U.S. patent application number 15/015940 was filed with the patent office on 2016-06-02 for lipopeptide inhibitors of ras oncoproteins.
The applicant listed for this patent is 1058638 B.C. LTD., The Board of Trustees of the University of Illinois, The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv, Vanderbilt University. Invention is credited to Michael C. Dean, Vadim Gaponenko, Alla Ivanova, Joseph Kates, Nadya I. Tarasova, Sergey G. Tarasova.
Application Number | 20160151456 15/015940 |
Document ID | / |
Family ID | 46210444 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151456 |
Kind Code |
A1 |
Tarasova; Nadya I. ; et
al. |
June 2, 2016 |
LIPOPEPTIDE INHIBITORS OF RAS ONCOPROTEINS
Abstract
A method of inhibiting Ras activity in a cell comprising
introducing a peptide or peptidomimetic into the cell, wherein the
peptide or peptidomimetic is derived from or based upon the amino
acid sequence of the C-terminal .alpha.-helix or hypervariable
region (HVR) or a Ras protein.
Inventors: |
Tarasova; Nadya I.;
(Frederick, MD) ; Tarasova; Sergey G.; (Frederick,
MD) ; Gaponenko; Vadim; (Naperville, IL) ;
Kates; Joseph; (San Francisco, CA) ; Ivanova;
Alla; (Brentwood, TN) ; Dean; Michael C.;
(Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv
The Board of Trustees of the University of Illinois
1058638 B.C. LTD.
Vanderbilt University |
Bethesda
Urbana
Vancouver
Nashville |
MD
IL
TN |
US
US
CA
US |
|
|
Family ID: |
46210444 |
Appl. No.: |
15/015940 |
Filed: |
February 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14119596 |
Jan 30, 2014 |
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PCT/US2012/039623 |
May 25, 2012 |
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15015940 |
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61489919 |
May 25, 2011 |
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Current U.S.
Class: |
514/19.4 ;
435/375; 514/19.3; 514/19.5; 514/44R |
Current CPC
Class: |
C07K 7/06 20130101; C07K
7/08 20130101; A61K 38/08 20130101; A61K 38/1709 20130101; C07K
14/82 20130101; A61K 38/10 20130101; A61K 38/005 20130101; A61P
35/00 20180101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/10 20060101 A61K038/10; A61K 38/08 20060101
A61K038/08; A61K 38/00 20060101 A61K038/00 |
Claims
1. A method of inhibiting Ras activity in a cell comprising
introducing a peptide or peptidomimetic into the cell, whereby
activity of Ras is inhibited; wherein the peptide or peptidomimetic
comprises: (a) the amino acid sequence
X.sup.1YTLVRX.sup.2X.sup.3RX.sup.4X.sup.5 (SEQ ID NO: 5) or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (b) the amino acid
sequence KTPGX.sup.1VKIKK (SEQ ID NO: 6) or the inverse sequence
thereof, wherein the peptide or peptidomimetic comprises about 30
or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID
NO: 7) or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids; (d) the
amino acid sequence SGPGX.sup.1X.sup.2SX.sup.3K (SEQ ID NO: 8) or
the inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (e) the amino acid
sequence GTQGX.sup.1X.sup.2GLP (SEQ ID NO: 9) or the inverse
sequence thereof, wherein the peptide or peptidomimetic comprises
about 30 or fewer amino acids; (f) eight or more contiguous amino
acids of the C-terminal .alpha.-helix of a Ras protein or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises a total of about 30 or fewer amino acids; or (g) five or
more contiguous amino acids of the hypervariable region (HVR) of a
Ras protein or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises a total of about 30 or fewer amino
acids.
2. The method of claim 1, wherein the peptide or peptidomimetic is
introduced into the cell by contacting the cell with the peptide or
peptidomimetic.
3. The method of claim 1, wherein the peptide or peptidomimetic is
introduced into the cell by contacting the cell with a nucleic acid
encoding the peptide or peptidomimetic, whereby the peptide or
peptidomimetic is expressed in the cell.
4. The method of claim 1, wherein the cell is a cancer cell.
5. The method of claim 1, wherein the cell is in a mammal.
6. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence
X.sup.1YTLVRX.sup.2X.sup.3RX.sup.4X.sup.5 (SEQ ID NO: 5) or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids.
7. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence KTPGX.sup.1VKIKK (SEQ ID NO: 6)
or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids.
8. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence KKSKTK (SEQ ID NO: 7) or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids.
9. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence SGPGX.sup.1X.sup.2SX.sup.3K (SEQ
ID NO: 8) or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids.
10. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence GTQGX.sup.1X.sup.2GLP (SEQ ID NO:
9) or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids.
11. The method of claim 1, wherein the peptide or peptidomimetic
comprises eight or more contiguous amino acids of the C-terminal
.alpha.-helix of a Ras protein or the inverse sequence thereof,
wherein the peptide or peptidomimetic comprises a total of about 30
or fewer amino acids.
12. The method of claim 1, wherein the peptide or peptidomimetic
comprises five or more contiguous amino acids of the hypervariable
region (HVR) of a Ras protein or the inverse sequence thereof,
wherein the peptide or peptidomimetic comprises a total of about 30
or fewer amino acids.
19. The method of claim 1, wherein the peptide or peptidomimetic
comprises the amino acid sequence of any one of SEQ ID NOs: 10-72
or 75-86, or the inverse sequence thereof.
13. The method of claim 1, wherein the peptide or peptidomimetic
comprises about 20 or fewer amino acids.
14. The method of claim 1, wherein the peptide or peptidomimetic
comprises one or more D-amino acids.
15. The method of claim 1, wherein the peptide or peptidomimetic
further comprises a cell-penetrating motif.
16. The method of claim 1, wherein the peptide or peptidomimetic
further comprises a terminal fatty acid group, an N-terminal
palmitoyl residue, an N-terminal myristoyl residue, an N-terminal
lauryl residue, or an N-terminal octanoyl residue.
20. The method of claim 1, wherein the peptide or peptidomimetic
interacts with the C-terminal .alpha.-helix or Hypervariable Region
(HVR) of Ras.
17. A method for inhibiting the growth or proliferation of a cancer
cell comprising administering a peptide or peptidomimetic to the
cancer cell, whereby the growth and proliferation of the cancer
cell is inhibited; wherein the peptide or peptidomimetic comprises:
(a) the amino acid sequence
X.sup.1YTLVRX.sup.2X.sup.3RX.sup.4X.sup.5 (SEQ ID NO: 5) or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (b) the amino acid
sequence KTPGX.sup.1VKIKK (SEQ ID NO: 6) or the inverse sequence
thereof, wherein the peptide or peptidomimetic comprises about 30
or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID
NO: 7) or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids; (d) the
amino acid sequence SGPGX.sup.1X.sup.2SX.sup.3K (SEQ ID NO: 8) or
the inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (e) the amino acid
sequence GTQGX.sup.1X.sup.2GLP (SEQ ID NO: 9) or the inverse
sequence thereof, wherein the peptide or peptidomimetic comprises
about 30 or fewer amino acids; (f) eight or more contiguous amino
acids of the C-terminal .alpha.-helix of a Ras protein or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises a total of about 30 or fewer amino acids; or (g) five or
more contiguous amino acids of the hypervariable region (HVR) of a
Ras protein or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises a total of about 30 or fewer amino
acids.
18. A method for treating cancer in a host comprising administering
to the host a peptide or peptidomimetic, or nucleic acid encoding a
peptide or peptidomimetic, whereby cancer is treated; wherein the
peptide or peptidomimetic comprises: (a) the amino acid sequence
X.sup.1YTLVRX.sup.2X.sup.2RX.sup.4X.sup.5 (SEQ ID NO: 5) or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (b) the amino acid
sequence KTPGX.sup.1VKIKK (SEQ ID NO: 6) or the inverse sequence
thereof, wherein the peptide or peptidomimetic comprises about 30
or fewer amino acids; (c) the amino acid sequence KKSKTK (SEQ ID
NO: 7) or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids; (d) the
amino acid sequence SGPGX.sup.1X.sup.2SX.sup.3K (SEQ ID NO: 8) or
the inverse sequence thereof, wherein the peptide or peptidomimetic
comprises about 30 or fewer amino acids; (e) the amino acid
sequence GTQGX.sup.1X.sup.2GLP (SEQ ID NO: 9) or the inverse
sequence thereof, wherein the peptide or peptidomimetic comprises
about 30 or fewer amino acids; (f) eight or more contiguous amino
acids of the C-terminal .alpha.-helix of a Ras protein or the
inverse sequence thereof, wherein the peptide or peptidomimetic
comprises a total of about 30 or fewer amino acids; or (g) five or
more contiguous amino acids of the hypervariable region (HVR) of a
Ras protein or the inverse sequence thereof, wherein the peptide or
peptidomimetic comprises a total of about 30 or fewer amino
acids.
19. The method of claim 18, wherein the cancer is colon cancer,
pancreatic cancer, leukemia, bladder cancer, salivary gland cancer,
melanoma, myeloid malignancy, or germ cell tumors.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 14/119,596, filed Jan. 30, 2014, which is a
U.S. National Phase of International Patent Application No.
PCT/US2012/039623, filed May 25, 2012, which claims the benefit of
U.S. Provisional Patent Application No. 61/489,919, filed May 25,
2011, each of which is incorporated by reference in its entirety
herein.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 36,556 Byte
ASCII (Text) file named "722500_ST25.TXT," dated Jan. 21, 2016.
BACKGROUND OF THE INVENTION
[0003] Ras proteins are small GTPases that act as signal
transducers between cell surface receptors and several
intracellular signaling cascades. These molecules regulate such
essential cellular functions as cell survival, proliferation,
motility, and cytoskeletal organization (see Karnoub et al., Nat.
Rev. Mol. Cell Biol., 9: 517-531 (2008)).
[0004] Ras proteins function as GDP/GTP-regulated binary switches
in signal transduction cascades that can lead to cell growth,
proliferation, differentiation, or survival. In its active form,
Ras is bound to GTP. This causes a conformational change that
allows it to interact and bind to several effector molecules, most
notably the members of the Raf family, the RalGDS family, and
Phosphoinositide 3-kinases (PI3 Kinase). Ras then cleaves GTP to
GDP resulting in its inactivation. In its oncogenic, mutated state,
Ras is unable to hydrolyze GTP to GDP, thus staying in an active
state and activating numerous pathways.
[0005] The Ras superfamily has at least five major branches that
include Ras, Rho, Ran, Arf/Sar, Rab. The four classical p21 Ras
proteins are H-Ras (Harvey sarcoma viral oncogene), N-Ras
(neuroblastoma oncogene), and the splice variants K-Ras4A and
K-Ras4B (Kirsten sarcoma viral oncogene) (see Karnoub et al.,
supra). They are collectively referred to as Ras.
[0006] The p21 Ras proteins share 85% of sequence homology and
activate very similar signaling pathways. However, recent studies
clearly demonstrate that each Ras isoform functions in a unique,
radically different way from the other Ras proteins in normal
physiological processes as well as in pathogenesis (Quinlan et al.,
Future Oncol., 5: 105-116 (2009)). According to Catalogue of
Somatic Mutations in Cancer
(www.sanger.ac.uk/genetics/CGP/cosmic/), K-Ras mutations were
detected in 22.1% of analyzed human tumors, N-Ras in 8.2%, and
H-Ras in 3.3%.
[0007] Mutations in cellular Ras have been found to be present in a
large percentage of all human cancers, such as leukemias, colon
cancers, and lung cancer (see Quinlan et al., supra, and Boissel et
al., Leukemia, 20: 965-970 (2006)). More specifically, K-Ras
mutations occur frequently in lung, pancreatic, and colon cancers,
where as H-Ras mutations are prevalent in bladder, kidney, thyroid,
and salivary gland cancers (see Shulz, Int. J. Cancer, 119:
1513-1518 (2006), and Yoo et al., Arch. Pathol. Lab Med., 124:
836-839 (2000)), and N-Ras mutations are associated with myeloid
malignancies, germ cell tumors, melanoma, hepatocellular carcinoma,
and leukemia. Additionally, K-Ras mutation is predictive of
response to EGFR antagonists therapy in colorectal cancer (see
Lopez-Chavez et al., Curr. Opin. Investig. Drugs, 10: 1305-1314
(2009)).
[0008] Despite the central role of ras proteins in oncogenesis and
wide-spread efforts to develop ras-directed anti-cancer
therapeutics, no selective, specific inhibitor of the ras pathway
is available for clinical use, and ras mutant cancers remain among
the most refractory to available treatments (Adjei, J. Thorac.
Oncol., 3: S160-163 (2008); Graham et al., Recent Results Cancer
Res., 172: 125-153 (2007); and Saxena et al., Cancer Invest., 26:
948-955 (2008)). Moreover, no inhibitors acting directly on ras
oncogenes have been developed even for in vitro use. Consequently,
ras proteins are considered to be non-druggable targets. Therefore,
there is a desire to identify inhibitors of ras oncoproteins.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides a peptide or peptidomimetic
comprising the amino acid sequence comprising eight or more
contiguous amino acids of the C-terminal .alpha.-helix of a Ras
protein (e.g., eight or more contiguous amino acids of one of SEQ
ID NOs: 66-68, or inverse thereof), wherein the peptide or
peptidomimetic comprises a total of about 30 or fewer amino acids.
In a related aspect, the invention provides a peptide or
peptidomimetic comprising X.sub.1YTLVRX.sub.2X.sub.3RX.sub.4X.sub.5
(SEQ ID NO: 5) or the inverse thereof, wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids.
[0010] The invention also provides a peptide or peptidomimetic
comprising five or more contiguous amino acids of the hypervariable
region (HVR) of a Ras protein (e.g., five or more contiguous amino
acids of one of SEQ ID NOs: 69-72, or inverse thereof), wherein the
peptide or peptidomimetic comprises a total of about 30 or fewer
amino acids. In a related aspect, the invention provides a peptide
or peptidomimetic comprising the amino acid sequence
KTPGX.sub.1VKIKK (SEQ ID NO: 6) or inverse thereof, the amino acid
sequence KKSKTK (SEQ ID NO: 7) or the inverse thereof, the amino
acid sequence SGPGX.sub.1X.sub.2SX.sub.3X.sub.4 (SEQ ID NO: 8) or
the inverse thereof, or the amino acid sequence
GTQGX.sub.1X.sub.2GLP (SEQ ID NO: 9) or the inverse thereof,
wherein the peptide or peptidomimetic comprises about 30 or fewer
amino acids.
[0011] The invention further provides a method of inhibiting the
activity of a Ras protein in a cell comprising introducing a
peptide or peptidomimetic described herein into the cell, whereby
the activity of the Ras protein is inhibited.
[0012] The invention also provides a method for inhibiting the
growth or proliferation of a cancer cell comprising administering a
peptide or peptidomimetic described herein to the cancer cell,
whereupon the growth or proliferation of the cancer cell is
inhibited.
[0013] In addition, the invention provides a method of treating
cancer in a host comprising administering to the host a peptide or
peptidomimetic described herein or nucleic acid encoding same,
whereby the cancer is treated.
[0014] Related compounds, compositions, and methods also are
provided, as will be apparent from the detailed description of the
invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0015] FIG. 1 depicts the primary structure alignment of four human
Ras proteins with secondary structure elements shown above the
sequences. The helix 6 and C-terminal hypervariable region (HVR)
are shaded. Positions of the sequences that are conserved are
marked with an asterisk. Positions of the sequences showing
variation are marked with dots indicating the relative similarity
between the residues that occupy a given position in the sequence.
Two dots below a given position in the sequence indicate
substitution by more closely related residues and one dot or no
dots indicates substitution by less similar residues.
[0016] FIG. 2 is a graph illustrating growth inhibition in human
lung tumor cell lines (A549, H460, and H1944) and a mouse
astrocytoma cell line (187436 tu2) expressing mutated K-Ras by
treatment with a Ras inhibitor (KR-H6-23). The concentration (nM)
of inhibitor is represented on the x-axis and the cell number
(percent of control) is represented on the y-axis.
[0017] FIG. 3 is a graph illustrating growth inhibition in human
lung tumor cell lines (A549, H460, and H1944) and a mouse
astrocytoma cell line (187436 tu2) expressing mutated K-Ras by
treatment with a Ras inhibitor (KR-4A-4). The concentration (nM) of
inhibitor is represented on the x-axis and the cell number (percent
of control) is represented on the y-axis.
[0018] FIG. 4 is a graph illustrating growth inhibition in a breast
epithelial cell line (BJ-5ta, immortalized by transfection of
normal cells with human Telomerase Reverse Transcriptase
(hTERT)-expressing plasmid) and a human lung tumor cell line (A549)
by treatment with a Ras inhibitor (kR-H6-30). The concentration
(nM) of inhibitor is represented on the x-axis and the cell number
(percent of control) is represented on the y-axis.
[0019] FIG. 5 is a graph illustrating the inhibition of HCC15 cell
motility by treatment with Ras inhibitors (kR-4A-1 and kR-4B-1).
The average closure in .mu.m is indicated on the y-axis for
control.
[0020] FIG. 6 is a graph illustrating the inhibition of NCC15 cell
migration by treatment with Ras inhibitors (kR-4A-1 and kR-4B-1).
The average number of cells migrated is indicated on the
y-axis.
[0021] FIG. 7 is a graph illustrating the inhibition of HCC15 cell
invasion by treatment with Ras inhibitors (kR-4A-1 and kR-4B-1).
The average number of cells migrated is indicated on the
y-axis.
[0022] FIG. 8 is a graph illustrating the suppression of growth of
an H358 human tumor in nude mice. Tumor volume change (mm.sup.3)
for control (DMSO vehicle) (0) or 10 mg/kg kR-4B-8 treated (B8)
(.quadrature.) mice is indicated on the y-axis. The number of days
is indicated on the x-axis.
[0023] FIG. 9 is a graph illustrating the effect of a Ras inhibitor
(kR-H6-48) on Lewis Lung Carcinoma isograft growth in female mice.
Tumor volume (mm.sup.3) for control (PBS) (.diamond.) or 10 mg/kg
KR-H6-48 treated (.quadrature.) mice is indicated on the y-axis.
The number of days is indicated on the x-axis.
[0024] FIG. 10 is a graph illustrating the reduction in activated
(GTP-bound) Ras in lung cancer cells by Ras inhibitors. GTP-bound
Ras (fraction of control) for kR-H6-48 (.diamond.), HR-1
(.quadrature.), kR-4A-4 (.DELTA.), and kR-48-2 (x) is indicated on
the y-axis. The number of hours is indicated on the x-axis.
[0025] FIGS. 11A-B are graphs illustrating the growth inhibition of
Ras-dependent cancer cells by kR-H6-48 (A) and HR-1 (B). The cell
number (percent of control) of H358 (.diamond.), SKBR3
(.quadrature.), SKOV3 (.DELTA.), MPR-178 (x), and ID8 (.cndot.)
cells is indicted on the y-axis. The concentration (nM) is
indicated on the x-axis.
[0026] FIG. 12 illustrates the direct interaction of fluorescent
lipopeptide analogs of Ras. The bound fraction of kR-H6-57
(.quadrature.), kR-H6-58 (.diamond.), kR-4B-14 (.DELTA.), HR-6
(.smallcircle.), and kR-4A-11 (-) in dodecylphosphocholine (DPC)
micelles is indicated on the y-axis. The K-Ras-GDP concentration
(nM) is indicated on the x-axis. The K.sub.D for each inhibitor is
as follows: 1.44.+-.0.16 .mu.M (kR-H6-57), 0.89.+-.0.13 M
(kR-H6-58), 10.8.+-.1.6 .mu.M (kR-4B-14), >100 .mu.M (HR-6), and
11.8.+-.1.3 .mu.M (kR-4A-11).
[0027] FIGS. 13A-D illustrate that the affinity of Ras inhibitors
towards recombinant K-Ras depends on the membrane-mimicking
environment and is higher in bicelles than in micelles. The bound
fraction of kR-48-14 in DMPC/DHPC bicelles (A), kR-4B-14 in DPC
micelles (B), kR-H6-57 in DMPC/DHPC bicelles (C), and kR-H6-57 in
DPC micelles (D) is indicated on the y-axis. The K-Ras-GDP
concentration (nM) is indicated on the x-axis. The K.sub.D for each
inhibitor is as follows: 1.3.+-.0.1 .mu.M (kR-4B-14 in DMPC/DHPC
bicelles), 10.8.+-.1.6 .mu.M (kR-4B-14 in DPC micelles),
86.3.+-.7.6 .mu.M (kR-H6-57 in DMPC/DHPC bicelles), and 1.4.+-.1.6
.mu.M (kR-H6-57 in DPC micelles).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention provides a peptide or peptidomimetic that is
derived from or based upon the amino acid sequence of the
C-terminal .alpha.-helix of a Ras protein, particularly K-Ras-4a,
K-Ras-4b, N-Ras, or H-Ras. The C-terminal .alpha.-helix of K-Ras-4a
and N-Ras comprises the amino acid sequence of SEQ ID NO: 66. The
C-terminal .alpha.-helixes of K-Ras-4b and H-Ras comprise the amino
acid sequences of SEQ ID NO: 67 and SEQ ID NO: 68, respectively. In
one aspect, the peptide or peptidomimetic comprises about eight or
more (e.g., about nine or more, about ten or more, about eleven or
more, or even about 12 or more) contiguous amino acids of the
C-terminal .alpha.-helix of Ras or inverse thereof, provided that
the peptide or peptidomimetic comprises a total of about 30 or
fewer amino acids.
[0029] In a related aspect, the peptide or peptidomimetic comprises
the amino acid sequence
X.sub.1YTLVRX.sub.2X.sub.3RX.sub.4X.sub.5(SEQ ID NO: 5) or the
inverse thereof, wherein the peptide or peptidomimetic comprises
about 30 or fewer amino acids. Positions X.sub.1-X.sub.5 of SEQ ID
NO: 5 can be any suitable amino acid. Desirably, X.sub.1 is
phenylalanine or tryptophan, X.sub.2 is glutamic acid or glutamine,
X.sub.3 is isoleucine, valine, or lysine, X.sub.4 is glutamine or
lysine, and/or X.sub.5 is tyrosine or histidine. Other amino acids
also can be used, especially those having properties (e.g., size,
polar or non-polar characteristics, charge, and or acid/base
properties) similar to the amino acids provided above for any given
position. More specific examples of a peptide or peptidomimetic
comprising SEQ ID NO: 5 or the inverse thereof include, but are not
limited to, those comprising any of SEQ ID NOs: 10-46, 66-68, and
75-84.
[0030] The invention provides a peptide or peptidomimetic that is
derived from or based upon the amino acid sequence of the
hypervariable region (HVR) of a Ras protein, particularly K-Ras-4a,
K-Ras-4b, N-Ras, or H-Ras. The HVRs of K-Ras-4a, K-Ras-4b, H-Ras,
and N-Ras comprise the amino acid sequences of SEQ ID NOs: 69-72,
respectively. According to one aspect, the peptide or
peptidomimetic comprises about five or more (e.g., about six or
more, about seven or more, about eight or more, or about nine or
more) contiguous amino acids of the HVR of Ras or inverse thereof,
wherein the peptide or peptidomimetic comprises a total of about 30
or fewer amino acids.
[0031] In another aspect, the peptide or peptidomimetic comprises
the amino acid sequence KTPGX.sub.1VKIKK (SEQ ID NO: 6) or the
inverse thereof, wherein the peptide or peptidomimetic comprises
about 30 or fewer amino acids. X.sub.1 of SEQ ID NO: 6 can be any
suitable amino acid. Desirably, X.sub.1 is serine, norleucine, or
alanine. Other amino acids also can be used, especially those
having similar properties to serine, norleucine, or alanine. More
specific examples of a peptide or peptidomimetic comprising SEQ ID
NO: 6 or the inverse thereof include, but are not limited to, those
comprising any of SEQ ID NOs: 47-54 and 69.
[0032] Alternatively, the peptide or peptidomimetic comprises the
amino acid sequence KKSKTK (SEQ ID NO: 7) or the inverse thereof,
wherein the peptide or peptidomimetic comprises about 30 or fewer
amino acids. Particular examples of a peptide or peptidomimetic
comprising SEQ ID NO: 7 or the inverse thereof include, but are not
limited to, those comprising any of SEQ ID NOs: 55-63 and 70.
[0033] In yet another embodiment, the peptide or peptidomimetic
comprises the amino acid sequence
SGPGX.sub.1X.sub.2SX.sub.3X.sub.4(SEQ ID NO: 8) or the inverse
thereof, wherein the peptide or peptidomimetic comprises about 30
or fewer amino acids. X.sub.1-X.sub.4 of SEQ ID NO: 8 can be any
suitable amino acid. In one embodiment, X.sub.1-X.sub.3 are
non-polar amino acids, and/or X.sub.4 is a non-polar or basic amino
acid. Desirably, X.sub.1 is cysteine or norleucine, X.sub.2 is
methionine or norleucine, X.sub.3 is cysteine or norleucine, and/or
X.sub.4 is lysine or norleucine. More specific examples of a
peptide or peptidomimetic comprising SEQ ID NO: 8 or the inverse
thereof include, but are not limited to, those comprising any of
SEQ ID NOs: 64, 71, 85, or 86.
[0034] According to still another aspect, the peptide or
peptidomimetic comprises the amino acid sequence
GTQGX.sub.1X.sub.2GLP (SEQ ID NO: 9), wherein the peptide or
peptidomimetic comprises about 30 or fewer amino acids. X.sub.1 and
X.sub.2 can be any suitable amino acid. According to one
embodiment, X.sub.1 and/or X.sub.2 is a non-polar amino acid. In
another embodiment, X.sub.1 is cysteine or norleucine, and/or
X.sub.2 is methionine or norleucine. Particular examples of a
peptide or peptidomimetic comprising SEQ ID NO: 9 or the inverse
thereof include, but are not limited to, SEQ ID NOs: 65 and 72.
[0035] Preferably, the peptide or peptidomimetic inhibits Ras
activity. The term "Ras" is sometimes used herein to refer to the
superfamily of Ras proteins collectively and individually, and is
intended to encompass any such protein, especially H-Ras, N-Ras,
K-Ras4A, and K-Ras4B. For the purposes of this invention, a peptide
or peptidomimetic is considered to inhibit Ras activity if it
inhibits any biological function of a Ras protein. Biological
functions of Ras proteins include, for example, signal transduction
activity. Thus, for instance, a peptide or peptidomimetic is
considered to inhibit Ras activity if, in the presence of the
peptide or peptidomimetic, the signal transduction activity of a
Ras protein is reduced to any degree as compared to the signal
transduction activity of the Ras protein in the absence of the
peptide or peptidomimetic. Preferably, the peptide or
peptidomimetic inhibits Ras activity to a degree sufficient to
reduce the rate of cell growth of a cancer cell, reduce malignant
transformation of a host, and/or induce cell death of a cancer
cell. Suitable assays to test for such a reduction in the
biological activity of Ras are known in the art, including cell
growth and cytotoxicity assays, migration and invasion assays,
phosphorylation/de-phosphorylation of Ras downstream targets, and
gene regulation assays (e.g., luciferase reporter assay).
[0036] The invention is not limited with respect to any particular
mechanism of action. The peptide or peptidomimetic may inhibit Ras
by binding to Ras or its targets, thereby interfering with Ras
signal transduction. Alternatively, or in addition, the peptide or
peptidomimetic may inhibit the multimerization of Ras and/or
inhibit Ras' membrane binding activity. Of course, the peptide or
peptidomimetic may act by some other mechanism, such as by
increasing the rate of Ras protein degradation or enhancing
expression of small RNA molecules (miRNAs) that interfere with Ras
transcription.
[0037] The inventive peptide or peptidomimetic can further comprise
one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15) flanking residues. The flanking residues should be chosen so as
not to interfere with the ability of the peptide to inhibit Ras
activity. Guidance for the selection of such residues is provided
by the relevant sequence of the C-terminal .alpha.-helix or HVR of
Ras itself. For instance, one can choose residues for use in the
peptide that are identical to, or have properties similar to, the
residues at the corresponding positions of a given Ras protein
(preferably a human Ras protein). The peptide or peptidomimetic
can, for example, be a fragment of a Ras protein or have an amino
acid sequence of a fragment of a Ras protein or the inverse
sequence thereof.
[0038] Variant sequences other than those specifically mentioned
herein are contemplated, which comprise significant sequence
identity (e.g., 80%, 85%, 90%, 95%, 98%, or 99% sequence identity)
to the amino acid sequence of the C-terminal .alpha.-helix (e.g.,
SEQ ID NOs: 66-68) or HVR (e.g., SEQ ID NOs: 69-72) or fragment
thereof, provided that such variants retain the ability to inhibit
Ras activity. Such variants can comprise one or more (e.g., 2, 3,
4, or 5) amino acid substitutions, deletions, or insertions as
compared to the parent amino acid sequence. Conservative amino acid
substitutions are known in the art, and include amino acid
substitutions in which one amino acid having certain physical
and/or chemical properties is exchanged for another amino acid that
has the same or similar chemical or physical properties. For
instance, the conservative amino acid substitution can be an acidic
amino acid substituted for another acidic amino acid (e.g., Asp or
Glu), an amino acid with a nonpolar side chain substituted for
another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val,
Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid
substituted for another basic amino acid (Lys, Arg, etc.), an amino
acid with a polar side chain substituted for another amino acid
with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.),
etc.
[0039] The peptide or peptidomimetic also can comprise synthetic,
non-naturally occurring amino acids. Such synthetic amino acids
include, for example, aminocyclohexane carboxylic acid, norleucine,
.alpha.-amino n-decanoic acid, homoserine,
S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,
4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,
4-carboxyphenylalanine, .beta.-phenylscrine
.beta.-hydroxyphenylalanine, phenylglycine,
.alpha.-naphthylalanine, cyclohexylalanine, cyclohexylglycine,
indoline-2-carboxylic acid,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic
acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine,
N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,
.alpha.-aminocyclopentane carboxylic acid, .alpha.-aminocyclohexane
carboxylic acid, .alpha.-aminocycloheptane carboxylic acid,
.alpha.-(2-amino-2-norbornane)-carboxylic acid,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, homophenylalanine, and
.alpha.-tert-butylglycine. The properties of such synthetic amino
acids are well-documented. Any natural amino acid of one or more of
the sequences discussed herein can be substituted with a synthetic
amino acid having similar properties.
[0040] The term "peptidomimetic" as used herein refers to a
compound that comprises the same general structure of a
corresponding polypeptide, but which includes modifications that
increase its stability or biological function. For instance, the
peptidomimetic can be a "retro" or "reverso" analogue of a given
peptide, which means that the peptidomimetic comprises the reverse
sequence of the peptide. In addition, or instead, the
peptidomimetic can comprise one or more amino acids in a "D"
configuration (e.g., D-amino acids), providing an "inverso"
analogue. Peptides comprising both a reverse sequence and D-amino
acids are referred to as "retro-inverso" peptides. Peptidomimetics
also include peptoids, wherein the sidechain of each amino acid is
appended to the nitrogen atom of the amino acid as opposed to the
alpha carbon. Peptoids can, thus, be considered as N-substituted
glycines which have repeating units of the general structure of
NRCH.sub.2CO and which have the same or substantially the same
amino acid sequence as the corresponding polypeptide. In this
respect, the peptide or peptidomimetic can comprise any of the
sequences described herein in reverse order.
[0041] Smaller peptides and peptidomimetics are believed to be
advantageous for inhibiting Ras function and to facilitate entry
into a cell. Thus, the peptide or peptidomimetic preferably
comprises about 30 or fewer amino acids, such as about 25 or fewer
amino acids, about 20 or fewer amino acids, or about 15 or fewer
amino acids or even about 12 or fewer amino acids. Generally,
however, the peptide or peptidomimetic will comprise about 8 or
more amino acids, such as about 10 or more amino acids, about 12 or
more amino acids, or about 15 or more amino acids.
[0042] The peptide or peptidominietic can comprise, consist
essentially of, or consist of, any of foregoing sequences or
variants thereof. The peptide or peptidomimetic consists
essentially of the foregoing sequences if it does not comprise
other elements, such as other amino acid sequences, that prevent
the peptide from inhibiting Ras activity.
[0043] The peptide or peptidomimetic coupled to a cell penetrating
motif or other moiety so as to more efficiently facilitate the
delivery of the peptide to the interior of a cell, anchor the
peptide to the cell membrane of a cell, and/or promote folding of
the peptide. Thus, the peptide or peptidomimetic can be provided as
part of a composition comprising the peptide and cell penetrating
motif or other moiety. Any of various cell penetrating motifs and
or other moieties useful for these purposes can be used. By way of
illustration, suitable cell penetrating motifs and other relevant
moieties (e.g., cell-membrane anchoring moieties) include lipids
and fatty acids, peptide transduction domains (e.g., HIV-TAT, HSV
Transcription Factor (VP22), and penetratin), and other types of
carrier molecules (e.g., Pep-1).
[0044] According to one aspect of the invention, the cell
penetrating motif or other moiety comprises a fatty acid or lipid
molecule. The fatty acid or lipid molecule can be, for example, a
palmitoyl group, farnesyl group (e.g., farnesyl diphosphate), a
geranylgeranyl group (e.g., geranylgeranyl diphosphate), a
phospholipid group, glycophosphatidylinositol, phosphatidylserine,
phosphatidylethanolamine, sphingomyelin, phosphatidylcholine,
cardiolipin, phosphatidylinositol, phosphatidic acid,
lysophosphoglyceride, a cholesterol group, and the like.
Preferably, the fatty acid molecule is a C.sub.1 to C.sub.24 fatty
acid or C.sub.6 to C.sub.14 fatty acid. Desirably, the fatty acid
comprises three or more, four or more, five or more, six or more,
seven or more, eight or more, nine or more or ten or more carbon
atoms. Typically, the fatty acid will comprise 22 or fewer, 20 or
fewer, 18 or fewer, or 16 or fewer carbon atoms. Specific examples
of fatty acids include, without limitation, lauric acid, myristic
acid, stearic acid, oleic acid, linoleic acid, .alpha.-linoleic
acid, linolenic acid, arachidonic acid, timnodonic acid,
docosohexenoic acid, erucic acid, arachidic acid, behenic acid,
aminoisobutiric acid (Aib), caprylic acid (Cap), and octanoic acid
(Oct).
[0045] The fatty acid or lipid molecule can be attached to any
suitable part of the peptide or peptidomimetic. In a preferred
embodiment of the invention, the fatty acid or lipid molecule is
attached at the amino (N-) terminus, the carboxyl (C-) terminus, or
both the N- and C-termini of the peptide or peptidomimetic.
Typically, the fatty acid or lipid molecule is attached via an
amide or ester linkage. When the fatty acid or lipid molecule is
attached at the C-terminus of the polypeptide or peptidomimetic,
the fatty acid or lipid molecule preferably is modified, e.g., to
include an amino group such as NH.sub.2(CH.sub.2).sub.nCOOH or
CH.sub.3(CH.sub.2).sub.mCH(NH.sub.2)COOH, wherein each of n and m
is, independently, 1 to 24, preferably 6 to 14. The fatty acid or
lipid residue can advantageously be attached to a terminal lysine
in the epsilon (c) position.
[0046] According to another aspect of the invention, the cell
penetrating motif is a peptide transduction domain (also known as
protein transduction domains or PTDs). PTDs typically are fused to
the Ras-inhibitory peptide or peptidomimetic. Thus, the peptide or
peptidomimetic can be a fusion protein comprising the peptide or
peptidomimetic and a PTD. Often, the fusion protein is cleaved
inside of a cell to remove the cell penetrating motif.
[0047] The peptide or peptidomimetic can further comprise linking
residues disposed between the amino acid sequence derived from or
based upon the C-terminal .alpha.-helix or HVR of Ras and the cell
penetrating motif or other moiety. Illustrative examples of such
linking residues include K, KK, RK, RQ, KQ, RQI, KQI, RQIK (SEQ ID
NO: 73), and KQIK (SEQ ID NO: 74).
[0048] The peptide or peptidomimetic can be prepared by any method,
such as by synthesizing the peptide or peptidomimetic, or by
expressing a nucleic acid encoding an appropriate amino acid
sequence in a cell and harvesting the peptide from the cell. Of
course, a combination of such methods also can be used. Methods of
de novo synthesizing peptides and peptidomimetics, and methods of
recombinantly producing peptides and peptidomimetics are known in
the art (see, e.g., Chan et al., Fmoc Solid Phase Peptide
Synthesis, Oxford University Press, Oxford, United Kingdom, 2005;
Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,
Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University
Press, Oxford, United Kingdom, 2000; Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring Harbor
Press, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
John Wiley & Sons, NY, 1994).
[0049] The invention also provides a nucleic acid encoding the
amino acid sequence of the peptide or peptidomimetic. The nucleic
acid can comprise DNA or RNA, and can be single or double stranded.
Furthermore, the nucleic acid can comprise nucleotide analogues or
derivatives (e.g., inosine or phophorothioate nucleotides and the
like). The nucleic acid can encode the amino acid sequence of the
peptide or peptidomimetic alone, or as part of a fusion protein
comprising such sequence and a cell penetrating motif, as described
herein. The nucleic acid encoding the amino acid sequence of the
peptide or peptidomimetic can be provided as part of a construct
comprising the nucleic acid and elements that enable delivery of
the nucleic acid to a cell, and/or expression of the nucleic acid
in a cell. Such elements include, for example, expression vectors
and transcription and/or translation sequences. Suitable vectors,
transcription/translation sequences, and other elements, as well as
methods of preparing such nucleic acids and constructs, are known
in the art (e.g., Sambrook et al., supra; and Ausubel et al.,
supra). Accordingly, a recombinant vector comprising the nucleic
acid encoding the amino acid sequence of the peptide or
peptidomimetic also is encompassed by the invention.
[0050] The invention further provides an antibody to the peptide or
peptidomimetic, or an antigen binding fragment or portion thereof
(e.g., Fab, F(ab').sub.2, dsFv, sFv, diabodies, and triabodies).
The antibody can be monoclonal or polyclonal, and of any isotype,
e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be a
naturally-occurring antibody, e.g., an antibody isolated and/or
purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,
hamster, human, etc. Alternatively, the antibody can be a synthetic
or genetically-engineered antibody, e.g., a humanized antibody or a
chimeric antibody. The antibody can be in monomeric or polymeric
form. The antibody, or antigen binding portion thereof, can be
modified to comprise a detectable label, such as, for instance, a
radioisotope, a fluorophore (e.g., fluorescein isothiocyanate
(FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase,
horseradish peroxidase), or element particles (e.g., gold
particles). Such antibodies can be used for any purpose, such as to
facilitate the detection or purification of a peptide or
peptidomimetic described herein. Suitable methods of making
antibodies are known in the art, including standard hybridoma
methods, EBV-hybridoma methods, bacteriophage vector expression
systems, and phage-display systems (see, e.g., Kohler and Milstein,
Eur. J. Immunol., 5, 511-519 (1976); Harlow and Lane (eds.),
Antibodies: A Laboratory Manual, CSH Press (1988); C. A. Janeway et
al. (eds.), Immunobiology, 5.sup.th Ed., Garland Publishing, New
York, N.Y. (2001); Haskard and Archer, J. Immunol. Methods, 74(2),
361-67 (1984); Roder et al., Methods Enzymol., 121, 140-67 (1986);
Huse et al., Science, 246, 1275-81 (1989); Sambrook et al., supra;
Ausubel et al., supra; and Knappik et al., J. Mol. Biol. 296: 57-86
(2000)).
[0051] The peptide or peptidomimetic, nucleic acid, or antibody can
be isolated. The term "isolated" as used herein encompasses
compounds or compositions that have been removed from a biological
environment (e.g., a cell, tissue, culture medium, body fluid,
etc.), or otherwise increased in purity to any degree (e.g.,
isolated from a synthesis medium). Isolated compounds and
compositions, thus, can be synthetic or naturally produced.
[0052] A cell comprising the peptide or peptidomimetic, nucleic
acid encoding the amino acid sequence of the peptide or
peptidomimetic, or recombinant vector comprising the nucleic acid
also is provided herein. Such a cell includes, for example, a cell
engineered to express a nucleic acid encoding the amino acid
sequence of the peptide or peptidomimetic. Suitable cells include
prokaryotic and eukaryotic cells, e.g., mammalian cells, yeast,
fungi, and bacteria (such as E. coli). The cell can be in vitro, as
is useful for research or for production of the peptide or
peptidomimetic, or the cell can be in vivo, for example, in a
transgenic mammal that expresses the peptide.
[0053] The peptide or peptidomimetic can be used for any purpose,
but is especially useful for inhibiting Ras activity in a cell.
Thus, provided herein is a method of inhibiting Ras activity in a
cell, which method comprises administering a peptide or
peptidomimetic described herein to a cell in an amount sufficient
to inhibit Ras activity.
[0054] The peptide or peptidomimetic can be administered to the
cell by any method. For example, the peptide or peptidomimetic can
be administered to a cell by contacting the cell with the peptide
or peptidomimetic, typically in conjunction with a reagent or other
technique (e.g., microinjection or electroporation) that
facilitates cellular uptake. Alternatively, and preferably, the
peptide or peptidomimetic is administered by contacting the cell
with a composition comprising the peptide or peptidomimetic and a
cell penetrating motif, as discussed herein. The peptide or
peptidomimetic additionally or alternatively can be encapsulated in
nanoparticles (e.g., using the methods described in International
Patent Application Publication 2008/058125) prior to administration
to the cell.
[0055] The peptide or peptidomimetic also can be administered by
introducing a nucleic acid encoding the amino acid sequence of the
peptide into the cell such that the cell expresses a peptide
comprising the amino acid sequence. The nucleic acid encoding the
peptide can be introduced into the cell by any of various
techniques, such as by contacting the cell with the nucleic acid or
a composition comprising the nucleic acid as part of a construct,
as described herein, that enables the delivery and expression of
the nucleic acid. Specific protocols for introducing and expressing
nucleic acids in cells are known in the art (see, e.g., Sambrook et
al. (eds.), supra; and Ausubel et al., supra).
[0056] The peptide, peptidomimetic, or nucleic acid can be
administered to a cell in vivo by administering the peptide,
peptidomimetic, nucleic acid encoding the peptide or
peptidomimetic, or recombinant vector comprising the nucleic acid.
The host can be any host, such as a mammal, preferably a human.
Suitable methods of administering peptides, peptidomimetics, and
nucleic acids to hosts are known in the art, and discussed in
greater detail in connection with the pharmaceutical composition
comprising such compounds, below.
[0057] The cell can be any type of cell that comprises Ras.
Preferably, the cell is of a type that is related to a disease or
condition mediated by Ras activity. For example, the cell can be an
engineered cell that is designed to mimic a condition or disease
associated with Ras activity, or the cell can be a cell of a
patient afflicted with a disease or condition associated with Ras
activity. Diseases mediated by Ras include diseases characterized
by Ras overexpression or overactivity. Cancer cells are one example
of a cell type that can be used. The cell can be in vitro or in
vivo in any type of animal, such as a mammal, preferably a
human.
[0058] The method of inhibiting Ras activity in a cell can be used
for any purpose, such as for the research, treatment, or prevention
of diseases or conditions mediated by Ras. Ras activity has been
linked to a large variety of cancers. Thus, according to one aspect
of the method of the invention, the peptide or peptidomimetic is
administered to a cancer cell, in vitro or in vivo, and
administration of the peptide or peptidomimetic to the cancer cell
inhibits the growth or survival of the cancer cell.
[0059] The cancer cell can be a cell of any type of cancer, in
vitro or in vivo, particularly those associated with Ras activity,
such as those associated with Ras overexpression, up-regulation of
Ras, and/or increased activation of Ras (e.g., constitutive
activation of Ras). Non-limiting examples of specific types of
cancers include cancer of the head and neck, eye, skin, mouth,
throat, esophagus, chest, bone, lung, colon, sigmoid, rectum,
stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain,
intestine, heart or adrenals. More particularly, cancers include
solid tumor, sarcoma, carcinomas, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor, acute lymphoblastic leukemia,
acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell
leukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acutenonlymphocyctic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia, chronic lymphocytic
leukemia, hairy cell leukemia, or multiple myeloma. See, e.g.,
Harrison's Principles of Internal Medicine, Eugene Braunwald et
al., eds., pp. 491 762 (15th ed. 2001). The methods of the
invention are believed to be especially useful for the treatment of
leukemia, pancreatic cancer, colon (colorectal) cancer, ovarian
cancer, lung cancer, bladder cancer, cancer of the salivary gland,
myeloid malignancies, germ cell tumors, and melanoma, as well as
any other cancer known to be responsive to Ras inhibitors.
[0060] Ras activity also has been linked to other diseases,
including Costello syndrome, Noonan syndrome, and autoimmune
diseases, such as Alopecia areata, Ankylosing spondylitis, Crohns
Disease, Graves' disease, Dermatomyositis, Diabetes mellitus type
1, Goodpasture's syndrome, Guillain-Barre syndrome (GBS),
Hashimoto's disease, Idiopathic thrombocytopenic purpura, Lupus
erythematosus, Mixed Connective Tissue Disease, Multiple Sclerosis,
Myasthenia gravis, Narcolepsy, Pemphigus vulgaris, Pernicious
anaemia, Psoriasis, Psoriatic Arthritis, Polymyositis, Primary
biliary cirrhosis, Relapsing polychondritis, Rheumatoid arthritis,
Sjogren's syndrome, Temporal arteritis (also known as "giant cell
arteritis"), Ulcerative Colitis (one of two types of idiopathic
inflammatory bowel disease "IBD"), Vasculitis, and Wegener's
granulomatosis. Thus, the methods of the invention are believed to
be useful for the treatment of such diseases, as well.
[0061] Peptides and peptidomimetics, as described herein, include
salts, esters, alkylated (e.g., methylated), and acetylated
peptides. Any one or more of the compounds or compositions of the
invention described herein (e.g., peptide or peptidomimetic,
nucleic acid, antibody, or cell) can be formulated as a
pharmaceutical composition, comprising a compound of the invention
and a pharmaceutically acceptable carrier. Furthermore, the
compounds or compositions of the invention can be used in the
methods described herein alone or as part of a pharmaceutical
formulation.
[0062] The pharmaceutical composition can comprise more than one
compound or composition of the invention. Alternatively, or in
addition, the pharmaceutical composition can comprise one or more
other pharmaceutically active agents or drugs. Examples of such
other pharmaceutically active agents or drugs that may be suitable
for use in the pharmaceutical composition include anticancer
agents. Suitable anticancer agents include, without limitation,
alkylating agents; nitrogen mustards; folate antagonists; purine
antagonists; pyrimidine antagoinists; spindle poisons;
topoisomerase inhibitors; apoptosis inducing agents; angiogenesis
inhibitors; podophyllotoxins; nitrosoureas; cisplatin; carboplatin;
interferon; asparginase; tamoxifen; leuprolide; flutamide;
megestrol; mitomycin; bleomycin; doxorubicin; irinotecan; and
taxol, geldanamycin (e.g., 17-AAG), and various anti-cancer
peptides and antibodies.
[0063] The carrier can be any of those conventionally used and is
limited only by physio-chemical considerations, such as solubility
and lack of reactivity with the active compound(s), and by the
route of administration. The pharmaceutically acceptable carriers
described herein, for example, vehicles, adjuvants, excipients, and
diluents, are well-known to those skilled in the art and are
readily available to the public. It is preferred that the
pharmaceutically acceptable carrier be one which is chemically
inert to the active agent(s) and one which has no detrimental side
effects or toxicity under the conditions of use.
[0064] The choice of carrier will be determined in part by the
particular compound or composition of the invention and other
active agents or drugs used, as well as by the particular method
used to administer the compound and/or inhibitor. Accordingly,
there are a variety of suitable formulations of the pharmaceutical
composition of the present inventive methods. The following
formulations for oral, aerosol, parenteral, subcutaneous,
intravenous, intramuscular, interperitoneal, rectal, and vaginal
administration are exemplary and are in no way limiting. One
skilled in the art will appreciate that these routes of
administering the compound of the invention are known, and,
although more than one route can be used to administer a particular
compound, a particular route can provide a more immediate and more
effective response than another route.
[0065] Injectable formulations are among those formulations that
are preferred in accordance with the present invention. The
requirements for effective pharmaceutical carriers for injectable
compositions are well-known to those of ordinary skill in the art
(See, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott
Company, Philadelphia, Pa., Banker and Chalmers, eds., pages
238-250 (1982), and ASHP Handbook on Injectable Drugs. Toissel, 4th
ed., pages 622-630 (1986)).
[0066] Topical formulations are well-known to those of skill in the
art. Such formulations are particularly suitable in the context of
the present invention for application to the skin.
[0067] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the inhibitor
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant. Capsule forms
can be of the ordinary hard- or soft-shelled gelatin type
containing, for example, surfactants, lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and corn
starch. Tablet forms can include one or more of lactose, sucrose,
mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible excipients. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to
the active ingredient, such excipients as are known in the art.
[0068] The compounds and compositions of the invention, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an atomizer.
Such spray formulations also may be used to spray mucosa.
[0069] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The compounds and
compositions of the invention can be administered in a
physiologically acceptable diluent in a pharmaceutical carrier,
such as a sterile liquid or mixture of liquids, including water,
saline, aqueous dextrose and related sugar solutions, an alcohol,
such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, dimethylsulfoxide,
glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol,
ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a
fatty acid ester or glyceride, or an acetylated fatty acid
glyceride with or without the addition of a pharmaceutically
acceptable surfactant, such as a soap or a detergent, suspending
agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or
emulsifying agents and other pharmaceutical adjuvants.
[0070] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0071] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-b-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0072] Preservatives and buffers may be used. In order to minimize
or eliminate irritation at the site of injection, such compositions
may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5% to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0073] Additionally, the compounds of the invention, or
compositions comprising such compounds, can be made into
suppositories by mixing with a variety of bases, such as
emulsifying bases or water-soluble bases. Formulations suitable for
vaginal administration can be presented as pessaries, tampons,
creams, gels, pastes, foams, or spray formulas containing, in
addition to the active ingredient, such carriers as are known in
the art to be appropriate.
[0074] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLES
[0075] Except otherwise stated, the peptides referenced in the
following examples were prepared and analyzed as follows:
Peptide Synthesis and Purification
[0076] The peptides were synthesized on a 433A Peptide Synthesizer
(Applied Biosystems) using Fmoc chemistry. The peptides were
cleaved from the resin and deprotected with a mixture of 90.0%
(v/v) trifluoroacetic acid (TFA) with 2.5% water, 2.5%
triisopmrpyl-silane, and 5% thioanisol. The resin and deprotection
mixture were pre-chilled to -5.degree. C. and reacted for 15
minutes at -5.degree. C. with stirring. The reaction was allowed to
continue at room temperature for 1 hour and 45 minutes. The resin
was filtered off and the product was precipitated with cold diethyl
ether. The resin was washed with neat TFA. Peptide suspended in
diethyl ether was centrifuged at -20.degree. C. and the precipitate
was washed with diethyl ether four more times and left to dry in a
vacuum overnight. The dried crude peptide was dissolved in DMSO and
purified on a preparative (25 mm.times.250 mm) Atlantis C18 reverse
phase column (Agilent Technologies) in a 90 minute gradient of 0.1%
(v/v) trifluoroacetic acid in water and 0.1% trifluoroacetic acid
in acetonitrile with a 10 mL/min flow rate. The fractions
containing peptides were analyzed on Agilent 1100 LC/MS
spectrometer with the use of a Zorbax 300SB-C3 Poroshell column and
a gradient of 5% acetic acid in water and acetonitrile. Fractions
that were more than 95% pure were combined and freeze dried. Resin
preloaded with .alpha.-Fmoc-.epsilon.-palmytoil-Lys was prepared as
described in Remsberg et al., J. Med. Chem., 50: 4534-4538
(2007).
Cell Toxicity Assay
[0077] MCF-7 (breast cancer), T47D (breast cancer), Cole 205 (colon
cancer), JM-1 (rat hepatoma), Sk Mel-2 (melanoma), PLC (human
hepatoma), and HepG2 (human hepatoma) were obtained from American
Type Cell Culture Collection. MCF-7 cells were grown in RPMI medium
supplemented with 10% Fetal Bovine Serum. The remaining cell lines
were grown in DMEM medium supplemented with 10% Fetal Bovine Serum.
For the assay, cells were seeded into 96 well plates in medium
containing 1% Fetal Bovine Serum and 100 .mu.L of a cell suspension
containing 5000 cells per well were used for each well. Cell growth
was evaluated utilizing MTT
((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium). The
absorbance of the wells at 544 nm was determined by a
FLUOstar/POLARstar Galaxy (BMG Lab Technologies GmbH) microplate
reader.
Cell Invasion Assays
[0078] Cell invasion assays were performed using the BD Biocoat
Matrigel Invasion Chambers in accordance with the manufacturer's
protocol. Cells (1.times.10.sup.5/mL) were seeded onto 12-well cell
culture chamber using inserts with 8 .mu.m pore size polycarbonate
membrane over a thin layer of Matrigel Basement Membrane Matrix
without phenol red (BD Biosciences) diluted at 1:100 in PBS.
Following incubation of the plates for 48 hours at 37.degree. C.,
cells that invaded through the Matrigel and migrated to the lower
surface of the membrane were stained with Giemsa solution, counted
under the microscope in at least 10 different fields, and
photographed. Three wells were examined for each condition and cell
type, and the experiments were repeated in triplicate.
Example 1
[0079] This example demonstrates the identification of analogs of
the C-terminal .alpha.-helix of Ras.
[0080] The design of inhibitors was based on available x-ray
structures of K-Ras and H-Ras proteins. The structures have
suggested that the C-terminal .alpha.-helix of the proteins (helix
6 in K-Ras) occupies central position in the protein, is involved
in multiple intramolecular interactions, and, thus, is likely to
play a critical role in both structure stabilization and structural
rearrangements during signaling events.
[0081] A library of synthetic peptide analogs of helix 6 was
constructed. For structural stabilization of protein fragments and
membrane anchoring, all peptide analogs were equipped with
palmitate residues (see Table 1). The sequences of the peptides are
presented in Table 1, wherein amino acid substitutions in the
native sequences are in bold.
TABLE-US-00001 TABLE 1 Structure-activity relationships in
derivatives of the C-terminal .alpha.-helix of K-Ras. SEQ ID
Compound NO Structure GI.sub.50, nM kR-H6-1 10 Pal-RYQRIERVLTYFADEV
(All D) 1350 kR-H6-2 11 Pal-LRYQRIERVLTYFADEV (All D) 300 kR-H6-3
12 e-Pal-KRYQRIERVLTYFADEV (All D) >5000 kR-H6-4 13
Pal-KVEDAFYTLVREIRQYR >5000 kR-H6-5 14 Ac-Doa-VEDAFYTLVREIRQYR
3300 kR-H6-6 15 Pal-e-KAFTYLVREIRQYR 250 kR-H6-7 16
Ac-VEDAFYTLVREIRQYRK (e-Pal) 2700 kR-H6-8 17 Ac-AFYTLVREIRQYRK
(e-Pal) 5000 kR-H6-9 18 Ac-DAFYTLVREIRQYRK (e-Pal) 500 kR-H6-10 19
Pal-e-KAFYTLVREIRQY 300 kR-H6-12 20 Pal-e-DFYTLVREIRQYR 180
kR-H6-11 21 Ac-YQRIQRVLTYFK-e-Pal (all D) 10 kR-H6-13 22
Ac-YQRKQRVLTYFK-e-Pal (all D) 60 kR-H6-14 23 Pal-YQRKQRVLTYF (all
D) 40 kR-H6-16 24 Ac-VEDAFYTLVREIRQYR >5000 kR-H6-17 25
Ac-AFYTLVREIRQYR >5000 kR-H6-18 26 Pal-e-KAFYTLVREIRKHK >1000
kR-H6-19 27 Pal-RYQRIQRVLTYFA (all D) >1000 kR-H6-20 28
Pal-RYQRIQRVLTYF (all D) 250 kR-H6-21 29 Pal-YQRIQRBLTYFA (all D)
30 kR-H6-22 30 Pal-RYQRVQRVLTYFA (all D) In testing kR-H6-23 31
Ac-RYQRIEFVLTYFAK-e-Pal (all D) 25 .+-. 10 kR-H6-27 32
Pal-e-KAFYTLVREIRQYRL 280 .+-. 30 kR-H6-23 33 Pal-e-KAFYTLVRQIRQYRL
250 .+-. 50 kR-H6-30 34 Ac-RYQRIQRVLTYFAK-e-Pal (all D) 10 .+-. 5
kR-H6-31 35 Ac-LRYQRIQRVLTYFAK-e-Pal (all D) 25 .+-. 10 kR-H6-32 36
Ac-RYQRIQRVLTYFK-e-Pal (all D) 10 .+-. 4 kR-H6-33 37
Ac-YQRIQRVLTYFAK-e-Pal (all D) 4.5 .+-. 1 kR-H6-34 38
Pal-YQRIQRVLTYF (all D) 18 .+-. 5 kR-H6-35 39 Pal-Aib-YQRIQRVLTYF
(all D) 450 .+-. 50 kR-H6-36 40 Pal-YQRVQRVLTYF (all D) 10 .+-. 5
kR-H6-38 41 Lau-YQRVQRVLTYF (all D) 15 kR-H6-39 42 Lau-YQRVQRVLTYW
(all D) 90 .+-. 15 kR-H6-40 43 Myr-YQRVQRVLTYW (all D) 20 .+-. 10
kR-H6-41 44 Cap-YQRVQRVLTYW (all D) 180 .+-. 40 kR-H6-42 45
Lau-YKRVQRVLTYF (all D) 300 .+-. 50 kR-H6-46 46 Lau-HQRVQRVLTYF
(all D) 300 .+-. 50 kR-H6-75 75 Lau-WQRVQRVLTYF (all D) 1 .+-. 1
kR-H6-76 76 Lau-YQRVQRVLTYFC (all D) In testing kR-H6-77 77
Ac-YQRVQRVLTYFC (all D) In testing kR-H6-78 78 Lau-YQRVQRVLTYFA
(all D) 1 .+-. 1 kR-H6-79 79 Cap-YQRVQRVLTYFA (all D) 0.8 .+-. 0.5
kR-H6-80 80 Cap-YQRVQRVLTYF (all D) 0.8 kR-H6-81 81 Oct-YQRVQRVLTYF
(all D) 4 .+-. 1 kR-H6-82 82 Lau-YQRVQRVLTYFC(Fluo) (all D) In
testing kR-H6-83 83 Oct-YQRVQRVLTYFA (all D) In testing kR-H6-84 84
Oct-WQRVQRVLTYFA (all D) In testing Ac = acetylatation Aib =
amino-isobutiric acid Doa = 2-dodecyl-alanine Pal = palmtic acid
Urn = lauric or dodecanoic acid Cap = caprylic or decanoic acid Oct
= octanoic acid
[0082] The growth inhibitory activity of compounds was compared
using the A549 human lung cancer cell line harboring constitutively
active G12D Ras mutant with the help of an MTT assay. Peptide
kR-H6-12 was the most effective in inhibiting cell growth with
GI.sub.50=180 nM (see Table 1). Stepwise extensions and truncations
confirmed that the peptide had the optimal length (see Table
1).
[0083] Longer peptides (e.g., kR-H6-6) had lower potency. Further
extensions led to further reduction in activity, possibly due to
incorporation of negatively charged residues that interfere with
cell entry (e.g., kR-H6-4). Compounds with palmitate on the
N-terminal end were significantly more active than compounds with
palmitic acid on the C-terminus (kR-H6-6 compared to kR-H6-8).
Retro-inverso versions of the peptide turned out to be
significantly more potent. For example, the retroinverso version of
kR-H6-6 (kR-H6-23) was 10-fold more toxic to cancer cells (see
Table 1).
[0084] Analysis of Ras structures suggested that Glu162 is not
involved in formation of any salt bridges. Since negative charges
interfere with cell entry, Glu162 was replaced with uncharged Gln,
which resulted in additional 1.7-fold reduction in GI.sub.50
(kR-H6-30). Incorporation of aminoisobutiric acid (Aib) is known to
stabilize the helical fold of peptides and, therefore, was
incorporated on the termini of the helix 6 derivatives to improve
efficacy of inhibitors by facilitating more efficient folding and
thus better mimicking the parent structure.
Example 2
[0085] This example demonstrates the characterization of the
interaction of analogs of the C-terminal .alpha.-helix with Ras
protein.
[0086] To study the interactions of the catalytic domain of K-Ras
with kR-H6-6, .sup.15N-labeled truncated K-Ras (1-166) lacking the
hypervariable region was prepared as described in (Abraham et al.,
Protein Expression and Purification, 73(2): 125-131 (2010)). The
purified protein was concentrated to 200 .mu.M and titrated with a
solution of kR-H6-6. The titration was followed by NMR
.sup.15N-edited HSQC spectra acquired on a 600 MHz Bruker
spectrometer at 25.degree. C.
[0087] Analysis of NMR titration data revealed a localized
interaction of the peptide with the Switch I region of K-Ras with
the most significant chemical shift perturbations found in residues
Q25, N26, S32, T35, and E37. Intermolecular interactions between
helix 6 of GTP-.gamma.-S loaded K-Ras and the Switch I region of
the neighboring K-Ras molecule previously were observed by x-ray
crystallography. The Switch I region of Ras is critical for
nucleotide binding and hydrolysis, and for interactions with
effector proteins. Therefore, it is not surprising that kR-H6-6
binding to the Switch I region may interfere with K-Ras
signaling.
Example 3
[0088] This example demonstrates the identification of analogs of
the hypervariable region (HVR) of Ras.
[0089] The HVR of Ras protein has been suggested to be involved in
targeting proteins to certain regions of cellular membranes and in
interactions with effector proteins. HVR of Ras protein is
naturally lipidated on the C-terminal ends. To generate the
mimetics of HVR, peptides palmytilated on the C-terminal ends
through .epsilon.-amino group of Lys that replaced farnesylated Cys
of Ras were synthesized. The sequences of the peptides are
presented in Table 2.
[0090] Since there was no structural data for any HVR of Ras,
extensive structure-activity studies along with comparison of
equivalent sequences in all isoforms of human Ras had to be
undertaken (see FIG. 1 and Table 2). K-Ras-4A and N-Ras were
palmitoylated on Cys 180 and 181, respectively, and were
farnesylated on Cys 186, while H-Ras was palmitoylated on Cys 181
and 184 and farnesylated on Cys 186. For mimicking the
palmitoylated Cys 180, 181, and 184 without significant decrease in
peptide solubility, structurally similar, but less hydrophopic,
norleucine residues (L.sub.N) were introduced in the sequence of
peptides.
[0091] The growth inhibitory activity of compounds was compared
using the A549 human lung cancer cell line harboring constitutively
active G12D Ras mutant with the help of an MIT assay. Optimization
of compounds resulted in peptides that were significantly less
potent than derivatives of helix 6; however, the compounds showed
promising activity in
TABLE-US-00002 TABLE 2 Structure-activity relationships in
derivatives of the hypervariable regions of Ras. SEQ ID GI.sub.50,
Compound NO Structure .mu.M kR-4A-1 47
Ac-RLKKISKEEKTPGSVKIKKK-e-Pal 3.1 kR-4A-2 48
Ac-YRLKKISKEEKTPGK-e-Pal 1.65 kR-4A-4 49 Ac-KTPGL.sub.NVKIKKK-e-Pal
0.7 kR-4A-3 50 Ac-YRLKKISKEEKTPGL.sub.NVKIKKK-e-Pal 1.9 kR-4A-5 51
Ac-KTPGL.sub.NVKIKKK-e-Pal 5 kR-4A-6 52 Ac-TPGL.sub.NVKIKKK-e-Pal
>10 kR-4A-7 53 Ac-PGL.sub.NVKIKKK-e-Pal >10 kR-4A-8 54
Ac-GL.sub.NVKIKKK-e-Pal >10 kR-4B-1 55
Ac-KEKL.sub.NSKDGKKKKKKSKTKK-e-Pal 1.45 kR-4B-2 56
Ac-KL.sub.NSKDGKKKKKKSKTKK-e-Pal 1.4 kR-4B-3 57
Ac-KKKKKKSKTKK-e-Pal 1.75 kR-4B-4 58 Ac-KKKKKKSKTK-e-Pal 1.45 (0.5)
kR-4B-6 59 Pal-KTKSKKKKK-NH.sub.2 All-D 1.7 kR-4B-9 60
e-Pal-KKTKSKKKKK-NH.sub.2 All-D 1.4 kR-4B-7 61 Ac-KKKKSKTKK-e-Pal 2
kR-4B-8 62 Ac-KKKSKTKK-e-Pal 2.3 kR-4B-10 63 Ac-KKSKTKK-e-Pal In
testing HR-1 64 Ac-ESGPGL.sub.NL.sub.NSL.sub.NKK-e-Pal 0.25 HR-2 85
Pal-KL.sub.NL.sub.NSL.sub.NGPGSE-NH.sub.2 In testing HHR-3 86
Lau-KL.sub.NL.sub.NSL.sub.NGPGSE-NH.sub.2 In testing NR-1 65
Ac-DGTQGL.sub.NL.sub.NGLPK-e-Pal >10
Example 4
[0092] This example demonstrates the membrane binding activity of
analogs of the HVR of Ras.
[0093] Membrane binding is thought to be important for Ras
transforming activity. To assess the ability of K-Ras inhibitors to
accumulate on the membrane, stabilized phospholipid bilayer
nanodiscs were used as plasma membrane mimics.
[0094] Dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5%
dipalmitoyldiphosphatidylethanolamine (DPPE) stabilized by MSP1
scaffold protein were prepared. The DPPC lipids were chosen because
they are among the most common components of the plasma membrane.
The purified MSP1 protein was a generous gift from Dr. Sligar's
lab, University of Illinois at Urbana, which also provided the
procedure for preparation of nanodiscs. The preparation procedure
involved mixing DPPC lipids with MSP1 at a 90:1 molar ratio in the
presence of 100 mM cholate. Cholate was removed by slow dialysis.
The assembled nanodiscs were purified by size-exclusion
chromatography on a calibrated Superdex 200 column and immobilized
on a surface plasmon resonance (SPR) sensor chip.
[0095] The presence of a primary amine group in DPPE provided a
convenient site for cross-linking of nanodiscs to an SPR sensor
chip. The noncross-linked dextran in the sample and reference cells
was blocked by the coupling reagent. Four different peptides were
used to study membrane binding by SPR. The HVR peptide represents
the hypervariable region of K-Ras-4B and contains residues 165
through 183. The HVR peptide lacks post-translational modifications
present in K-Ras4B. The second peptide is the HVR peptide
conjugated to S-farnesyl L-cysteine methyl ester via a bifunctional
cross-linker
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC) (Pierce). The other two peptides were kR-4A-C1 and
kR-4B-C1.
[0096] In all cases, with the exception of the HVR peptide,
cooperative interaction with the phospholipid bilayers was
observed. In this situation determination of true dissociation
constants was not feasible. Instead, the data were analyzed using
the Hill equation. The Hill coefficient describes the degree of
binding cooperativity. While cooperativity for the HVR peptide was
low with the Hill coefficient of 1.106.+-.0.001, lipid modification
significantly increased membrane binding cooperativity with the
highest Hill coefficient of 6.15.+-.0.02 observed for kR-4B-C1. The
Hill coefficient for the HVR peptide modified with S-farnesyl
L-cysteine methyl ester was 5.725.+-.0.002. Among lipidated
peptides kR-4A-C1 exhibited the lowest membrane binding
cooperativity with the Hill coefficient of 3.606.+-.0.003.
[0097] Cooperative interaction of the peptides with the membrane
phospholipids not only enhances their binding affinity but also
facilitates their clustering on the plasma membrane for a more
potent effect on K-Ras. Among the peptides studied for their
binding to the phospholipid bilayers, the palmitoylated kR-4B-1
peptide showed the best cooperative interactions with membrane
lipids. The ability of kR-4B-1 to cluster on the phospholipid
bilayers was superior even to the HVR with native farnesyl and
methyl ester modifications.
Example 5
[0098] This example demonstrates the biological activity of Ras
inhibitors.
[0099] Activity of compounds towards different cell lines varied
significantly even if the cells expressed identical versions of
mutated Ras. Relative activities for different cells were similar
for compounds mimicking helix 6 and HVR of K-Ras (see FIGS. 2 and
3), suggesting that they may act by similar mechanisms in spite of
targeting different regions of Ras protein. Interestingly, mouse
cell line 187436 tu2 with well-defined genetic alterations was the
most sensitive to the compounds (see FIGS. 2 and 3). The cell line
was generated from astrocytoma developed in a transgenic mouse
harboring a G12D mutation in K-Ras and inactivation of the Rb
gene.
[0100] Human lung cancer cell lines, A549, H460, and HI 1944, are
known to have additional oncogenic mutations. Consequently, these
cell lines are likely to have populations of cells resistant to Ras
inhibitors and, thus, will appear less sensitive to the compounds.
Human lung cancer cell lines with identical Ras mutations also have
been reported to have different degree of "Ras addiction" or
sensitivity to Ras inhibition.
[0101] The inventive Ras inhibitors were much more toxic even to
less sensitive lung cancer cells than to regular immortalized
fibroblasts (see FIG. 4). No killing of epithelial cells was
observed even for micromolar concentrations of the inhibitors
suggesting that a therapeutic window should exist that will allow
selective elimination of tumor cells with the help of the inventive
helix 6 analogs.
[0102] Analogs of HVR were on average twice as potent in growth
inhibition of cells with mutated K-Ras as the cells with wild-type
protein (see Table 3).
TABLE-US-00003 TABLE 3 Growth inhibitor activity of HVR analogs
evaluated on thoracic malignancy cell lines. Cell K-Ras kR-4A-3
kR-4B-3 Tumor type line status GI.sub.50 (.mu.M) GI.sub.50 (.mu.M)
lung adenocarcinoma A549 Mutant 3.1 1.45 lung adenocarcinoma H2009
Mutant 2.2 2.9 lung adenocarcinoma H23 Mutant 2.6 2.4 lung
adenocarcinoma Calu 6 Mutant 3.0 2.7 broncheoalveolar H358 Mutant
2.2 2.0 lung squamous cell carcinoma H157 Mutant 2.4 1.5 large cell
carcinoma H460 Mutant 1.5 1.0 lung adenocarcinoma Calu3 WT 5.9 2.2
lung adenocarcinoma H2882 WT 4.0 3.0 lung squamous cell carcinoma
HCC95 WT 5.1 3.3 lung squamous cell carcinoma HCC15 WT 7.4 3.1 MPM
H2373 WT 6.1 4.8 MPM H2461 WT >10.0 >10.0 MPM H2596 WT 8.2
6.2 MPM HP-1 WT >10 7.3 MPM = malignant pleural mesothelioma WT
= wild-type
[0103] Although not wishing to be bound by any theory, the
inventors hypothesize that the differences in sensitivity are
relatively small because Ras gets activated in tumor cells by
multiple mechanisms including activation of receptor tyrosine
kinases. Since Ras functions downstream of important regulators of
tumor cell growth, such as epithelial growth factor receptor,
insulin-like growth factor 1 receptor, and MET, its activation is a
common event in tumors. Thus, wide-spread sensitivity of tumor cell
lines to Ras inhibitors is not surprising.
[0104] Using a "wound closure" assay on HCC15 cells, wherein the
closure of a "wound" or gap in a cell monolayer is monitored and
measured, demonstrated remarkable reduction in cell motility in the
presence of analogs of HVR and helix 6 (see FIG. 5). The rate of
migration and invasion was determined using Boyden Chambers
(Millipore) in accordance with the manufacturer's protocol. The
migration and invasion rate was dramatically reduced in the
presence of IC.sub.25 concentration of the analogs (see FIGS. 6 and
7). The data suggests that Ras inhibitors have an effect on tumor
growth and its metastatic ability.
[0105] A further experiment was undertaken to determine the effect
of analogs of helix 6 and HVR on the amount of K-Ras protein in
cancer cells. H2009 (lung adenocarcinoma cells) and H2592 (plural
mesothelioma cells) were exposed to varying concentrations (0-3.0
.mu.M) of kR-4A-3, kR-4B-3, or kR-H6-3 for 18 hours. The cells then
were lysed and the lysate was analyzed by Western blot using
anti-Ras mAb (Cell Biolabs, Inc.). Inhibitors of Ras reduced the
amount of Ras protein in lung cancer cells in a
concentration-dependent manner.
[0106] H2592 cells were exposed to GI.sub.50 concentrations of
kR-4A-3 or kR-H6-3 for 18 hours, fixed with 4-7% (w/v)
paraformaldehyde for 30 minutes, permeabilized with 0.1% (v/v)
Triton X-100, and immunostained with anti-Ras mAb (Cell Biolabs,
Inc.). Goat anti-mouse antibodies with Alexa Fluor.TM. 594 (red)
were used to visualize the anti-Ras mouse mAb. Immunohistochemistry
of treated cells showed not only reduction in protein levels
compared to control cells treated with vehicle (0.1% DMSO), but
also a lack of characteristic punctuate pattern of K-Ras
distribution.
Example 6
[0107] This example demonstrates the effect of Ras inhibitors on
tumor growth in mice.
[0108] 2.times.10.sup.6 H358 human lung cancer cells with mutated
K-Ras were implanted subcutaneously (s.c.) in nude mice. When
tumors reached measurable size, 10 mg/kg kR-4A-8 or control (DMSO
in buffer) was injected s.c. near the tumor every second day for 20
days. No toxicity was detected during treatment or necropsy.
[0109] Administration of kR-4B-8 abolished the growth of a very
aggressive tumor formed by broncheoalveolar carcinoma H358 cells
(see FIG. 8). Furthermore, tumors at the time of sacrifice were
much smaller in treated (e.g., 0.35 g) versus control (e.g., 1.21
g) mice.
Example 7
[0110] This example further demonstrates the effect of Ras
inhibitors on tumor growth in mice.
[0111] Lewis lung carcinoma LLJ2 (LLC1) (ATCC Catalog No. CRL-1642)
cells were grown in DMEM medium containing 10% FBS. Cells were
trypsinized and suspended in PBS. 3.times.10.sup.6 LLC1 cells in
200 .mu.l PBS were injected subcutaneously to the right flank of 8
weeks old female CB57Bl/6 mice.
[0112] Ras inhibitor injections started 2 weeks after LLC1
injections when tumors were at least 3 mm in diameter. For
injections, solid kR-H6-48 was initially dissolved in DMSO to yield
20 mg/mL stock solution. DMSO stocks were diluted 20-fold in PBS
pre-warmed to 35-37.degree. C. 200 .mu.l of the resulting solution
(10 mg/kg dose) was injected subcutaneously near the tumor(s) every
second day. The control group received 200 .mu.l of 5% DMSO in PBS.
Tumor size was measured with a caliper before each injection. The
mice were sacrificed when the tumors reached maximal allowed
size.
[0113] Administration of kR-H6-48 inhibited LLC1 isograft growth in
female mice (see FIG. 9).
Example 8
[0114] This example demonstrates that Ras inhibitors reduce the
amount of activated Ras in lung cancer cells.
[0115] H358 human lung cancer cells were grown in 6-well plates in
DMEM medium containing 10% FBS. When the cells were about 70%
confluent, the medium was replaced with DMEM containing 1% FBS. Two
hours later, Ras inhibitors (kR-H6-48, HR-1, kR-4A-4, or kR-4B-2)
were added to provide a final concentration of 5 .mu.M.
[0116] After varying exposure times, the cells were rinsed with PBS
and lysed. The lysates were cleared by centrifugation, incubated
with immobilized Raf-RBD beads (which bind Ras) and analyzed by
Western blot using manufacturer's protocols (Cytoskeleton, Inc.,
Denver, Colo.; Catalog No. BK008). The bands corresponding to
active (GTP-bound) Ras were quantified with the help of MIPAV
software.
[0117] Administration of the inhibitors reduced the amount of
activated (GTP-bound) Ras in lung cancer cells (see FIG. 10).
Example 9
[0118] This example demonstrates that Ras inhibitors reduce growth
of Ras-dependent cancer cells.
[0119] Ras-dependent cancer cells (H358 human lung cancer cells,
SKBR3 human breast cancer cells, SKOV3 human ovarian cancer cells,
ID8 murine ovarian cancer cells, and MPR-178 murine prostate cancer
cells) were grown in DMEM medium containing 1% FBS and exposed to
varying concentrations of Ras inhibitors (kR-H6-48 and HR-1) for 48
hours. Cell growth was evaluated utilizing an MTT
((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. The
absorbance of the wells at 544 nm was determined by a
FLUOstar/POLARstar Galaxy (BMG Lab Technologies GmbH, Ortenberg,
Germany) microplate reader. The assays were performed on untreated
(control) and test cells. Cellular responses were calculated from
the data using the following formula:
100.times.[(T-T.sub.0)/(C-T.sub.0)] for T>T.sub.0 and
100.times.[(T-T0)/T0] for T<T.sub.0, wherein T.sub.0 corresponds
to cell density at the time of drug addition.
[0120] Administration of the inhibitors potently inhibited growth
of Ras-dependent cancer cells (see FIGS. 11A-B).
Example 10
[0121] This experiment demonstrates high affinity binding of
lipopeptide analogs of HVR and helix 6 to recombinant K-Ras.
[0122] Recombinant truncated K-Ras protein (1-166) was prepared as
described in Abraham et al., Protein Expr. Purif, 73(2): 125-31
(2010). Inhibitors labeled with fluorescein were dissolved in
DMPC/DHPC bicelles (q=2.7, lipids=5% w/v) to an approximate
concentration of 1 mg/mL. The exact concentration was determined by
measuring UV absorbance in the range of 480-496 nm. Fluorescein
extinction coefficient equal to 68,000 M.sup.-1cm.sup.-1 value was
used to calculate the exact concentration of the inhibitor.
TABLE-US-00004 TABLE 4 Inhibitors used in microscale thermophoresis
experiments. SEQ Compound ID NO Structure kR-H6-57 87
K(.epsilon.-Pal)C(Fluo)FYTLVREIRQYR kR-H6-58 88
Ac-C(Fluo)FYTLVREIRQYR kR-4B-14 89
Ac-C(Fluo)KKKKKSKTKK(.epsilon.-Pal) kR-4A-11 90
Ac-C(Fluo)KTPGL.sub.NVKIKKK(.epsilon.-Pal) HR-6 91
Ac-C(Fluo)ESGPGL.sub.NL.sub.NSL.sub.NKK(.epsilon.-Pal)
[0123] Inhibitors were diluted to an appropriate concentration with
bicelles solution. Titration series (16 binding mixtures) were
prepared containing constant amounts of fluorescent peptide (5 nM
for kR-H6-57 and 40 nM for other inhibitors) in each sample and
varying concentrations of recombinant Ras protein. The buffer
composition for the protein dilution contained 25 mM Tris-citrate
pH 6.5, 150 mM NaCl, 10 mM MgCl.sub.2, 5% Glycerol, 1 mM EDTA, and
1 mM .beta.-mercaptoethanol. Measurements were taken in standard
treated capillaries on a Monolith NT. 115 instrument (NanoTemper
Technologies GmbH, Germany) using 20% IR-laser power and LED
excitation source with .lamda.=470 nm at ambient temperature.
NanoTemper Analysis 1.2.20 software was used to fit the data and
determine the apparent KD values.
[0124] Microscale thermophoresis performed in the presence of
membrane-mimicking micelles and/or bicelles confirmed direct
interaction of fluorescent lipopeptide analogs of the HVR and helix
6 with recombinant truncated GDP-loaded K-Ras (see FIGS. 12 and
13A-D). The affinity of inhibitors towards recombinant K-Ras
depended on the membrane-mimicking environment and was higher in
bicelles than micelles (see FIG. 13A-D).
[0125] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0126] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0127] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
911189PRTHomo sapiens 1Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala
Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln
Asn His Glu Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser
Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu
Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met
Arg Asp Gln Tyr Asn Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val
Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90
95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val
100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp
Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro
Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp
Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg
Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val
Lys Ile Lys Lys Cys Ile Ile Met 180 185 2188PRTHomo sapiens 2Met
Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10
15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr
20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile
Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly
Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr
Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys
Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg
Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn
Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala
Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140
Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145
150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly
Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met
180 185 3189PRTHomo sapiens 3Met Thr Glu Tyr Lys Leu Val Val Val
Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu
Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu
Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys
Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser
Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70
75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln
Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val
Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg
Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr
Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly
Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg
Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly
Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 4189PRTHomo
sapiens 4Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val
Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe
Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys
Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu
Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln
Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile
Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu
Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115
120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu
Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr
Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys
Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu
Pro Cys Val Val Met 180 185 511PRTArtificial SequenceSynthetic 5Xaa
Tyr Thr Leu Val Arg Xaa Xaa Arg Xaa Xaa 1 5 10 610PRTArtificial
SequenceSynthetic 6Lys Thr Pro Gly Xaa Val Lys Ile Lys Lys 1 5 10
76PRTArtificial SequenceSynthetic 7Lys Lys Ser Lys Thr Lys 1 5
89PRTArtificial SequenceSynthetic 8Ser Gly Pro Gly Xaa Xaa Ser Xaa
Xaa 1 5 99PRTArtificial SequenceSynthetic 9Gly Thr Gln Gly Xaa Xaa
Gly Leu Pro 1 5 1016PRTArtificial SequenceSynthetic 10Xaa Tyr Gln
Arg Ile Glu Arg Val Leu Thr Tyr Phe Ala Asp Glu Val 1 5 10 15
1117PRTArtificial SequenceSynthetic 11Xaa Arg Tyr Gln Arg Ile Glu
Arg Val Leu Thr Tyr Phe Ala Asp Glu 1 5 10 15 Val 1217PRTArtificial
SequenceSynthetic 12Xaa Arg Tyr Gln Arg Ile Glu Arg Val Leu Thr Tyr
Phe Ala Asp Glu 1 5 10 15 Val 1317PRTArtificial SequenceSynthetic
13Xaa Val Glu Asp Ala Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln Tyr 1
5 10 15 Arg 1417PRTArtificial SequenceSynthetic 14Xaa Val Glu Asp
Ala Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln Tyr 1 5 10 15 Arg
1514PRTArtificial SequenceSynthetic 15Xaa Ala Phe Tyr Thr Leu Val
Arg Glu Ile Arg Gln Tyr Arg 1 5 10 1617PRTArtificial
SequenceSynthetic 16Xaa Glu Asp Ala Phe Tyr Thr Leu Val Arg Glu Ile
Arg Gln Tyr Arg 1 5 10 15 Xaa 1714PRTArtificial SequenceSynthetic
17Xaa Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln Tyr Arg Xaa 1 5 10
1815PRTArtificial SequenceSynthetic 18Xaa Ala Phe Tyr Thr Leu Val
Arg Glu Ile Arg Gln Tyr Arg Xaa 1 5 10 15 1913PRTArtificial
SequenceSynthetic 19Xaa Ala Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln
Tyr 1 5 10 2013PRTArtificial SequenceSynthetic 20Xaa Phe Tyr Thr
Leu Val Arg Glu Ile Arg Gln Tyr Arg 1 5 10 2112PRTArtificial
SequenceSynthetic 21Xaa Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe Xaa
1 5 10 2212PRTArtificial SequenceSynthetic 22Xaa Gln Arg Lys Gln
Arg Val Leu Thr Tyr Phe Xaa 1 5 10 2311PRTArtificial
SequenceSynthetic 23Xaa Gln Arg Lys Gln Arg Val Leu Thr Tyr Phe 1 5
10 2416PRTArtificial SequenceSynthetic 24Xaa Glu Asp Ala Phe Tyr
Thr Leu Val Arg Glu Ile Arg Gln Tyr Arg 1 5 10 15 2513PRTArtificial
SequenceSynthetic 25Xaa Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln Tyr
Arg 1 5 10 2614PRTArtificial SequenceSynthetic 26Xaa Ala Phe Tyr
Thr Leu Val Arg Glu Ile Arg Lys His Lys 1 5 10 2713PRTArtificial
SequenceSynthetic 27Xaa Tyr Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe
Ala 1 5 10 2812PRTArtificial SequenceSynthetic 28Xaa Tyr Gln Arg
Ile Gln Arg Val Leu Thr Tyr Phe 1 5 10 2912PRTArtificial
SequenceSynthetic 29Xaa Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe Ala
1 5 10 3013PRTArtificial SequenceSynthetic 30Xaa Tyr Gln Arg Val
Gln Arg Val Leu Thr Tyr Phe Ala 1 5 10 3114PRTArtificial
SequenceSynthetic 31Xaa Tyr Gln Arg Ile Glu Arg Val Leu Thr Tyr Phe
Ala Xaa 1 5 10 3215PRTArtificial SequenceSynthetic 32Xaa Ala Phe
Tyr Thr Leu Val Arg Glu Ile Arg Gln Tyr Arg Leu 1 5 10 15
3315PRTArtificial SequenceSynthetic 33Xaa Ala Phe Tyr Thr Leu Val
Arg Gln Ile Arg Gln Tyr Arg Leu 1 5 10 15 3414PRTArtificial
Sequencesynthetic 34Xaa Tyr Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe
Ala Xaa 1 5 10 3515PRTArtificial SequenceSynthetic 35Xaa Arg Tyr
Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe Ala Xaa 1 5 10 15
3613PRTArtificial SequenceSynthetic 36Xaa Tyr Gln Arg Ile Gln Arg
Val Leu Thr Tyr Phe Xaa 1 5 10 3713PRTArtificial SequenceSynthetic
37Xaa Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe Ala Xaa 1 5 10
3811PRTArtificial SequenceSynthetic 38Xaa Gln Arg Ile Gln Arg Val
Leu Thr Tyr Phe 1 5 10 3912PRTArtificial SequenceSynthetic 39Xaa
Tyr Gln Arg Ile Gln Arg Val Leu Thr Tyr Phe 1 5 10
4011PRTArtificial SequenceSynthetic 40Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe 1 5 10 4111PRTArtificial SequenceSynthetic 41Xaa
Gln Arg Val Gln Arg Val Leu Thr Tyr Phe 1 5 10 4211PRTArtificial
SequenceSynthetic 42Xaa Gln Arg Val Gln Arg Val Leu Thr Tyr Trp 1 5
10 4311PRTArtificial SequenceSynthetic 43Xaa Gln Arg Val Gln Arg
Val Leu Thr Tyr Trp 1 5 10 4411PRTArtificial SequenceSynthetic
44Xaa Gln Arg Val Gln Arg Val Leu Thr Tyr Trp 1 5 10
4511PRTArtificial SequenceSynthetic 45Xaa Lys Arg Val Gln Arg Val
Leu Thr Tyr Phe 1 5 10 4611PRTArtificial SequenceSynthetic 46Xaa
Gln Arg Val Gln Arg Val Leu Thr Tyr Phe 1 5 10 4720PRTArtificial
SequenceSynthetic 47Xaa Leu Lys Lys Ile Ser Lys Glu Glu Lys Thr Pro
Gly Ser Val Lys 1 5 10 15 Ile Lys Lys Xaa 20 4815PRTArtificial
SequenceSynthetic 48Xaa Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys Thr
Pro Gly Xaa 1 5 10 15 4911PRTArtificial SequenceSynthetic 49Xaa Thr
Pro Gly Xaa Val Lys Ile Lys Lys Xaa 1 5 10 5020PRTArtificial
SequenceSynthetic 50Xaa Leu Lys Lys Ile Ser Lys Glu Glu Lys Thr Pro
Gly Xaa Val Lys 1 5 10 15 Ile Lys Lys Xaa 20 5111PRTArtificial
SequenceSynthetic 51Xaa Thr Pro Gly Ala Val Lys Ile Lys Lys Xaa 1 5
10 5210PRTArtificial SequenceSynthetic 52Xaa Pro Gly Xaa Val Lys
Ile Lys Lys Xaa 1 5 10 539PRTArtificial SequenceSynthetic 53Xaa Gly
Xaa Val Lys Ile Lys Lys Xaa 1 5 548PRTArtificial SequenceSynthetic
54Xaa Xaa Val Lys Ile Lys Lys Xaa 1 5 5519PRTArtificial
SequenceSynthetic 55Xaa Glu Lys Xaa Ser Lys Asp Gly Lys Lys Lys Lys
Lys Lys Ser Lys 1 5 10 15 Thr Lys Xaa 5617PRTArtificial
SequenceSynthetic 56Xaa Xaa Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys
Ser Lys Thr Lys 1 5 10 15 Xaa 5711PRTArtificial SequenceSynthetic
57Xaa Lys Lys Lys Lys Lys Ser Lys Thr Lys Xaa 1 5 10
5810PRTArtificial SequenceSynthetic 58Xaa Lys Lys Lys Lys Ser Lys
Thr Lys Xaa 1 5 10 599PRTArtificial SequenceSynthetic 59Xaa Thr Lys
Ser Lys Lys Lys Lys Lys 1 5 6010PRTArtificial SequenceSynthetic
60Xaa Lys Thr Lys Ser Lys Lys Lys Lys Lys 1 5 10 619PRTArtificial
SequenceSynthetic 61Xaa Lys Lys Lys Ser Lys Thr Lys Xaa 1 5
628PRTArtificial SequenceSynthetic 62Xaa Lys Lys Ser Lys Thr Lys
Xaa 1 5 637PRTArtificial SequenceSynthetic 63Xaa Lys Ser Lys Thr
Lys Xaa 1 5 6411PRTArtificial SequenceSynthetic 64Xaa Ser Gly Pro
Gly Xaa Xaa Ser Xaa Lys Xaa 1 5 10 6511PRTArtificial
SequenceSynthetic 65Xaa Gly Thr Gln Gly Xaa Xaa Gly Leu Pro Xaa 1 5
10 6612PRTArtificial SequenceSynthetic 66Ala Phe Tyr Thr Leu Val
Arg Glu Ile Arg Gln Tyr 1 5 10 6712PRTArtificial SequenceSynthetic
67Ala Phe Tyr Thr Leu Val Arg Glu Ile Arg Lys His 1 5 10
6812PRTArtificial SequenceSynthetic 68Ala Phe Tyr Thr Leu Val Arg
Glu Ile Arg Gln His 1 5 10 699PRTArtificial SequenceSynthetic 69Thr
Pro Gly Cys Val Lys Ile Lys Lys 1 5 709PRTArtificial
SequenceSynthetic 70Lys Lys Lys Lys Lys Ser Lys Thr Lys 1 5
719PRTArtificial SequenceSynthetic 71Ser Gly Pro Gly Cys Met Ser
Cys Lys 1 5 729PRTArtificial SequenceSynthetic 72Gly Thr Gln Gly
Cys Met Gly Leu Pro 1 5 734PRTArtificial SequenceSynthetic 73Arg
Gln Ile Lys 1 744PRTArtificial SequenceSynthetic 74Lys Gln Ile Lys
1 7511PRTArtificial SequenceSynthetic 75Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe 1 5 10 7612PRTArtificial SequenceSynthetic 76Xaa
Gln Arg Val Gln Arg Val Leu Thr Tyr Phe Cys 1 5 10
7712PRTArtificial SequenceSynthetic 77Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe Cys 1 5 10 7812PRTArtificial SequenceSynthetic
78Xaa Gln Arg Val Gln Arg Val Leu Thr Tyr Phe Ala 1 5 10
7912PRTArtificial SequenceSynthetic 79Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe Ala 1 5 10 8011PRTArtificial SequenceSynthetic
80Xaa Gln Arg Val Gln Arg Val Leu Thr Tyr Phe 1 5 10
8111PRTArtificial SequenceSynthetic 81Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe 1 5 10 8212PRTArtificial SequenceSynthetic 82Xaa
Gln Arg Val Gln Arg Val Leu Thr Tyr Phe Cys 1 5 10
8312PRTArtificial SequenceSynthetic 83Xaa Gln Arg Val Gln Arg Val
Leu Thr Tyr Phe Ala 1 5 10 8412PRTArtificial SequenceSynthetic
84Xaa Gln Arg Val Gln Arg Val Leu Thr Tyr Phe Ala 1 5 10
8510PRTArtificial SequenceSynthetic 85Xaa Xaa Xaa Ser Xaa Gly Pro
Gly Ser Glu 1 5 10 8610PRTArtificial SequenceSynthetic 86Xaa Xaa
Xaa Ser Xaa Gly Pro Gly Ser Glu 1 5 10 8714PRTArtificial
SequenceSynthetic 87Xaa Xaa Phe Tyr Thr Leu Val Arg Glu Ile Arg Gln
Tyr Arg 1 5 10 8813PRTArtificial SequenceSynthetic 88Xaa Phe Tyr
Thr Leu Val Arg Glu Ile Arg Gln Tyr Arg 1 5 10 8911PRTArtificial
SequenceSynthetic 89Xaa Lys Lys Lys Lys Lys Ser Lys Thr Lys Xaa 1 5
10 9012PRTArtificial SequenceSynthetic 90Xaa Lys Thr Pro Gly Xaa
Val Lys Ile Lys Lys Xaa 1 5 10 9112PRTArtificial SequenceSynthetic
91Xaa Glu Ser Gly Pro Gly Xaa Xaa Ser Xaa Lys Xaa 1 5 10
* * * * *