers to the
concentration of inhibitor needed to induce nuclear translocation
of PIR in 50% of the treated cells. This test indicated that in our
assay, the EC.sub.50 values for MG132 and Bortezomib were 0.5
.mu.M, and 0.05 .mu.M, respectively. These values favorably compare
with those reported for other detection systems, such as the
commercial Living Colors HEK 293 ZsGreen Proteasome Sensor system
(Clontech), which detects MG132 at 2.5 .mu.M (after 20 hours of
treatment using flow cytometry) [Andreatta C et al., Biotechniques
30: 656-660] or for the Ubi[G76V]-GFP-based reporter system
(Biolmage), in which the reported EC.sub.50 value for MG-132 was
approximately 1.0 .mu.M [Dantuma et al, 2000 Nat Biotechnol 18:
538-543]. Thus, H1299-PIR cells appear to be sensitive reporters,
capable of detecting the activity of proteasome inhibitors in a
cell-based assay.
Nuclear Accumulation of Endogenous MDM2 in Response to Proteasome
Inhibition is Responsible for PIR Nuclear Translocation
[0232] To further characterize the mode of PIR nuclear
translocation upon proteasome inhibition, the present inventors
considered the possibility that proteasome-sensitive p53 binding
proteins, are responsible for carrying PIR into the nucleus.
Towards this end, MDM2, a p53 E3 ubiquitin ligase and a known
target of proteasome-dependent degradation, was transfected into
PIR cells, and its localization was assessed by immunofuorescence
microscopy. As expected, endogenous MDM2 labeling in the PIR-H1299
cells was relatively faint and mostly nuclear while PIR was mainly
localized to the cytoplasm (FIGS. 3A-H). In contrast, in the cells
transfected with wild type MDM2, both the fluorescent PIR and MDM2
translocated to the nucleus. This suggests that MDM2 can transport
NLS-deficient PIR from the cytoplasm into the nucleus, perhaps via
the NLS of MDM2, consistently with previous studies suggesting that
MDM2 can promote the nuclear import of ANLS p53. Interestingly, PIR
remained cytoplasmic in cells over-expressing a mutated MDM2
lacking the p53 binding site (MDM2 .DELTA.9-58), suggesting that
the interaction between the two proteins is needed for their
cotranslocation to the nucleus. On the other hand, MDM2 mutant with
point mutation that abolishes its E3 ubiquitin ligase function
(MDM2 Ser440) induced PIR nuclear localization similar to the wild
type molecule.
[0233] To check whether MDM2 expression is critical for PIR nuclear
translocation, siRNA-mediated knockdown of MDM2 expression was
performed in PIR-cells, and then treated the cells with proteasome
inhibitors (FIGS. 4A-H). It was found that when MDM2 levels in the
knocked-down cells were reduced, PIR remained cytoplasmic even
following treatment with proteasome inhibitors, indicating that
MDM2 is an essential player in the nuclear localization of
NLS-deficient PIR.
Screening for Novel Proteasome Inhibitors in the Diversity Set of
the NIH/NCI Chemical Library
[0234] To assess the potential use of the cytoplasm-to-nucleus
translocation of PIR in high-throughput, microscopy-based screening
for novel proteasome inhibitors, 1,992 low molecular weight
compounds comprising the NCI Diversity Set chemical library were
tested. A flow chart depicting the screening procedure is shown in
FIG. 5, and described in the Experimental Procedures. Following the
initial automated screen, the images of the affected cells were
inspected manually and a secondary screen was performed, in which
hit compounds were tested at multiple concentrations, and directly
compared to the well-established proteasome inhibitor MG132. This
procedure resulted in the discovery of four compounds that induced
nuclear translocation of PIR, indicating a hit rate of .about.0.2%.
As summarized in FIG. 6, all four compounds detected in the primary
screen were confirmed by manual inspection.
Biochemical Validation of the Inhibitory Effects of the Hit
Compounds
[0235] One characteristic outcome of proteasome inhibition is the
accumulation of ubiquitinated proteins in the treated cells. To
monitor the levels of ubiquitinated proteins that accumulated upon
incubation with the novel inhibitors detected in the present
screen, H1299-PIR cells were treated with each of the inhibitors
for 6 hours, at doses comparable to those that were used in the
screen. Following incubation, cell extracts were analyzed by
Western blot, using anti-ubiquitin and anti-.beta.-catenin
antibodies. As shown in FIGS. 7A-B, accumulation of endogenous
polyubiquitinated proteins, as well as elevated levels of
.beta.-catenin (a known target of the proteasome), at varying
degrees, were caused by all four inhibitors, confirming their
inhibitory effect on proteasomal degradation. The hit compounds
NSC321206 (at a concentration of 0.15 .mu.M) and NSC310551 (0.3
.mu.M) were the most effective, demonstrating inhibitory activity
comparable to that of 5 .mu.M MG132. NSC99671 and NSC3907 (50 .mu.M
and 20 .mu.M, respectively) displayed less of an inhibitory effect.
It is noteworthy that the same concentrations that induced nuclear
transport in the PIR assay, also resulted in accumulation of
polyubiquitinated proteins and stabilization of .beta.-catenin.
Moreover, the potency of proteasomal inhibition, judged by these
criteria, coincides nicely with the magnitude of the nuclear
fluorescence signal detected in the PIR cell-based assay, upon
inhibition with the different hit compounds.
[0236] To directly test the capacity of the four compounds to
inhibit activity in mammalian proteasomes, an in vitro activity
assay was performed in which the hit compounds were tested for
their effects on the degradation of the model fluorogenic
tetrapeptide LLVY-AMC by purified rabbit 26S proteasomes. As seen
in FIG. 8, all four compounds inhibited proteasomal degradation to
varying degrees. Both NSC310551 and NSC321206 showed levels of
inhibition comparable to that of MG132, with NSC321206 being the
most effective inhibitor. NSC99671 displayed a moderate inhibitory
effect, and NSC3907 had only a minor effect. The low potency of
NSC3907 in inhibiting the purified proteasome was consistent with
previous findings, showing that this molecule (8-Quinolinol
salicylate) can specifically inhibit the chymotryptic activity of
the proteasome only in complex with intracellular copper. The fact
that this compound was still picked up by the present screen
reflects an advantage of this cell-based assay.
Effect of the Novel Proteasome Inhibitors on Cell Viability
[0237] Proteasome inhibitors are known to be particularly cytotoxic
to malignant cells via multiple mechanisms. To directly test the
effects of the new proteasome inhibitors discovered in this study
on cell viability, PIR-expressing H1299 cells were treated for 48
hours with each of the four compounds, at a wide range of
concentrations, ranging from 0.1 to 100 .mu.M. The cells were then
subjected to an Alamar Blue viability assay, which quantifies the
number of metabolically active cells. As shown in FIG. 9, all four
compounds affect cell viability, or inhibit the growth of PIR-H1299
cells (independent of the presence of PIR), at different
concentrations. NSC3907 and NSC99671 exhibited a relatively weak
growth inhibition effect, with GI.sub.50 values of 47 .mu.M and 96
.mu.M, respectively, while NSC310551 and NSC321206 displayed a
considerably stronger effect, with GI.sub.50 values of 0.27 .mu.M
and 0.17 .mu.M, respectively. For all proteasome inhibitors
examined in this screen, there was a high correlation between
proteasomal inhibitory activity and cell viability.
[0238] In view of previous reports, indicating that malignant cells
are significantly more sensitive to proteasome inhibition than
their normal counterparts, the present inventors compared the
effect of the most effective inhibitory compound, NSC321206, toward
normal breast epithelial cell line (MCF10A) and malignant breast
carcinoma cells (MDM-MB-231) cell lines. As shown in FIG. 10
NSC321206 effectively eliminated all MDA-MB-231 cells at a
concentration of 1 .mu.M (GI.sub.50 value of 0.4 .mu.M), while the
non-malignant breast epithelial cell line (MCF10A) were only
partially affected, at a considerably higher concentration of this
compound.
[0239] To gain insights into the effects of the present compounds
on a wide variety of cells, published information on the effects of
these compounds on the NCI-60 panel of human tumor cell lines used
in the NCI Developmental Therapeutics Program (DTP)
(http://dtpdotncidotnihdotgov) were explored. As seen in FIG. 11,
the four hit compounds showed cytotoxic effects (log.sub.10
GI.sub.50<-4.0) against a variety of cell lines, whereas
NSC321206 and NSC310551 demonstrated high cytotoxicity in vitro
against all tested human cancer cell lines in the panel, with
average negative log.sub.10 GI.sub.50 values of 7.2 and 6.6,
respectively. The activity of NSC3907 was much lower, with a mean
overall -log.sub.10 GI.sub.50 value of 5.3. NSC99671 was non-toxic
for most of the lines (overall -log10 GI.sub.50 of 4.1). The most
sensitive cell lines for all hit compounds were the leukemia cells,
with overall -log.sub.10 GI.sub.50 equaling 7.73 for NSC321206,
7.028 for NSC310551, 6.249 for NSC3907, and 4.473 for NSC9967.
These initial findings corroborate the present in vitro results,
and directly demonstrate the use of the presently identified novel
proteasomal inhibitors as potential therapeutic agents in
cancer.
[0240] Characterization of the possible mode of action of the
proteasome inhibitor NSC321206, discovered using the PIR system
[0241] To gain an insight into the inhibitory mechanism of
NSC321206, a structural analysis of potential binding sites in the
proteasome at large, and around its catalytic centers, in
particular, was conducted. This survey addressed both the
possibility that the Cu.sup.++ ions play a role in the process, and
the location of potential docking site for the entire NSC321206
molecule. A search for putative Cu.sup.++ ion binding site (using
CHED server) in the yeast 20S proteosome 3D structure revealed
candidate binding sites for Cu.sup.++ ions (without the organic
ligand) on the outer surface of the a-ring, This was deemed
irrelevant for inhibition. The search was then optimized,
computationally, for the predicated 3D model of NSC321206. The
optimized structure was similar to known crystallographic one. Once
established these coordinates were used for searching for
energetically favorable docking sites that may provide a structural
bases for the observed inhibition of the Tryptic like (.beta.2),
Caspase like (.beta.1) and Chymotrypsin like (.beta.5) activities
of the proteasome, that were found for NSC321206. The best 250
potential docking sites, located in the vicinity of each of these
active sites were selected and grouped into clusters. A potential
docking site for NSC321206 was identified. The site has a deep
hydrophobic pocket and two Cys residues positioned in the ideal
conformation with regards to the Cu atom of the inhibitor (FIGS.
12A-B).
Discussion
[0242] Presently, few approaches for the high-throughput discovery
of proteasome inhibitors exist, those that do, are mostly based on
the use of biochemical techniques. Cell-based/image-based assays
enables evaluation of potential proteasomal inhibitors that may not
be detected using purified proteasomes and have several other
advantages such as demonstrating that active compounds are
cell-permeable and are sensitive to effects at multiple targets and
nodes within a given pathway, as opposed to a strict cell-free
assay that focuses on one particular target, such as degradation of
a particular substrate by a purified proteasome. The main
motivation directing the development of PIR was to establish a
method enabling the assessment of proteasomal inhibition in a
cellular context, based on the unequivocal translocation of a
fluorescent reporter protein from the cytoplasm to the nucleus upon
proteasomal inhibition, without grossly affecting its overall
levels. This approach was found to be highly specific, with
essentially no false positives, in contrast to existing cell-based
screens for proteasomal inhibitors that monitor the accumulation of
fluorescent signals from direct proteasomal substrates, that appear
to be sensitive to autofluorescence and to the
fluorescence-quenching effects of the screening molecules, as well
as to variations in cell geometry, some of which may be induced,
directly or indirectly, by proteasomal inhibition.
[0243] The design of the PIR reporter protein is based on a p53
R175H mutant which, in contrast to the short-lived, WT p53, has a
significantly longer half-life (several hours), presumably due to
its reduced susceptibility to proteasomal degradation. As a result,
the overall concentration of PIR in cells is only marginally
affected by treatment with proteasomal inhibitors such as MG132
(FIG. 21). In the PIR assay, monitoring proteasomal inhibition is
based on intracellular translocation of the reporter protein from
the cytoplasm to the nucleus, in response to proteasomal
inhibition. It is noteworthy that PIR was found to be particularly
suitable for high throughput screening for proteasomal inhibitors,
due to the unambiguous quantification of nuclear vs. cytoplasmic
fluorescence.
[0244] In conclusion, the novel cell-based screen described here
appears to be a robust and highly sensitive tool for the
identification on new proteasome inhibitors. It is based on the
stabilized MDM2-dependent accumulation of the PIR molecule in the
nucleus, and is compatible with microscopy-based high throughput
screening technology.
[0245] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0246] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
71360PRTHomo sapiensmisc_featureHuman p53 polypeptide 1Met Glu Glu
Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu
Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25
30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp
35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu
Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala
Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser
Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln
Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr
Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys
Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp
Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155
160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys
165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro
Pro Gln 180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu
Tyr Leu Asp Asp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val
Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile
His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly
Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp
Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg
Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280
285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr
290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro
Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln
Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn
Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro
Gly 355 360 2393PRTArtificial sequenceHuman derived mutant p53
mutated on L319A, L320A and L321A 2Met Glu Glu Pro Gln Ser Asp Pro
Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu
Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro
Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile
Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60
Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65
70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser
Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly
Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val
Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln
Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr
Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr
Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro
His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185
190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp
195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro
Pro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr
Met Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg
Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn
Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys
Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys
Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys
Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Ala Ala 305 310
315 320 Ala Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg
Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu
Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg
Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr
Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp
Ser Asp 385 390 3392PRTArtificial sequenceR175H, L319A, L320A and
L321A p53 mutant polypeptide 3Met Glu Glu Pro Gln Ser Asp Pro Ser
Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp
Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser
Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu
Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg
Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70
75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser
Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe
Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr
Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu
Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro
Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys
Gln Ser Gln His Met Thr Glu Val Val Arg His Cys 165 170 175 Pro His
His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190
His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195
200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro
Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met
Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro
Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu
Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro
Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys
Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg
Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Ala Ala 305 310 315
320 Ala Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu
325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu
Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala
His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser
Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser
385 390 41179DNAArtificial sequencemutant p53 coding portion of the
p53-YFP construct 4atggaggagc cgcagtcaga tcctagcgtc gagccccctc
tgagtcagga aacattttca 60gacctatgga aactacttcc tgaaaacaac gttctgtccc
ccttgccgtc ccaagcaatg 120gatgatttga tgctgtcccc ggacgatatt
gaacaatggt tcactgaaga cccaggtcca 180gatgaagctc ccagaatgcc
agaggctgct ccccccgtgg cccctgcacc agcagctcct 240acaccggcgg
cccctgcacc agccccctcc tggcccctgt catcttctgt cccttcccag
300aaaacctacc agggcagcta cggtttccgt ctgggcttct tgcattctgg
gacagccaag 360tctgtgactt gcacgtactc ccctgccctc aacaagatgt
tttgccaact ggccaagacc 420tgccctgtgc agctgtgggt tgattccaca
cccccgcccg gcacccgcgt ccgcgccatg 480gccatctaca agcagtcaca
gcacatgacg gaggttgtga ggcactgccc ccaccatgag 540cgctgctcag
atagcgatgg tctggcccct cctcagcatc ttatccgagt ggaaggaaat
600ttgcgtgtgg agtatttgga tgacagaaac acttttcgac atagtgtggt
ggtgccctat 660gagccgcctg aggttggctc tgactgtacc accatccact
acaactacat gtgtaacagt 720tcctgcatgg gcggcatgaa ccggaggccc
atcctcacca tcatcacact ggaagactcc 780agtggtaatc tactgggacg
gaacagcttt gaggtgcgtg tttgtgcctg tcctgggaga 840gaccggcgca
cagaggaaga gaatctccgc aagaaagggg agcctcacca cgagctgccc
900ccagggagca ctaagcgagc actgcccaac aacaccagct cctctcccca
gccagcggcg 960gcaccactgg atggagaata tttcaccctt cagatccgtg
ggcgtgagcg cttcgagatg 1020ttccgagagc tgaatgaggc cttggaactc
aaggatgccc aggctgggaa ggagccaggg 1080gggagcaggg ctcactccag
ccacctgaag tccaaaaagg gtcagtctac ctcccgccat 1140aaaaaactca
tgttcaagac agaagggcct gactcagac 117951923DNAArtificial sequenceYFP
tagged mutant p53 coding sequence 5atggaggagc cgcagtcaga tcctagcgtc
gagccccctc tgagtcagga aacattttca 60gacctatgga aactacttcc tgaaaacaac
gttctgtccc ccttgccgtc ccaagcaatg 120gatgatttga tgctgtcccc
ggacgatatt gaacaatggt tcactgaaga cccaggtcca 180gatgaagctc
ccagaatgcc agaggctgct ccccccgtgg cccctgcacc agcagctcct
240acaccggcgg cccctgcacc agccccctcc tggcccctgt catcttctgt
cccttcccag 300aaaacctacc agggcagcta cggtttccgt ctgggcttct
tgcattctgg gacagccaag 360tctgtgactt gcacgtactc ccctgccctc
aacaagatgt tttgccaact ggccaagacc 420tgccctgtgc agctgtgggt
tgattccaca cccccgcccg gcacccgcgt ccgcgccatg 480gccatctaca
agcagtcaca gcacatgacg gaggttgtga ggcactgccc ccaccatgag
540cgctgctcag atagcgatgg tctggcccct cctcagcatc ttatccgagt
ggaaggaaat 600ttgcgtgtgg agtatttgga tgacagaaac acttttcgac
atagtgtggt ggtgccctat 660gagccgcctg aggttggctc tgactgtacc
accatccact acaactacat gtgtaacagt 720tcctgcatgg gcggcatgaa
ccggaggccc atcctcacca tcatcacact ggaagactcc 780agtggtaatc
tactgggacg gaacagcttt gaggtgcgtg tttgtgcctg tcctgggaga
840gaccggcgca cagaggaaga gaatctccgc aagaaagggg agcctcacca
cgagctgccc 900ccagggagca ctaagcgagc actgcccaac aacaccagct
cctctcccca gccagcggcg 960gcaccactgg atggagaata tttcaccctt
cagatccgtg ggcgtgagcg cttcgagatg 1020ttccgagagc tgaatgaggc
cttggaactc aaggatgccc aggctgggaa ggagccaggg 1080gggagcaggg
ctcactccag ccacctgaag tccaaaaagg gtcagtctac ctcccgccat
1140aaaaaactca tgttcaagac agaagggcct gactcagacg cggccgcgga
tccttcaggt 1200actggcagtg tgagcaaggg cgaggagctg ttcaccgggg
tggtgcccat cctggtcgag 1260ctggacggcg acgtaaacgg ccacaagttc
agcgtgtccg gcgagggcga gggcgatgcc 1320acctacggca agctgaccct
gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 1380cccaccctcg
tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac
1440atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
ggagcgcacc 1500atcttcttca aggacgacgg caactacaag acccgcgccg
aggtgaagtt cgagggcgac 1560accctggtga accgcatcga gctgaagggc
atcgacttca aggaggacgg caacatcctg 1620gggcacaagc tggagtacaa
ctacaacagc cacaacgtct atatcaccgc cgacaagcag 1680aagaacggca
tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
1740ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
gctgcccgac 1800aaccactacc tgagctacca gtccgccctg agcaaagacc
ccaacgagaa gcgcgatcac 1860atggtcctgc tggagttcgt gaccgccgcc
gggatcactc tcggcatgga cgagctgtac 1920aag 19236641PRTArtificial
sequenceYFP tagged mutant p53 6Met Glu Glu Pro Gln Ser Asp Pro Ser
Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp
Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser
Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu
Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg
Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70
75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser
Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe
Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr
Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu
Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro
Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys
Gln Ser Gln His Met Thr Glu Val Val Arg His Cys 165 170 175 Pro His
His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190
His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195
200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro
Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met
Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro
Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu
Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro
Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys
Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg
Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Ala Ala 305 310 315
320 Ala Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu
325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu
Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala
His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser
Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser
Asp Ala Ala Ala Asp Pro Ser Gly 385 390 395 400 Thr Gly Ser Val Ser
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro 405 410 415 Ile Leu Val
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val 420 425 430 Ser
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys 435 440
445 Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
450 455 460 Thr Thr Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro
Asp His 465 470 475 480 Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
Pro Glu Gly Tyr Val 485 490 495 Gln Glu Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg 500 505 510 Ala Glu Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu 515 520 525 Lys Gly Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu 530 535 540 Glu Tyr Asn
Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln 545 550 555 560
Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp 565
570 575 Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly 580 585 590 Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Tyr Gln Ser 595 600 605 Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu 610
615 620 Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
Tyr 625 630 635 640 Lys 751PRTArtificial sequenceProteasome
Inhibitor Reporter (PIR), Bipartite Nuclear Localization Signal
7Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro 1
5 10 15 Gln Pro Ala Ala Ala Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln
Ile 20 25 30 Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn
Glu Ala Leu 35 40 45 Glu Leu Lys 50
* * * * *
References