U.S. patent application number 13/956628 was filed with the patent office on 2013-11-28 for smoothened polypeptides and methods of use.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is 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. Invention is credited to Michael Dean, Hong Lou, Nadya Tarasova.
Application Number | 20130316957 13/956628 |
Document ID | / |
Family ID | 39492945 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316957 |
Kind Code |
A1 |
Tarasova; Nadya ; et
al. |
November 28, 2013 |
SMOOTHENED POLYPEPTIDES AND METHODS OF USE
Abstract
Disclosed is an isolated or purified polypeptide or
peptidomimetic comprising an amino acid sequence of a portion of a
Smoothened (SMO) protein, wherein the portion comprises an amino
acid sequence of any of the intracellular loops of the SMO protein,
a functional fragment thereof, or a functional variant of either
the portion or the functional fragment, wherein the functional
fragment comprises at least 7 contiguous amino acids of the
intracellular loops, and wherein the functional fragment or
functional variant inhibits proliferation of a diseased cell, or a
fatty acid derivative thereof. Related conjugates, nucleic acids,
recombinant expression vectors, host cells, and pharmaceutical
compositions are further provided. Methods of inhibiting
proliferation of a diseased cell, treating or preventing cancer,
treating a neoplasm or psoriasis, and inhibiting the expression of
genes involved in the Hedgehog signaling pathway, thereby
inhibiting the Hedgehog signaling pathway, are furthermore provided
by the invention.
Inventors: |
Tarasova; Nadya; (Frederick,
MD) ; Dean; Michael; (Frederick, MD) ; Lou;
Hong; (Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
39492945 |
Appl. No.: |
13/956628 |
Filed: |
August 1, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13470965 |
May 14, 2012 |
8546524 |
|
|
13956628 |
|
|
|
|
12513091 |
Aug 11, 2009 |
8198402 |
|
|
PCT/US2007/083027 |
Oct 30, 2007 |
|
|
|
13470965 |
|
|
|
|
60855422 |
Oct 31, 2006 |
|
|
|
Current U.S.
Class: |
514/18.7 ;
435/375; 514/19.2; 514/19.3; 514/19.4; 514/19.5 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 21/00 20180101; A61P 15/00 20180101; A61P 11/00 20180101; A61P
1/18 20180101; A61P 25/00 20180101; A61P 17/06 20180101; A61P 19/00
20180101; A61P 1/16 20180101; A61P 1/04 20180101; A61P 17/00
20180101; A61P 35/02 20180101; C07K 14/705 20130101; A61P 15/14
20180101; A61P 5/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/18.7 ;
435/375; 514/19.3; 514/19.4; 514/19.5; 514/19.2 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Claims
1-32. (canceled)
33. A method of inhibiting proliferation of a diseased cell, the
method comprising contacting the diseased cell with a
pharmaceutical composition comprising (i) an isolated or purified
polypeptide, peptidomimetic, or fatty acid derivative of the
polypeptide or peptidomimetic comprising an amino acid sequence
comprising SEQ ID NO: 3, a functional fragment thereof, or a
functional variant of either SEQ ID NO: 3 or the functional
fragment, wherein the functional variant comprises an amino acid
sequence (a) which has at least 90% sequence identity with SEQ ID
NO: 3 or (b) which is a retroinverso analogue of SEQ ID NO: 3 or a
functional fragment thereof, wherein the polypeptide or
peptidomimetic is less than or equal to 50 amino acids in length,
wherein the functional fragment comprises at least 7 contiguous
amino acids of SEQ ID NO: 3, and wherein the functional fragment or
functional variant inhibits proliferation of a diseased cell, (ii)
optionally a lipid, and a (iii) pharmaceutically acceptable carrier
in an amount effective to inhibit proliferation of the diseased
cell.
34. The method of claim 33, wherein the diseased cell is in a
host.
35. The method of claim 34, wherein the host is a mammal.
36. The method of claim 35, wherein the mammal is a human.
37. The method of claim 33, wherein the method treats or prevents a
cancer of the host.
38. The method of claim 37, wherein the cancer is breast cancer,
prostate cancer, ovarian cancer, stomach cancer, colon cancer,
liver cancer, melanoma, basal cell carcinoma, rhabdomyosarcoma, a
medulloblastoma, pancreatic cancer, lung cancer, thyroid cancer, a
myeloma, a lymphoma, a glioma, or a sarcoma.
39. The method of claim 38, wherein the stomach cancer is a gastric
adenocarcinoma.
40. The method of claim 33, wherein the method treats or prevents a
neoplasm of the host.
41. The method of claim 33, wherein the pharmaceutical composition
is topically administered to the host.
42. The method of claim 33, wherein the pharmaceutical composition
is intratumorally administered to the host.
43. A method of treating or preventing cancer, psoriasis, or a
neoplasm in a host, the method comprising administering to the host
a pharmaceutical composition comprising (i) an isolated or purified
polypeptide, peptidomimetic, or fatty acid derivative of the
polypeptide or peptidomimetic comprising an amino acid sequence
comprising SEQ ID NO: 3, a functional fragment thereof, or a
functional variant of either SEQ ID NO: 3 or the functional
fragment, wherein the functional variant comprises an amino acid
sequence (a) which has at least 90% sequence identity with SEQ ID
NO: 3 or (b) which is a retroinverso analogue of SEQ ID NO: 3 or a
functional fragment thereof, wherein the polypeptide or
peptidomimetic is less than or equal to 50 amino acids in length,
wherein the functional fragment comprises at least 7 contiguous
amino acids of SEQ ID NO: 3, and wherein the functional fragment or
functional variant inhibits proliferation of a diseased cell, (ii)
optionally a lipid, and a (iii) pharmaceutically acceptable carrier
in an amount effective to treat or prevent the cancer, psoriasis,
or a neoplasm.
44-45. (canceled)
46. A method of inhibiting the expression of a gene selected from
the group consisting of Gli-1, Gli-2, Gli-3, Ptch, Shh, Smo, NES,
and a combination thereof, in a diseased cell, the method
comprising contacting the diseased cell with a pharmaceutical
composition comprising (i) an isolated or purified polypeptide,
peptidomimetic, or fatty acid derivative of the polypeptide or
peptidomimetic comprising an amino acid sequence comprising SEQ ID
NO: 3, a functional fragment thereof, or a functional variant of
either SEQ ID NO: 3 or the functional fragment, wherein the
functional variant comprises an amino acid sequence (a) which has
at least 90% sequence identity with SEQ ID NO: 3 or (b) which is a
retroinverso analogue of SEQ ID NO: 3 or a functional fragment
thereof, wherein the polypeptide or peptidomimetic is less than or
equal to 50 amino acids in length, wherein the functional fragment
comprises at least 7 contiguous amino acids of SEQ ID NO: 3, and
wherein the functional fragment or functional variant inhibits
proliferation of a diseased cell, (ii) optionally a lipid, and a
(iii) pharmaceutically acceptable carrier in an amount effective to
inhibit the expression of the gene.
47. A method of inhibiting the Hedgehog signal transduction pathway
in a diseased cell, the method comprising contacting the diseased
cell with a pharmaceutical composition comprising (i) an isolated
or purified polypeptide, peptidomimetic, or fatty acid derivative
of the polypeptide or peptidomimetic comprising an amino acid
sequence comprising SEQ ID NO: 3, a functional fragment thereof, or
a functional variant of either SEQ ID NO: 3 or the functional
fragment, wherein the functional variant comprises an amino acid
sequence (a) which has at least 90% sequence identity with SEQ ID
NO: 3 or (b) which is a retroinverso analogue of SEQ ID NO: 3 or a
functional fragment thereof, wherein the polypeptide or
peptidomimetic is less than or equal to 50 amino acids in length,
wherein the functional fragment comprises at least 7 contiguous
amino acids of SEQ ID NO: 3, and wherein the functional fragment or
functional variant inhibits proliferation of a diseased cell, (ii)
optionally a lipid, and a (iii) pharmaceutically acceptable carrier
in an amount effective to inhibit the Hedgehog signal transduction
pathway.
48-50. (canceled)
51. The method of claim 33, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 17 to 33.
52. The method of claim 33, wherein the functional variant
comprises the amino acid sequence of SEQ ID NO: 23 with one amino
acid substitution at any of positions 1-7,9,11, and 12 of SEQ ID
NO: 23 in which the amino acid at the position is substituted with
Ala.
53. The method of claim 33, wherein the functional variant
comprises a retroinverso analogue of SEQ ID NO: 23, 26, or 33.
54. The method of claim 33, wherein the functional variant
comprises the amino acid sequence of any of SEQ ID NOs: 34 to 37,
68, 84, and 92-94.
55. The method of claim 33, wherein the functional variant
comprises the amino acid sequence of any of SEQ ID NOs: 38-59.
56. The method of claim 33, wherein the fatty acid derivative
comprises a fatty acid molecule at the amino (N-) terminus, the
carboxyl (C-) terminus, or both the N- and C-termini, the fatty
acid molecule optionally containing at least one amino group.
57. The method of claim 33, wherein the functional variant
comprises the amino acid sequence of SEQ ID NO: 68 (all D-amino
acids), the functional variant is optionally amidated, and the
amino acid at position 1 of the functional variant is optionally
acetylated.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 60/855,422, filed Oct. 31, 2006,
which is incorporated by reference.
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 53,510 Byte
ASCII (Text) file named "701943 Sequence_ST25.TXT," created on Oct.
24, 2007.
BACKGROUND OF THE INVENTION
[0003] Cancer is caused by dysregulations of signal transduction
pathways. One such pathway is the Hedgehog (HH) signal transduction
pathway, which involves the Patch (Ptch) and Smoothened (SMO)
proteins. The HH pathway is essential for embryonic cell growth
(Beachy et al., Nature, 432: 324-331 (2004)) and was found to be
dysregulated in several cancers, including breast cancer (Katano et
al., Cancer Lett., 227: 99-104 (2005)), prostate cancer (Sanchez et
al., Proc. Natl. Acad. Sci. U.S.A., 101: 12561-12566 (2004)),
stomach cancer (Berman et al., Nature, 425: 846-851 (2003)), colon
cancer (Douard et al., Surgery, 139: 665-670 (2006)), liver cancer
(Sicklick et al., Carcinogenesis, 27: 748-757 (2006)), melanoma
(Pons et al., Clin. Transl. Oncol., 8: 466-474 (2006)), basal cell
carcinoma (Lam et al., Oncogene, 18, 833-836 (1999)), and
medulloblastoma (Berman et al., Science, 297, 1559-1561 (2002) and
Romer et al., Cancer Res., 65, 4975-4978 (2005)). There is a desire
for inhibitors of the HH pathway for use in treatment of
cancers.
BRIEF SUMMARY OF THE INVENTION
[0004] The invention provides an isolated or purified polypeptide
or a peptidomimetic, as well as a fatty acid derivative thereof.
The polypeptide or peptidomimetic comprises an amino acid sequence
corresponding to a portion of a SMO protein, wherein the portion
comprises an amino acid sequence of any of SEQ ID NOs: 2 to 4, each
sequence of which generally corresponds to an intracellular loop of
the SMO protein. The polypeptide or peptidomimetic can be a
functional fragment of the portion, which functional fragment
comprises at least 7 contiguous amino acids of SEQ ID NO: 2, 3, or
4. The polypeptide or peptidomimetic can be a functional variant of
the portion or of the functional fragment. The inventive
polypeptides and peptidomimetics (including fatty acid derivatives
thereof, functional fragments and functional variants) inhibit the
HH pathway and/or proliferation of a diseased cell.
[0005] The invention also provides conjugates comprising any of the
inventive polypeptides or peptidomimetics, or fatty acid
derivatives thereof. Further provided are nucleic acids encoding
the inventive polypeptides, as well as related recombinant
expression vectors and host cells. Pharmaceutical compositions
comprising any of the inventive polypeptides, peptidomimetics,
fatty acid derivatives, conjugates, nucleic acids, and recombinant
expression vectors are furthermore provided by the invention.
[0006] The inventive pharmaceutical compositions are useful for
inhibiting proliferation of a diseased cell, such that the
invention moreover provides a method of inhibiting proliferation of
a diseased cell. The method comprises contacting the diseased cell
with an inventive pharmaceutical composition in an amount effective
to inhibit proliferation of the diseased cell.
[0007] The invention provides other methods of use of the inventive
pharmaceutical compositions, including a method of treating or
preventing cancer in a host, a method of treating psoriasis in a
host, a method of treating a neoplasm in a host, and a method of
inhibiting expression of a gene selected from the group consisting
of Gli-1, Gli-2, Gli-3, Ptch, Shh, Smo, and NES in a diseased
cell.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0008] FIG. 1 depicts the % of viable MCF-7 breast cancer cells
(relative to a control) upon a 48-hour treatment as a function of
concentration of SMOi1-1, SMOi2-1, and SMOi3-1 lipidated
polypeptides, in accordance with an embodiment of the
invention.
[0009] FIG. 2 depicts the viable cell mass of gastric
adenocarcinoma cells upon treatment with 0, 5, or 10 .mu.M (white,
black, lined bars, respectively) of cyclopamine, SMOi3-1, or
SMOi2-1.
[0010] FIG. 3 depicts the % of viable MCF-7 cells (relative to a
control) upon a 48-hour treatment with polypeptides comprising
amino acid sequences based on the third intracellular loop of the
SMO protein, in accordance with an embodiment of the invention.
[0011] FIG. 4 depicts the % of viable SK-Mel2 cells upon a 48-hour
treatment with SMOi2-8 or retroinverso analogues, SMOi2-16 and
SMOi2-17, in accordance with an embodiment of the invention.
[0012] FIG. 5 depicts the relative expression of genes of the HH
pathway in DU145 cells upon a 48-hour treatment with cyclopamine,
SMOi3-1, or SMOi2-1 polypeptides, in accordance with an embodiment
of the invention.
[0013] FIG. 6 depicts the % of viable SK-Mel2 cells upon a 60-hour
treatment with SMOi2-8 or peptidomimetics containing a
4-benzoylphenylalanine (BPA) residue in place of the Trp residue at
position 5 of SMOi2-8, in accordance with an embodiment of the
invention.
[0014] FIG. 7 depicts the toxicity of the second intracellular loop
derivatives (SMOi2-12 (circles) and SMOi2-17 (squares)) as
determined by MTT assay in SK-Mel2 melanoma cells after 48 h of
exposure to the peptide compounds, in accordance with an embodiment
of the invention.
[0015] FIG. 8 depicts the fluorescence emission intensity measured
for probes with increasing SMOi2-8/WMC-77 ratio, in accordance with
an embodiment of the invention.
[0016] FIG. 9 depicts the growth inhibition of breast cancer,
melanoma, heptaoma, and pancreatic cancer cells upon exposure to
SMOi2-12 (diamonds) or SMOi2-20 (squares), in accordance with an
embodiment of the invention.
[0017] FIG. 10 depicts the circular dichroism spectrum of SMOi2-8
(diamonds) and SMOi2-16 (triangles) peptides.
DETAILED DESCRIPTION OF THE INVENTION
[0018] SMO proteins are transmembrane proteins which function in
the Hedgehog (HH) signal transduction pathway (see, for instance,
Huangfu and Anderson, Development 133: 3-14 (2006)), which, as
discussed is related to several cancers, e.g., breast cancer,
prostate cancer, stomach cancer, etc. These proteins comprise an
extracellular domain, seven transmembrane domains, three
intracellular loops, and an intracellular domain. SMO proteins
resemble a G-protein coupled receptor (GPCR) in general topology
but appear to signal differently from the GPCRs. Examples of SMO
proteins include human SMO proteins (e.g., GenBank Accession No.
NP.sub.--005622 (SEQ ID NO: 1)), as well as orthologs thereof, such
as mouse SMO proteins (e.g., GenBank Accession No.
NP.sub.--795970), rat SMO proteins (e.g., GenBank Accession No.
NP.sub.--036939), fruit fly SMO proteins (e.g., GenBank Accession
No. NP.sub.--523443), zebra fish SMO proteins (e.g., GenBank
Accession No. NP.sub.--571102), chicken SMO proteins, (e.g.,
GenBank Accession No. AAB84389), African clawed frog SMO proteins
(e.g., GenBank Accession No. AAK15464).
[0019] The invention provides an isolated or purified polypeptide
comprising an amino acid sequence corresponding to a portion of a
SMO protein, wherein the portion comprises an amino acid sequence
of any of SEQ ID NOs: 2 to 4, each of which is identical to or
substantially identical to an intracellular loop of the SMO
protein. For example, SEQ ID NO: 2 contains one additional amino
acid (Leu) at the N-terminus of the second intracellular loop of
SMO.
[0020] As used herein, the term "polypeptide" refers to a single
chain of amino acids connected by one or more peptide bonds. In
this regard, the term encompasses peptides, oligopeptides, and
polypeptides of any length, provided that there is at least one
peptide bond. For purposes herein, the polypeptide of the invention
comprises at least 6 peptide bonds, e.g., 10 or more peptide
bonds.
[0021] The inventive polypeptides comprise an amino acid sequence
of a portion of a SMO protein. That is to say that the polypeptides
of the invention do not encompass any full-length, wild-type SMO
proteins, e.g., SEQ ID NO: 1. In this respect, the inventive
polypeptides comprise less than about 780 amino acids of a
wild-type SMO protein. For example, the inventive polypeptides
comprise less than about 500 amino acids of a wild-type SMO
protein. Most preferably, the inventive polypeptides comprise less
than about 75, e.g., about 50, amino acids of a wild-type SMO
protein. Also preferred is that the inventive polypeptides comprise
about 10 to about 12 amino acids (excluding any CPP, as discussed
herein).
[0022] The portion of the SMO protein, in accordance with an
embodiment of the invention, comprises an amino acid sequence of
any of SEQ ID NOs: 2 to 4. In this regard, the inventive
polypeptides can comprise, consist essentially of, or consist of an
amino acid sequence of SEQ ID NO: 2, 3, or 4.
[0023] Alternatively, the portion of the SMO protein comprises an
amino acid sequence of any of SEQ ID NOs: 5 to 8. In this regard,
the inventive polypeptides can comprise, consist essentially of, or
consist of an amino acid sequence of SEQ ID NO: 5, 6, 7, or 8.
[0024] Included in the scope of the invention are functional
fragments of the inventive polypeptides described herein. The term
"functional fragment" when used in reference to an inventive
polypeptide refers to any part or portion of the polypeptide of the
invention, which part or portion retains the biological activity of
the polypeptide of which it is a part (the parent polypeptide). The
functional fragment can be any fragment comprising contiguous amino
acids of the polypeptide of which it is a part, provided that the
functional fragment inhibits proliferation of a diseased cell.
Functional fragments encompass, for example, those parts of an
inventive polypeptide that retain the ability to inhibit
proliferation, or treat or prevent a disease (e.g., cancer,
neoplasm, psoriasis), to a similar extent, the same extent, or to a
higher extent, as the parent polypeptide. In reference to the
parent polypeptide, the functional fragment can comprise, for
instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of
the parent polypeptide.
[0025] The functional fragment can comprise additional amino acids
at the amino or carboxy terminus, or at both termini, e.g., amino
acids not found in the amino acid sequence of the parent
polypeptide. Desirably, the additional amino acids do not interfere
with the biological function of the functional fragment, e.g.,
inhibit proliferation, or treat or prevent a disease (e.g., cancer,
neoplasm, psoriasis). More desirably, the additional amino acids
enhance the biological activity, as compared to the biological
activity of the parent polypeptide.
[0026] In a preferred embodiment of the invention, the functional
fragment comprises at least 5 contiguous amino acids of SEQ ID NO:
2, 3, or 4 and inhibits proliferation of a diseased cell. In a more
preferred embodiment of the invention, the functional fragment
comprises at least 7 contiguous amino acids of SEQ ID NO: 2, 3, or
4. In a further preferred embodiment of the invention, the
functional fragment comprises at least 4 contiguous amino acids of
SEQ ID NO: 2, 3, or 4 and inhibits proliferation of a diseased
cell. The functional fragment of the invention can, for example,
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs: 5 to 8. For instance, the functional fragment can
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs: 9 to 33. Also, the functional fragment of the
invention can consist essentially of or consist of an amino acid
sequence of any of SEQ ID NOs: 9 to 33.
[0027] Further included in the scope of the invention are
functional variants of the inventive polypeptides, as well as
functional variants of the inventive functional fragments described
herein. The term "functional variant" as used herein refers to a
polypeptide having substantial or significant sequence identity or
similarity to a parent polypeptide or parent functional fragment,
which functional variant retains the biological activity of the
polypeptide or functional fragment of which it is a variant.
Functional variants encompass, for example, those variants of the
inventive polypeptide described herein (the parent polypeptide) and
those variants of the functional fragment described herein (the
parent functional fragment) that retain the ability to inhibit
proliferation of a diseased cell to a similar extent, the same
extent, or to a higher extent, as the parent polypeptide or parent
functional fragment. In reference to the parent polypeptide or
parent functional fragment, the functional variant can, for
instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more
identical in amino acid sequence to the parent polypeptide or
parent functional fragment.
[0028] The functional variant can, for example, comprise the amino
acid sequence of the parent polypeptide or parent functional
fragment with at least one conservative amino acid substitution. In
this regard, the functional variant can comprise the amino acid
sequence of the parent polypeptide or parent functional fragment
with two, three, four, five, or more conservative amino acid
substitutions. Alternatively or additionally, the functional
variants can comprise the amino acid sequence of the parent
polypeptide or parent functional fragment with at least one
non-conservative amino acid substitution. In this regard, the
functional variant can comprise the amino acid sequence of the
parent polypeptide or parent functional fragment with two, three,
four, five, or more non-conservative amino acid substitutions. In
this case, it is preferable for the non-conservative amino acid
substitution to not interfere with or inhibit the biological
activity of the functional variant. Preferably, the
non-conservative amino acid substitution enhances the biological
activity of the functional variant, such that the biological
activity of the functional variant is increased as compared to the
parent polypeptide or parent functional fragment.
[0029] The functional variants preferably comprise one or more
conservative amino acid substitutions. 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 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, Gln, Ser, Thr, Tyr, etc.), an aromatic amino
acid (Trp, Phe, Tyr, etc.) for another aromatic amino acid,
etc.
[0030] Desirably, the functional variants of the invention comprise
at least 4 contiguous amino acids of SEQ ID NO: 2, 3, or 4, have at
least 75% sequence identity (e.g., 80%, 85%, 90%, 95% sequence
identity) to the parent polypeptide or parent functional fragment,
and inhibit proliferation of a diseased cell.
[0031] For example, the functional variant can be a functional
variant of any of SEQ ID NOs: 17 to 21 and 23 to 33. In this
regard, the functional variants can comprise the amino acid
sequence of any of SEQ ID NOs: 38 to 54 and 57 to 59, wherein Xaa
is selected from a group consisting of Tyr, Phe, or BPA.
[0032] Also, for example, the functional variant can be a
functional variant of SMOi2-8 (SEQ ID NO: 23) comprising the amino
acid sequence of SMOi2-8 with one of the amino acids at any of
positions 1-7,9,11, and 12 is substituted with Ala.
[0033] Alternatively, the functional variant can comprise a
retroinverso analogue of any of the inventive polypeptides or
functional fragments described herein. The term "retroinverso
analogue" refers to a polypeptide comprising a reversed amino acid
sequence of a parent polypeptide, such that the amino acid sequence
of the retroinverso analogue (when read from the N-terminus to the
C-terminus) is the same as the amino acid sequence of the parent
polypeptide when read from the C-terminus to the N-terminus.
Furthermore, with respect to a retroinverso analogue, each of the
amino acids is the D isomer of the amino acid, as opposed to the L
isomer. For example, the retroinverso analogue of the tripeptide
Val-Ala-Gly has an amino acid sequence Gly-Ala-Val, in which each
amino acid is the D isomer. With respect to the invention, the
functional variant preferably comprises a retroinverso analogue of
SEQ ID NO: 23, 26, or 33. In this regard, the functional variant
comprises the amino acid sequence of any of SEQ ID NOs: 34 to
37.
[0034] The polypeptides of the invention (including functional
fragments and functional variants) can be obtained by methods known
in the art. Suitable methods of de novo synthesizing polypeptides
are described in references, such as 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; and U.S. Pat. No.
5,449,752. Also, polypeptides can be recombinantly produced using
the nucleic acids described herein using standard recombinant
methods. See, for instance, 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. Further, some of the polypeptides of the
invention (including functional fragments and functional variants
thereof) can be isolated and/or purified from a source, such as a
plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
Methods of isolation and purification are well-known in the art.
Alternatively, the polypeptides described herein (including
functional fragments and functional variants thereof) can be
synthesized or obtained commercially from companies such as Synpep
(Dublin, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.),
and Multiple Peptide Systems (San Diego, Calif.). In this respect,
the inventive polypeptides can be synthetic, recombinant, isolated,
and/or purified.
[0035] Also provided by the invention are peptidomimetics of any of
the inventive polypeptides (including functional fragments and
functional variants) described herein. The term "peptidomimetic" as
used herein refers to a compound which has essentially the same
general structure of a corresponding polypeptide with modifications
that increase its stability or biological function. A
peptidomimetic includes, for example, those compounds comprising
the same amino acid sequence of a corresponding polypeptide with an
altered backbone between two or more of the amino acids.
Additionally, the peptidomimetic can comprise synthetic or
non-naturally occurring amino acids in place of naturally-occurring
amino acids.
[0036] In a preferred embodiment, the peptidomimetic is a peptoid.
The term "peptoid" as used herein refers to a peptidomimetic in
which the sidechains of each amino acid is appended to the nitrogen
atom of the amino acid as opposed to the alpha carbon. For example,
peptoids can 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.
[0037] In another preferred embodiment, the peptidomimetic
comprises an altered backbone in which the bond between each amino
acid is methylated. In this regard, the peptidomimetic can comprise
a methylated peptide backbone of the following structure:
##STR00001##
[0038] The polypeptides (including functional fragments and
functional variants) and peptidomimetics of the invention can be of
any length, i.e., can comprise any number of amino acids, provided
that the polypeptide (or functional fragment or functional variant
thereof) or peptidomimetic retains their biological activity, e.g.,
the ability to inhibit proliferation of a diseased cell, treat or
prevent disease (e.g., cancer, neoplasm, psoriasis) in a host, etc.
For example, the inventive polypeptide or peptidomimetic can be 50
to 5000 amino acids long, such as 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 20, 25, 30, 40, 50, 70, 75, 100, 125, 150, 175, 200,
300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in
length. Preferably, the polypeptides of the invention are 5 to 50
amino acids in length.
[0039] The polypeptides (including functional fragments and
functional variants) and peptidomimetics of the invention can
comprise synthetic amino acids in place of one or more
naturally-occurring amino acids. Such synthetic amino acids are
known in the art, and 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-benzoylphenylalanine, 4-nitrophenylalanine,
4-chlorophenylalanine, 4-carboxyphenylalanine, .beta.-phenylserine,
.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.
[0040] The polypeptides (including functional fragments and
functional variants) and peptidomimetics of the invention can be
lipidated (e.g., fatty acidated), glycosylated, amidated,
carboxylated, phosphorylated, esterified, N-acylated, cyclized via,
e.g., a disulfide bridge, or converted into an acid addition salt
and/or optionally dimerized or polymerized, or conjugated.
[0041] In this regard, the invention further provides lipidated
derivatives of any of the polypeptides (including functional
fragments and functional variants) and peptidomimetics of the
invention. Lipidated derivatives of the invention encompass any of
the polypeptides and peptidomimetics described herein comprising a
lipid molecule. As used herein, the term "lipid molecule" refers to
any molecule comprising a hydrophobic moiety which facilitates the
entry of the polypeptide (including functional fragments and
functional variants) or peptidomimetic across the cell membrane and
into the cell. The lipid can be any lipid known in the art, such
as, for example, a fatty acid, a 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, and a cholesterol
group.
[0042] Preferably, the lipidated derivative is a fatty acid
derivative in which the polypeptide or peptidomimetic described
herein comprises a fatty acid molecule. The fatty acid molecule can
be any C.sub.8 to C.sub.20 fatty acid. The fatty acid molecule can
be, e.g., lauric acid, palmitic acid, myristic acid, stearic acid,
oleic acid, linoleic acid, .alpha.-linoleic acid, linolenic acid,
arachidonic acid, timnodonic acid, docosohexenoic acid, erucic
acid, arachidic acid, or behenic acid. The fatty acid may
optionally contain additional functional groups, e.g., one or more
amino groups on any of the carbon atoms. In a preferred embodiment,
the fatty acid molecule is a C.sub.8 to C.sub.16 fatty acid, for
example, a C.sub.16 fatty acid. In a more preferred embodiment, the
fatty acid is palmitate.
[0043] As is true with respect to the lipid molecule, the fatty
acid molecule can be attached to any suitable part of the inventive
polypeptide (including functional fragment and functional variant)
or peptidomimetic. In a preferred embodiment of the invention, the
fatty acid derivative of the inventive polypeptide (including
functional fragment and functional variant) or peptidomimetic
comprises a fatty acid molecule at the amino (N-) terminus, the
carboxyl (C-) terminus, or both the N- and C-termini.
[0044] The fatty acid molecule can be attached to the inventive
polypeptide (including functional fragment and functional variant)
or peptidomimetic directly or through a linker. In an embodiment of
the invention, when the fatty acid molecule is at the C-terminus of
the inventive polypeptide or peptidomimetic, the fatty acid
molecule is attached through an amino acid linker selected from the
group consisting of Lys, Cys, homocysteine (homoCys), Orn,
.alpha.,.gamma.-diaminobutyric acid, and
.alpha.,.beta.-diaminopropionic acid. In a preferred embodiment of
the invention, the fatty acid molecule is attached through a Lys.
In a more preferred embodiment of the invention, the fatty acid
molecule is attached through the epsilon carbon of Lys.
[0045] In another preferred embodiment, when the fatty acid
molecule is at the C-terminus of the inventive polypeptide or
peptidomimetic, the fatty acid molecule is modified e.g., to
include an amino group such as in a modified molecule of Formula I
or Formula H, wherein Formula I is NH.sub.2(CH.sub.2).sub.nCOOH and
Formula II is CH.sub.3(CH.sub.2).sub.mCH(NH.sub.2)COOH, wherein
each of n and in is 1 to 24. In this regard, the fatty acid
molecule is attached to the carboxyl group of the C-terminal amino
acid of the polypeptide or peptidomimetic. Preferably, n or m is 8
to 16. More preferably, n or m is 16.
[0046] Alternatively, the inventive polypeptides (including
functional fragments and functional variants) or peptidomimetics
described herein can comprise a cell-penetrating peptide (CPP).
Such a CPP facilitates the entry of the inventive polypeptide or
peptidomimetic across the cell membrane and into the cell. CPPs are
known in the art. See, for example, Deshayes et al., Cell. Mol.
Life. Sci. 62: 1839-1849 (2005); El-Andaloussi et al., Curr. Pharm.
Design 11: 3597-3611 (2005); and Mae and Langel, Curr. Opin.
Pharmacol. 6: 509-514 (2006)). The CPP can be any of those known in
the art, e.g., Transportan, VP22, Pep1, and the like. Preferably,
the CPP comprises an amino acid sequence of SEQ ID NO: 78 or 79,
which corresponds to the amino acid sequence of penetratin and Tat
(48-60), respectively.
[0047] The polypeptides (including functional fragments and
functional variants) and peptidomimetics, including fatty acid
derivatives thereof, of the invention can be a monomer peptide, or
can be a dimer or multimer peptide. For example, the polypeptide
can be a dimer of the following general structure:
##STR00002##
wherein Sequence X is selected from the group consisting of
Ac-LAKFSTHWAYTL (all-D) (SEQ ID NO: 85); Ac-AKFSTHWAYTL (All-D)
(SEQ ID NO: 86); and Ac-KFSTHWAYTL (All-D) (SEQ ID NO: 87); wherein
each of Linker 1 and Linker 2 is optionally present and each
independently is Gly, beta-Ala, aminopropionic acid,
gamma-aminobutyric acid, aminocaproic acid, or aminohexanoic acid;
wherein n and m is between 0 and 6; wherein Y is K, C, homoCys,
Orn, diaminopropanoic acid (DPA), diaminobutyric acid (DBA); and
wherein the fatty acid is a stearic, palmitic, myristic, lauric,
capric or caprilic acid. In a preferred embodiment, Sequence X is
SEQ ID NO: 87, n is 4, m is 0, each of Linker 1 and Linker 2 is
beta-Ala, and the fatty acid is palmitate. Methods of making
dimeric and multimeric polypeptides are known in the art. See, for
example, Wrighton et al., Nature Biotechnology 15: 1261-1265
(1997). A preferred method of making a dimeric polypeptide also is
set forth herein as Example 1.
[0048] When the polypeptides (including functional fragments and
functional variants) and peptidomimetics, including fatty acid
derivatives thereof, of the invention are in the form of a salt,
preferably, the polypeptides or peptidomimetcs are in the form of a
pharmaceutically acceptable salt. Suitable pharmaceutically
acceptable acid addition salts include those derived from mineral
acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric, and sulphuric acids, and organic acids,
such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic acids, for example,
p-toluenesulphonic acid.
[0049] Further provided by the invention is a nucleic acid encoding
any of the inventive polypeptides, including functional fragments
and functional variants, described herein. As used herein, the term
"nucleic acid" encompasses "polynucleotide," "oligonucleotide," and
"nucleic acid molecule," and generally refers to a polymer of DNA
or RNA, which can be single-stranded or double-stranded,
synthesized or obtained (e.g., isolated and/or purified) from
natural sources, which can contain natural, non-natural or altered
nucleotides, and which can contain a natural, non-natural or
altered internucleotide linkage, such as a phosphoroamidate linkage
or a phosphorothioate linkage, instead of the phosphodiester found
between the nucleotides of an unmodified oligonucleotide. It is
generally preferred that the nucleic acid does not comprise any
insertions, deletions, inversions, and/or substitutions. However,
it may be suitable in some instances, as discussed herein, for the
nucleic acid to comprise one or more insertions, deletions,
inversions, and/or substitutions.
[0050] Preferably, the nucleic acids of the invention are
recombinant. As used herein, the term "recombinant" refers to (i)
molecules that are constructed outside living cells by joining
natural or synthetic nucleic acid segments to nucleic acid
molecules that can replicate in a living cell, or (ii) molecules
that result from the replication of those described in (i) above.
For purposes herein, the replication can be in vitro replication or
in vivo replication.
[0051] The nucleic acids can be constructed based on chemical
synthesis and/or enzymatic ligation reactions using procedures
known in the art. See, for example, Sambrook et al., supra, and
Ausubel et al., supra. For example, a nucleic acid can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed upon hybridization (e.g., phosphorothioate
derivatives and acridine substituted nucleotides). Examples of
modified nucleotides that can be used to generate the nucleic acids
include, but are not limited to, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N.sup.2-carboxypropyl) uracil, and 2,6-diaminopurine.
Alternatively, one or more of the nucleic acids of the invention
can be purchased from companies, such as Macromolecular Resources
(Fort Collins, Colo.) and Synthegen (Houston, Tex.).
[0052] The nucleic acid can comprise any nucleotide sequence which
encodes any of the inventive polypeptides, including functional
fragments and functional variants. For example, the nucleic acid
can comprise a nucleotide sequence encoding any of SEQ ID NOs: 2 to
29, 32, 33, 38 to 48, 53 to 56, 59 to 66, 70 to 72, 76, and 80. The
nucleic acid alternatively can comprise a nucleotide sequence which
is degenerate to any of these sequences or a combination of
degenerate sequences. The invention also provides an isolated or
purified nucleic acid comprising a nucleotide sequence which is
complementary to the nucleotide sequence of any of the nucleic
acids described herein or a nucleotide sequence which hybridizes
under stringent conditions to the nucleotide sequence of any of the
nucleic acids described herein.
[0053] The nucleic acids of the invention can be incorporated into
a recombinant expression vector. In this regard, the invention
provides recombinant expression vectors comprising any of the
nucleic acids of the invention. For purposes herein, the term
"recombinant expression vector" means a genetically-modified
oligonucleotide or polynucleotide construct that permits the
expression of an mRNA, protein, polypeptide, or peptide by a host
cell, when the construct comprises a nucleotide sequence encoding
the mRNA, protein, polypeptide, or peptide, and the vector is
contacted with the cell under conditions sufficient to have the
mRNA, protein, polypeptide, or peptide expressed within the cell.
The vectors of the invention are not naturally-occurring as a
whole. However, parts of the vectors can be naturally-occurring.
The inventive recombinant expression vectors can comprise any type
of nucleotides, including, but not limited to DNA and RNA, which
can be single-stranded or double-stranded, synthesized or obtained
in part from natural sources, and which can contain natural,
non-natural or altered nucleotides. The recombinant expression
vectors can comprise naturally-occurring, non-naturally-occurring
internucleotide linkages, or both types of linkages. Preferably,
the non-naturally occurring or altered nucleotides or
internucleotide linkages does not hinder the transcription or
replication of the vector.
[0054] The recombinant expression vector of the invention can be
any suitable recombinant expression vector, and can be used to
transform or transfect any suitable host. Suitable vectors include
those designed for propagation and expansion or for expression or
both, such as plasmids and viruses. The vector can be selected from
the group consisting of the pUC series (Fermentas Life Sciences),
the pBluescript series (Stratagene, LaJolla, Calif.), the pET
series (Novagen, Madison, Wis.), the pGEX series (Pharmacia
Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto,
Calif.). Bacteriophage vectors, such as .lamda.GT10, .lamda.GT11,
.lamda.ZapII (Stratagene), .lamda.EMBL4, and .lamda.NM1149, also
can be used. Examples of plant expression vectors include pBI01,
pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of
animal expression vectors include pEUK-Cl, pMAM and pMAMneo
(Clontech). Preferably, the recombinant expression vector is a
viral vector, e.g., a retroviral vector.
[0055] The recombinant expression vectors of the invention can be
prepared using standard recombinant DNA techniques described in,
for example, Sambrook et al., supra, and Ausubel et al., supra.
Constructs of expression vectors, which are circular or linear, can
be prepared to contain a replication system functional in a
prokaryotic or eukaryotic host cell. Replication systems can be
derived, e.g., from ColEl, 2.mu. plasmid, .lamda., SV40, bovine
papilloma virus, and the like.
[0056] Desirably, the recombinant expression vector comprises
regulatory sequences, such as transcription and translation
initiation and termination codons, which are specific to the type
of host (e.g., bacterium, fungus, plant, or animal) into which the
vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA- or RNA-based.
[0057] The recombinant expression vector can include one or more
marker genes, which allow for selection of transformed or
transfected hosts. Marker genes include biocide resistance, e.g.,
resistance to antibiotics, heavy metals, etc., complementation in
an auxotrophic host to provide prototrophy, and the like. Suitable
marker genes for the inventive expression vectors include, for
instance, neomycin/G418 resistance genes, hygromycin resistance
genes, histidinol resistance genes, tetracycline resistance genes,
and ampicillin resistance genes.
[0058] The recombinant expression vector can comprise a native or
normative promoter operably linked to the nucleotide sequence
encoding the modified TCR, polypeptide, or protein (including
functional portions and functional variants thereof), or to the
nucleotide sequence which is complementary to or which hybridizes
to the nucleotide sequence encoding the modified TCR, polypeptide,
or protein. The selection of promoters, e.g., strong, weak,
inducible, tissue-specific and developmental-specific, is within
the ordinary skill of the artisan. Similarly, the combining of a
nucleotide sequence with a promoter is also within the skill of the
artisan. The promoter can be a non-viral promoter or a viral
promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter,
an RSV promoter, and a promoter found in the long-terminal repeat
of the murine stem cell virus.
[0059] The inventive recombinant expression vectors can be designed
for either transient expression, for stable expression, or for
both. Also, the recombinant expression vectors can be made for
constitutive expression or for inducible expression.
[0060] Further, the recombinant expression vectors can be made to
include a suicide gene. As used herein, the term "suicide gene"
refers to a gene that causes the cell expressing the suicide gene
to die. The suicide gene can be a gene that confers sensitivity to
an agent, e.g., a drug, upon the cell in which the gene is
expressed, and causes the cell to die when the cell is contacted
with or exposed to the agent. Suicide genes are known in the art
(see, for example, Suicide Gene Therapy: Methods and Reviews,
Springer, Caroline J. (Cancer Research UK Centre for Cancer
Therapeutics at the Institute of Cancer Research, Sutton, Surrey,
UK), Humana Press, 2004) and include, for example, the Herpes
Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine daminase,
purine nucleoside phosphorylase, and nitroreductase.
[0061] The invention further provides a host cell comprising any of
the recombinant expression vectors described herein. As used
herein, the term "host cell" refers to any type of cell that can
contain the inventive recombinant expression vector. The host cell
can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or
can be a prokaryotic cell, e.g., bacteria or protozoa. The host
cell can be a cultured cell or a primary cell, i.e., isolated
directly from an organism, e.g., a human. The host cell can be an
adherent cell or a suspended cell, i.e., a cell that grows in
suspension. Suitable host cells are known in the art and include,
for instance, DH5.alpha. E. coli cells, Chinese hamster ovarian
cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
For purposes of amplifying or replicating the recombinant
expression vector, the host cell is preferably a prokaryotic cell,
e.g., a DH5.alpha. cell. For purposes of producing a recombinant
modified TCR, polypeptide, or protein, the host cell is preferably
a mammalian cell. Most preferably, the host cell is a human cell.
The host cell can be of any cell type, can originate from any type
of tissue, and can be of any developmental stage.
[0062] Also provided by the invention is a population of cells
comprising at least one host cell described herein. The population
of cells can be a heterogeneous population comprising the host cell
comprising any of the recombinant expression vectors described, in
addition to at least one other cell, e.g., a host cell (e.g., a T
cell), which does not comprise any of the recombinant expression
vectors, or a cell other than a T cell, e.g., a B cell, a
macrophage, a neutrophil, an erythrocyte, a hepatocyte, an
endothelial cell, an epithelial cells, a muscle cell, a brain cell,
etc. Alternatively, the population of cells can be a substantially
homogeneous population, in which the population comprises mainly of
host cells (e.g., consisting essentially of) comprising the
recombinant expression vector. The population also can be a clonal
population of cells, in which all cells of the population are
clones of a single host cell comprising a recombinant expression
vector, such that all cells of the population comprise the
recombinant expression vector. In one embodiment of the invention,
the population of cells is a clonal population comprising host
cells comprising a recombinant expression vector as described
herein.
[0063] Included in the scope of the invention are conjugates, e.g.,
bioconjugates, comprising any of the inventive polypeptides
(including any of the functional fragments or functional variants)
or peptidomimetics, nucleic acids, recombinant expression vectors,
or host cells. Conjugates, as well as methods of synthesizing
conjugates in general, are known in the art (See, for instance,
Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et
al., Inorg Chem. 44(15): 5405-5415 (2005)).
[0064] The inventive polypeptides (including functional fragments
and functional variants), peptidomimetics, fatty acid derivatives,
nucleic acids, recombinant expression vectors, and host cells
(including populations thereof) can be isolated, purified,
synthetic, and/or recombinant. The term "isolated" as used herein
means having been removed from its natural environment. The term
"purified" as used herein means having been increased in purity,
wherein "purity" is a relative term, and not to be necessarily
construed as absolute purity. For example, the purity can be at
least about 50%, can be greater than 60%, 70%, 80%, or 90%, or can
be 100%.
[0065] The inventive polypeptides (including functional fragments
and functional variants), peptidomimetics, fatty acid derivatives,
conjugates, nucleic acids, recombinant expression vectors, and host
cells (including populations thereof), all of which are
collectively referred to as "inventive materials" hereinafter, can
be formulated into a composition, such as a pharmaceutical
composition. In this regard, the invention provides a
pharmaceutical composition comprising any of the polypeptides
(including functional fragments and functional variants),
peptidomimetics, fatty acid derivatives, conjugates, nucleic acids,
recombinant expression vectors, and host cells (including
populations thereof), and a pharmaceutically acceptable carrier.
The inventive pharmaceutical compositions containing any of the
inventive materials can comprise more than one inventive material,
e.g., a polypeptide and a nucleic acid, or two or more different
polypeptides. Alternatively, the pharmaceutical composition can
comprise an inventive material in combination with another
pharmaceutically active agent or drug, such as a chemotherapeutic
agent, e.g., asparaginase, busulfan, carboplatin, cisplatin,
daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,
methotrexate, paclitaxel, rituximab, vinblastine, vincristine,
etc.
[0066] In a preferred embodiment of the invention, the
pharmaceutical composition comprises the inventive material in
combination with a lipid. The lipid can be any lipid, including,
for example, a fatty acid, a phospholipid, a sterol, a
sphingolipid, a terpene, a glycerolipid, a glycerophospholipid, a
prenol lipid, a saccharolipid, and a polyketide. Such lipids are
known in the art. See, for example, Fahy et al., J. Lipid Res. 46:
839-861 (2005). Preferably, the lipid is a cholesterol.
[0067] With respect to pharmaceutical compositions, the
pharmaceutically acceptable carrier can be any of those
conventionally used and is limited only by chemico-physical
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.
[0068] The choice of carrier will be determined in part by the
particular inventive material, as well as by the particular method
used to administer the inventive material. Accordingly, there are a
variety of suitable formulations of the pharmaceutical composition
of the invention. The following formulations for oral, aerosol,
parenteral, subcutaneous, intravenous, intramuscular,
intraarterial, intrathecal, interperitoneal, rectal, and vaginal
administration are exemplary and are in no way limiting. More than
one route can be used to administer the inventive materials, and in
certain instances, a particular route can provide a more immediate
and more effective response than another route. In a preferred
embodiment of the invention, the pharmaceutical composition is a
topical formulation, an intravenous formulation, or a subcutaneous
formulation.
[0069] In a preferred embodiment of the invention, the
pharmaceutical composition is a topical formulation. Topical
formulations are well-known to those of skill in the art. Such
formulations are particularly suitable in the context of the
invention for application to the skin. The topical formulation of
the invention can be, for instance, a cream, a lotion, an ointment,
a patch, an oil, a paste, a spray, e.g., an aerosol spray, a gel, a
mousse, a roll-on liquid, a solid stick, etc. Preferably, the
topical formulation of the invention is a cream, a lotion, an
ointment, or a patch. When the topical formulation is a lotion,
preferably, the lotion also includes an ultraviolet (UV) light
blocking agent, such as tocopheryl, aminobenzoic acid, Avobenzone,
Cinoxate, dioxybenzone, homosalate, menthyl anthranilate,
octocrylene, octyl methoxycinnamate, octisalate, oxybenzone,
padimate O, phenylbenzimidazole, sulfonic acid, sulisobenzone,
titanium dioxide, trolamine salicylate, and zinc oxide.
[0070] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the inventive
material 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
other pharmacologically compatible excipients. Lozenge forms can
comprise the inventive material in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the inventive
material in an inert base, such as gelatin and glycerin, or sucrose
and acacia, emulsions, gels, and the like containing, in addition
to, such excipients as are known in the art.
[0071] The inventive material, 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.
[0072] 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 inventive material 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 or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol, ketals such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol)
400, oils, fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides 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.
[0073] 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.
[0074] 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-.beta.-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0075] The parenteral formulations will typically contain from
about 0.5% to about 25% by weight of the inventive material in
solution. 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 glycol 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.
[0076] Injectable formulations are in accordance with the
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)). Preferably, when
administering cells, e.g., dendritic cells, the cells are
administered via injection.
[0077] Additionally, the inventive materials, or compositions
comprising such inventive materials, 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.
[0078] It will be appreciated by one of skill in the art that, in
addition to the above-described pharmaceutical compositions, the
inventive materials of the invention can be formulated as inclusion
complexes, such as cyclodextrin inclusion complexes, or
liposomes.
[0079] For purposes of the invention, the amount or dose of the
inventive material administered should be sufficient to effect,
e.g., a therapeutic or prophylactic response, in the subject or
animal over a reasonable time frame. For example, the dose of the
inventive material should be sufficient to inhibit proliferation of
a diseased cell, or treat or prevent a disease (e.g., cancer,
neoplasm, or psoriasis in a period of from about 2 hours or longer,
e.g., 12 to 24 or more hours, from the time of administration. In
certain embodiments, the time period could be even longer. The dose
will be determined by the efficacy of the particular inventive
material and the condition of the animal (e.g., human), as well as
the body weight of the animal (e.g., human) to be treated.
[0080] Many assays for determining an administered dose are known
in the art. For purposes of the invention, an assay, which
comprises comparing the extent to which diseased cells are
inhibited from proliferating, upon administration of a given dose
of an inventive material to a mammal among a set of mammals of
which is each given a different dose of the inventive material,
could be used to determine a starting dose to be administered to a
mammal. The extent to which diseased cells are inhibited from
proliferating upon administration of a certain dose can be assayed
by methods known in the art, including, for instance, the methods
described herein as Example 2.
[0081] The dose of the inventive material also will be determined
by the existence, nature and extent of any adverse side effects
that might accompany the administration of a particular inventive
material. Typically, the attending physician will decide the dosage
of the inventive material with which to treat each individual
patient, taking into consideration a variety of factors, such as
age, body weight, general health, diet, sex, inventive material to
be administered, route of administration, and the severity of the
condition being treated. By way of example and not intending to
limit the invention, the dose of the inventive material can be
about 0.001 to about 1000 mg/kg body weight of the subject being
treated/day, from about 0.01 to about 10 mg/kg body weight/day,
about 0.01 mg to about 1 mg/kg body weight/day.
[0082] One of ordinary skill in the art will readily appreciate
that the inventive materials of the invention can be modified in
any number of ways, such that the therapeutic or prophylactic
efficacy of the inventive materials is increased through the
modification. For instance, the inventive materials can be
conjugated either directly or indirectly through a linker to a
targeting moiety. The practice of conjugating compounds, e.g.,
inventive materials, to targeting moieties is known in the art.
See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995)
and U.S. Pat. No. 5,087,616. The term "targeting moiety" as used
herein, refers to any molecule or agent that specifically
recognizes and binds to a cell-surface receptor, such that the
targeting moiety directs the delivery of the inventive materials to
a population of cells on which surface the receptor is expressed.
Targeting moieties include, but are not limited to, antibodies, or
fragments thereof, peptides, hormones, growth factors, cytokines,
and any other natural or non-natural ligands, which bind to cell
surface receptors (e.g., Epithelial Growth Factor Receptor (EGFR),
T-cell receptor (TCR), B-cell receptor (BCR), CD28,
Platelet-derived Growth Factor Receptor (PDGF), nicotinic
acetylcholine receptor (nAChR), etc.). The term "linker" as used
herein, refers to any agent or molecule that bridges the inventive
materials to the targeting moiety. One of ordinary skill in the art
recognizes that sites on the inventive materials, which are not
necessary for the function of the inventive materials, are ideal
sites for attaching a linker and/or a targeting moiety, provided
that the linker and/or targeting moiety, once attached to the
inventive materials, do(es) not interfere with the function of the
inventive materials, i.e., the ability to inhibit proliferation of
a diseased cell, or to treat or prevent disease (e.g., cancer,
neoplasm, psoriasis).
[0083] Alternatively, the inventive materials can be modified into
a depot form, such that the manner in which the inventive materials
is released into the body to which it is administered is controlled
with respect to time and location within the body (see, for
example, U.S. Pat. No. 4,450,150). Depot forms of inventive
materials can be, for example, an implantable composition
comprising the inventive materials and a porous or non-porous
material, such as a polymer, wherein the inventive materials is
encapsulated by or diffused throughout the material and/or
degradation of the non-porous material. The depot is then implanted
into the desired location within the body and the inventive
materials are released from the implant at a predetermined
rate.
[0084] It is contemplated that the inventive pharmaceutical
compositions, polypeptides (including functional fragments and
functional variants), peptidomimetics, fatty acid derivatives,
nucleic acids, recombinant expression vectors, host cells, or
populations of cells can be used in methods of inhibiting the
proliferation of a diseased cell. In this regard, the invention
provides a method of inhibiting proliferation of a diseased cell.
The method comprises contacting the diseased cell with any of the
pharmaceutical compositions described herein in an amount effective
to inhibit proliferation of the diseased cell.
[0085] In a preferred embodiment of the host, the diseased cell is
in a host. The host referred to herein can be any host. Preferably,
the host is a mammal. As used herein, the term "mammal" refers to
any mammal, including, but not limited to, mammals of the order
Rodentia, such as mice and hamsters, and mammals of the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Carnivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simoids (monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.
[0086] The diseased cell can be a cell characteristic of or
inflicted with any disease. The disease can be any disease,
condition, or malady, especially any of those caused by or
involving the proliferation of a cell. The disease can be, for
example, a cancer or a non-cancerous tumor, e.g., a cyst, a
neoplasm, a fibroma, etc.
[0087] The cancer can be any cancer, including any of acute
lymphocytic cancer, acute myeloid leukemia, a sarcoma, e.g.,
alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast
cancer, cancer of the anus, anal canal, or anorectum, cancer of the
eye, cancer of the intrahepatic bile duct, cancer of the joints,
glioma, cancer of the neck, gallbladder, or pleura, cancer of the
nose, nasal cavity, or middle ear, cancer of the oral cavity,
cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid
cancer, colon cancer, esophageal cancer, cervical cancer,
gastrointestinal carcinoid tumor. Hodgkin lymphoma, hypopharynx
cancer, kidney cancer, larynx cancer, liver cancer, lung cancer,
malignant mesothelioma, melanoma, multiple myeloma, nasopharynx
cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,
peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate
cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma
(RCC)), small intestine cancer, soft tissue cancer, stomach cancer,
testicular cancer, thyroid cancer, ureter cancer, and urinary
bladder cancer. Preferably, the cancer is breast cancer, prostate
cancer, ovarian cancer, stomach cancer (e.g., gastric
adenocarcinoma), colon cancer, liver cancer, melanoma, basal cell
carcinoma, rhabdomyosarcoma, medulloblastoma, pancreatic cancer,
lung cancer, thyroid cancer, a myeloma, a lymphoma, a glioma, or a
sarcoma.
[0088] As the proliferation of cells can cause a number of
diseases, it is further contemplated that the inventive materials
described herein can be used in methods of treating or preventing
these diseases. In this regard, the invention provides a method of
treating or preventing cancer or a neoplasm (e.g., eye neoplasm) in
a host. The method comprises administering to the host any of the
pharmaceutical compositions described herein in an amount effective
to treat the cancer or neoplasm.
[0089] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the inventive methods can provide any amount of
any level of treatment or prevention of cancer or a neoplasm in a
mammal. Furthermore, the treatment or prevention provided by the
inventive method can include treatment or prevention of one or more
conditions or symptoms of the disease, e.g., cancer, being treated
or prevented. Also, for purposes herein, "prevention" can encompass
delaying the onset of the disease, or a symptom or condition
thereof.
[0090] In a preferred embodiment of the inventive methods, the
pharmaceutical composition is topically administered to the host.
In another preferred embodiment, the pharmaceutical composition is
administered directly to the tumor, e.g., delivered
intratumorally.
[0091] The invention furthermore provides a method of treating
psoriasis in a host comprising administering to the host any of the
pharmaceutical compositions described herein in an amount effective
to treat psoriasis in the host. Psoriasis is a common skin disease
characterized by thickened patches of inflamed, red skin covered
with thick, silvery scales. The psoriasis can be any form of
psoriasis including, for example, plaque psoriasis, or psoriasis
vulgaris, pustular psoriasis, guttate psoriasis, and inverse
psoriasis.
[0092] The invention also provides a method of inhibiting the
Hedgehog signal transduction pathway. The method comprises
contacting the diseased cell with any of the pharmaceutical
compositions described herein in an amount effective to inhibit the
Hedgehog signal transduction pathway. Since expression of certain
genes are activated for transcription upon activation of the
Hedgehog signal transduction pathway, the invention also provides a
method of inhibiting the expression of these genes in a diseased
cell. The gene can be one or a combination of: Gli-1 (e.g., GenBank
Accession No. NM.sub.--005269), Gli-2 (e.g., GenBank Accession No.
NM.sub.--005270), Gli-3 (e.g., GenBank Accession
No._NM.sub.--001034190), Ptch (e.g., GenBank Accession No.
NM.sub.--0000264), Shh (e.g., GenBank Accession No.
NM.sub.--000193), Smo (e.g., GenBank Accession No.
NM.sub.--005631), or NES (e.g., GenBank Accession No.
NM.sub.--016701), which genes are known in the art. The method of
inhibiting the expression of these genes comprises contacting the
diseased cell with any of the pharmaceutical compositions described
herein in an amount effective to inhibit the expression of the
gene.
[0093] For purposes herein, when a cell, e.g., a diseased cell is
contacted with a pharmaceutical composition comprising a nucleic
acid or recombinant expression vector, the method involves the
expression of the nucleic acid such that the encoded polypeptide
(or functional fragment or functional variant) is expressed inside
of the cell. When a cell, e.g., a diseased cell is contacted with a
pharmaceutical composition comprising a host cell (or a population
thereof), the method involves the expression of the nucleic acid
inside of the host cell and the secretion of the encoded
polypeptide (or functional fragment or functional variant) outside
of the host cell where the polypeptide is then available to contact
the diseased cell.
[0094] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0095] This example demonstrates a method of preparing polypeptides
(including functional fragments and functional variants) in
accordance with an embodiment of the invention.
[0096] Polypeptides having the amino acid sequences as set forth in
Table 1 are synthesized by solid phase peptide synthesis on a 433A
Peptide Synthesizer (Applied Biosystems, Foster City, Calif.)
equipped with a conductivity monitoring unit utilizing Fmoc amino
acid derivatives (AnaSpec, San Jose, Calif.). The synthesis is
performed with conditional blocking of unreacted amino groups with
acetic anhydride for easier purification of the resulting peptides.
Peptides are cleaved from the resin with 87.5% trifluoroacetic acid
containing 5% water, 5% thioanisol and 2.5% triisopropyl-silane,
precipitated with cold diethyl ether, washed five times with ether
and dried in vacuum overnight. Peptides dissolved in
dimethylformamide are purified by HPLC on a preparative
(25.times.250 mm) Atlantis C18 reverse phase column (Agilent, Palo
Alto, Calif.) in a gradient of 0.05% trifluoroacetic acid in water
and acetonitrile containing 0.05% trifluoroacetic acid. The
fractions are analyzed by electrospray LC/MS on Agilent 1100 series
instrument (Agilent Technologies, Palo Alto, Calif.) with the use
of Zorbax 300SB-C18 Poroshell column and a gradient of 5% acetic
acid in water and acetonitrile. Only fractions containing more than
95% pure product are combined and freeze-dried. Peptides are dried
from 5% acetic acid to ensure conversion into acetate salts. The
purity and structure are further confirmed by LC/MS with separation
on Zorbax 300SB-C18 analytical column.
TABLE-US-00001 TABLE 1 SEQ Peptide ID Name Amino acid sequence NO:
SMO-i3-1 PalRGVMTLFSIKSNHPGLLSEKAASKINETMLR 4 SMO-i3-2
PalRGVMTLFSIKSNHPGLLSEKA 9 SMO-i3-4 PalLFSIKSNHPGLLSEKAASKINETMLR
10 SMO-i3-5 RGVMTLFSIKSNHPGLLSEKAASKINETMLRK-.epsilon.
.quadrature.Pal 60 SMO-i3-6 LLSEKAASKINETMLRK-.quadrature.
.epsilon.-Pal 61 SMO-i3-7 LFSIKSNHPGLLSEKAASKINETMLRK-.quadrature.
.epsilon.-Pal 62 SMO-i3-8 PalRGVMTLFSIKSNHPGLLS 14 SMO-i3-9
PalHseARGVMTLFSIKSNHPGLLS 77 SMO-i3-10 PalRGVMTLFSIKSNH 15
SMO-i3-12 SEKAASKINETMLRK-.quadrature. .epsilon.-Pal 63 SMOi2-1
PalLTYAWHTSFKALGTTYQPLSGKYSY 3 SMOi2-2 PalLTYAWHTSFKALGTTYQPLSGKTSY
17 SMOi2-3 PalLTYAWHTSFKALGTTYQPLSG 18 SMOi2-4
AcLTYAWHTSFKALGTTYQPLSGKTSYK-.epsilon.-Pal 64 SMOi2-5
AcYAWHTSFKALGTTYQPLSGKTSYK-.quadrature. .epsilon.-Pal 65 SMOi2-6
PalLTYAWHTSFKALGTTYQP 21 SMOi2-7 GTTYQPLSGKTSYK-.quadrature.
.epsilon.-Pal 66 SMOi2-8 PalLTYAWHTSFKAL 23 SMOi2-9 AcLTYAWHTSFKAL
24 SMOi2-10 PalTYAWHTSFKAL 25 SMOi2-11 PalLTYAWHTSFKA 26 SMOi2-12
PalLTYAWHTSFK 27 SMOi2-13 AcTYAWHTSFKA 28 SMOi2-14
VWFVVLTYAWHTSFKAL 55 SMOi2-15 WFVVLTYAWHTSFKAL 56 SMOi2-16
AcLAKFSTHWAYTLK(.epsilon.-Pal)-All-D 67 SMOi2-17
AcAKFSTHWAYTLK(.epsilon.-Pal)-All-D 68 SMOi2-18 PalLTYABpaHTSFKAL
54 SMOi2-20 AcKFSTHWAYTLK(.epsilon.-Pal)All-D 69 SMOi2-21
Pal-LTYABpaHTSFKAL-Hcy-Biotin 81 SMOi2-22 AKFSTHWAYTL (All-D) 37
SMOi2-23 PalLTYAWHTSFKALGTTYQPLSGKTSYK(.epsilon.-Pal) 70 SMOi2-24
PalLTYAWHTSFKAL (All-D) 30 SMOi2-25 AcLTYAWHTSFKAL (All-D) 31
SMOi2-26 MyrLTYAWHTSFKAL 32 SMOi2-29 Ac-LTYAWHTSFKAL-Penetratin 82
SMOi2-30 Penetratin-LTYAWHTSFKAL 83 SMOi2-56 ##STR00003## 84
SMOi2-57 D-(LAKFSTHWAYTL)-K-(.epsilon.-Pal)-LTYAWHTSFKAL 92
SMOi2-58 D-(AKFSTHWAYTL)-K-(.epsilon.-Pal)-LTYAWHTSFKAL 93 SMOi2-59
D-(AKFSTHWAYTL)-K-(.epsilon.-Pal)-LTYAWHTSFKA 94 Pal = palmitate;
"all D" = each amino acid of the polypeptide is the D isomer; Ac =
acetate; Myr = myristate; PalHse = homoserine palmitate; Bpa =
4-benzoylphenylalanine; and HCy-Biotin, homocysteine-Biotin, in
which biotin is attached to the SH of homocysteine.
[0097] The peptides described herein can be made into a dimeric
form having the following general structure:
##STR00004##
wherein Sequence X is selected from the group consisting of
Ac-LAKFSTHWAYTL (all-D) (SEQ ID NO: 85); Ac-AKFSTHWAYTL (All-D)
(SEQ ID NO: 86); and Ac-KFSTHWAYTL (All-D) (SEQ ID NO: 87); wherein
each of Linker 1 and Linker 2 is optionally present and each
independently is Gly, beta-Ala, aminopropionic acid,
gamma-aminobutyric acid, aminocaproic acid, or aminohexanoic acid;
wherein n and m is between 0 and 6; wherein Y is K, C, homoCys,
Orn, diaminopropanoic acid (DPA), diaminobutyric acid (DBA); and
wherein the fatty acid is a stearic, palmitic, myristic, lauric,
capric or caprilic acid. In a preferred embodiment, Sequence X is
SEQ ID NO: 87, n is 4, m is 0, each of Linker 1 and Linker 2 is
beta-Ala, and the fatty acid is palmitate.
[0098] For the synthesis of such dimeric inhibitors, wherein Y is
Lys, resin preloaded with Fmoc-Lys with fatty acid attached to the
c-amino group is reacted with a corresponding diamino acid (e.g.,
Orn, Lys, diaminobutyric acid (DBA), or diaminopropionic acid
(DPA)) that has an Fmoc protection group on one amino group and a
DDE protection group on the other amino group. The DDE group is
selectively removed with a mixture of hydroxylamine and imidazole
in DMF. The resulting resin is coupled to a linker amino acid
(e.g., Fmoc-Gly, beta-Ala, aminopropionic acid, gamma-aminobutyric
acid, aminocaproic acid, or aminohexanoic acid) on an ABI433
peptide synthesizer. The remainder of each Sequence X is
simultaneously built on a peptide synthesizer using a standard
synthetic protocol. The dimeric product is cleaved, deprotected and
purified as in a standard synthetic protocol. SMOi2-56, which is
the dimeric form of SMOi2-17, is made in this manner.
[0099] SMOi2-29 and -30 are peptides based on SMOi2-9 fused to
penetratin, which is a peptide from Antennapedia used to introduce
a variety of biologically active molecules, such as DNA, peptides,
or proteins into cells (Granier et al., J. Biol. Chem. 279:
50904-50914 (2004)). Penetratin has the amino acid sequence
RQIKIWFPNRR-Nle-KWKK (SEQ ID NO: 78). Penetratin-containing
peptides are made as a single peptide chain using standard peptide
synthesis methods.
[0100] Polypeptides are lipidated as follows: for L-peptides
containing s-palmitoyl-Lys on the C-terminus, commercially
available Fmoc-s-palmitoyl-L-Lys (AnaSpac, San Jose, Calif.) is
utilized. Fmoc-s-palmitoyl-D-Lys is not commercially available. It
is synthesized on the resin utilizing orthogonally protected
Fmoc-D-Lys(ivDDE)
(N-.alpha.-Fmoc-N-.epsilon.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)--
3-methylbutyl-D-lysine) (Novabiochem, San Diego, Calif.). After
attachment of the amino acid to Rink-amide resin, ivDDE protection
group is removed by treatment with hydrazine/imidazole mixture in
NMP. The resin is washed with NMP and reacted with 10-fold excess
of palmitic acid/HBTU/HOBt in NMP for two hours. After washing of
the resin with NMP, the synthesis is continued utilizing standard
protocols on the peptide synthesizer. For peptides comprising
myristic acid or acetate at the N-terminus, the corresponding fatty
acid (10-fold excess) was dissolved in NMP or NMP/DCM mixture,
activated with HBTU/HOBt mixture and reacted with the peptide on
the resin. Subsequent cleavage and deprotection was carried out as
was done for lipidations with palmitic acid.
[0101] The molecular mass of each peptide is determined by
ion-spray mass spectrometry utilizing an Agilent1100 LC/MS system
(Agilent, Santa Clara, Calif.) and is shown in Tables 2 and 3.
TABLE-US-00002 TABLE 2 Mass Mass Compound (calculated) (found)
t.sub.R.sup.a(min) Purity SMO i2-1 3121.7 3121.0 16.67 95% SMO i2-2
3059.6 3059.0 16.51 95% SMO i2-3 2580.0 2580.0 17.13 96% SMO i2-4
3015.5 3015.0 16.12 95% SMO i2-5 3229.8 3230.0 16.34 95% SMO i2-6
2225.6 2225.5 17.13 96% SMO i2-7 1646.9 1647.0 18.48 98% SMO i2-8
1675.1 1675.0 17.59 96% SMO i2-9 1478.6 1478.0 14.64 100% SMO i2-10
1561.9 1562.0 18.65 98% SMO i2-11 1561.9 1562.0 18.99 98% SMO i2-12
1490.8 1491.0 19.04 97% SMO i2-13 1294.4 1294.0 13.97 99% SMO i2-14
2109.4 2109.0 18.46 96% SMO i2-15 2010.3 2010.0 17.86 96% SMO i2-16
1845.2 1845.0 18.96 97% SMO i2-17 1732.1 1732.0 18.63 97% SMOi2-18
1740.1 1740.0 17.75 96% SMOi2-20 1661.0 1661.0 17.11 98% SMOi2-21
2292.3 2292.0 17.08 96% SMOi2-23 3426.2 3426.0 18.00 95% SMOi2-24
1675.1 1674.9 17.01 96% SMOi2-25 1477.9 1477.9 13.85 98% SMOi2-29
3689.4 3689.0 13.95 95% .sup.aThe retention times are for Zorbax
300SB-C3 column (Agilent, Santa Clara, CA) determined in 0-100% 25
min gradient of 0.5% acetic acid in water and 0.5% acetic acid in
acetonitrile, flow rate of 0.3 mL per minute.
TABLE-US-00003 TABLE 3 Mass Mass Compound (calculated) (found)
t.sub.R.sup.a(min) Purity SMOi3-1 3630.9 3630.9 16.08 95% SMOi3-2
2504.1 2504.4 16.42 96% SMOi3-4 3104.8 3104.4 16.69 95% SMOi3-5
3801.0 3801.0 15.87 95% SMOi3-6 2193.4 2193.4 15.26 96% SMOi3-7
3274.9 3274.5 16.08 95% SMOi3-8 2176.7 2176.2 17.05 96% SMOi3-10
1709.1 1708.8 17.23 97% SMOi3-12 1967.4 1967.2 16.09 97% .sup.aThe
retention times are for Zorbax 300SB-C3 column (Agilent, Santa
Clara, CA) determined in 0-100% 25 min gradient of 0.5% acetic acid
in water and 0.5% acetic acid in acetonitrile, flow rate of 0.3 mL
per minute.
[0102] HPLC of the peptides is performed on a Microsorb-MW 300A C8
column (Varian, Palo Alto, Calif.) in 0-100% 20 min gradient of
0.1% trifluoroacetic acid in water/acetonitrile containing 0.1%
trifluoroacetic acid, flow rate 1 ml/min. Peptides are detected by
UV monitoring at 225, 256, and 280 nm. Data not shown.
Example 2
[0103] This example demonstrates a method of testing the inventive
polypeptides for toxicity.
[0104] DU145 prostate cancer cells, PC3 prostate cancer cells, MCF7
breast cancer cells, or MeI-SK-2 melanoma cells (American Type
Culture Collection, Manassas, Va.) are inoculated in 96 well plates
at 200-400 cells/well density in DMEM medium containing 10% fetal
bovine serum and allowed to attach for 24 hours. Cell suspension of
100 .mu.l is used for each well. Polypeptides in 100 .mu.l medium
at 2.times. concentration are added the next day and kept in the
CO.sub.2 incubator for 48 hours. While the polypeptides are added
at a final concentration between 1 nM and 10 .mu.M, assays are
performed on extra reference plates to determine the cell
population density at time 0 (T.sub.0). The cells are stained with
Promega Non-Radioactive Cell Proliferation Assay Kit (MTT)
according to manufacture's protocol. The absorbance of the wells is
determined at 544 nm by a FLUOstar/POLARstar.RTM. Galaxy
MicroplateReader (BMG Labtechnologies GmbH, Germany). The assays
are performed on control (C) and test (T) cells. Cellular responses
are 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-T.sub.0)/T.sub.0] for T<T.sub.0.
Example 3
[0105] This example demonstrates that polypeptides in accordance
with embodiments of the invention are able to inhibit proliferation
of diseased cells.
[0106] Polypeptides corresponding to the full lengths of all three
intracellular loops of SMO (SMOi1-1, SMOi2-1, SMOi3-1) having an
N-terminal palmitoyl residue are constructed as described in
Example 1. The polypeptides are then tested for toxicity (growth
inhibition) as described in Example 2 using MCF-7 breast cancer
cells and gastric adenocarcinoma cells. Activity of SMOi2-1 and
SMOi3-1 is compared to that of cyclopamine (5 .mu.M), a teratogen
isolated from the corn lily Veratrum califonicum.
[0107] As shown in FIG. 1, all three peptides inhibit the growth of
MCF-7 cells. The SMOi3-1 polypeptide has the most significant
effect on cell growth, followed by SMOi2-1, while SMOi1-1
demonstrates the least amount of inhibitory activity. As shown in
FIG. 2, SMOi3-1 and SMOi2-1 polypeptides are able to inhibit the
growth of gastric adenocarcinoma cells as well or better than
cyclopamine.
Example 4
[0108] This example demonstrates that functional fragments and
functional variants having an amino acid sequence based on the
second and third intracellular loops of the SMO protein in
accordance with an embodiment of the invention are able to inhibit
proliferation of diseased cells.
[0109] Polypeptides based on the second or third intracellular loop
of SMO (SMOi2 or i3 polypeptides) (as shown in Table 1) are
synthesized as described in Example 1 and are tested as described
in Example 2 using MCF-7 breast cancer cells or SK-Mel2 melanoma
cells. The IC.sub.50 of each peptide as determined by the MTT assay
in SK-Mel2 melanoma cells after 48 hour exposure to the peptide is
shown in Tables 4 and 5.
TABLE-US-00004 TABLE 4 Compound Structure IC.sub.50, .mu.M SMO i2-1
Pal-LTYAWHTSFKALGTTYQPLSGKYSY 0.45 .+-. 0.05 SMO i2-2
Pal-LTYAWHTSFKALGTTYQPLSGKTSY 0.45 .+-. 0.05 SMO i2-3
Pal-LTYAWHTSFKALGTTYQPLSG 1.4 .+-. 0.4 SMO i2-4
Ac-LTYAWHTSFKALGTTYQPLSGKTSYK-.epsilon.-Pal 1.0 .+-. 0.1 SMO i2-5
Ac-YAWHTSFKALGTTYQPLSGKTSYK-.epsilon. Pal 1.0 .+-. 0.1 SMO i2-6
Pal-LTYAWHTSFKALGTTYQP 0.3 .+-. 0.05 SMO i2-7
Ac-GTTYQPLSGKTSYK-.epsilon. Pal 2.7 .+-. 0.4 SMO i2-8
Pal-LTYAWHTSFKAL 0.08 .+-. 0.02 SMO i2-9 Ac-LTYAWHTSFKAL >10 SMO
i2-10 Pal-TYAWHTSFKAL 0.7 .+-. 0.1 SMO i2-11 Pal-LTYAWHTSFKA 0.09
.+-. 0.007 SMO i2-12 Pal-LTYAWHTSFK 0.06 .+-. 0.007 SMO i2-13
Ac-TYAWHTSFKA 2.8 .+-. 0.3 SMO i2-14 VWFVVLTYAWHTSFKAL >5 SMO
i2-15 WFVVLTYAWHTSFKAL >5 SMO i2-16
Ac-LAKFSTHWATYLK-.epsilon.-Pal (all D-) 0.006 .+-. 0.0005 SMO i2-17
Ac-AKFSTHWATYLK-.epsilon.Pal (all D-) 0.0004 .+-. 0.0001 SMO i2-18
Pal-LTYABpaHTSFKAL 0.1 .+-. 0.05 SMO i2-20
Ac-KFSTHWATYLK-.epsilon.PaL (all D-) 0.0003 .+-. 0.0001 SMO i2-21
Pal-LTYABpaHTSFKAL-Hcy-Biotin >15 SMO i2-23
Pal-LTYAWHTSFKALGTTYQPLSGKTSYK-.epsilon.-Pal 0.05 .+-. 0.02
SMOi2-24 PalLTYAWHTSFKAL (All D) 0.039 .+-. 0.004 SMOi2-25
AcLTYAWHTSFKAL (All D) >10 SMO i2-26 Myr-LTYAWHTSFKAL 0.2 .+-.
0.05 SMO i2-29 Ac-LTYAWHTSFKAL-Penetratin >15 SMO i2-30
Penetratin-LTYAWHTSFKAL >15 SMOi2-56 ##STR00005## 4.0 nm Pal,
palmitate; Ac, acetate; (All D), each amino acid of the polypeptide
is the D isomer; Myr, myristic acid; .epsilon.-Pal, palmitate added
on the .epsilon. carbon of Lys; (Bpa), 4-benzoylphenylalanine.
TABLE-US-00005 TABLE 5 Compound Structure IC.sub.50,.mu.M SMO i3-1
PalRGVMTLFSIKSNHPGLLSEKAASKINETML 0.64 .+-. 0.1 SMO i3-2
PalRGVMTLFSIKSNHPGLLSEKA 0.50 .+-. 0.1 SMO i3-4
PalLFSIKSNHPGLLSEKAASKINETMLR 1.5 .+-. 0.2 SMO i3-5
AcRGVMTLFSIKSNHPGLLSEKAASKINETMLRK- .epsilon. -Pal 0.9 .+-. 0.2 SMO
i3-6 Ac-LLSEKAASKINETMLRK-.quadrature..epsilon.-Pal 0.8 .+-. 0.1
SMO i3-7 Ac-LFSIKSNHPGLLSEKAASKINETMLRK-.quadrature. .epsilon. -Pal
0.95 .+-. 0.2 SMO i3-8 PalRGVMTLFSIKSNHPGLLS 0.5 .+-. 0.1 SMO i3-10
PalRGVMTLFSIKSNH 0.95 .+-. 0.2 SMO i3-12
Ac-LLSEKAASKINETMLRK.quadrature. .epsilon. -Pal 1.33 .+-. 0.2
[0110] As shown in Table 5 and FIG. 3, polypeptides based on the
third intracellular loop of SMO exhibit the ability to inhibit the
growth of MCF-7 cells. Also, peptides corresponding to fragments of
the third intracellular loop have activities that are comparable or
lower than the full-length loop (SMOi3-1).
[0111] As shown in Table 4, several of the polypeptides based on
the second intracellular loop of SMO (SMOi2 polypeptides) are able
to inhibit the growth of SK-Mel2 melanoma cells after 48 hours of
exposure to the polypeptides. Among the most potent inhibitors are
SMOi2-16, SMOi2-17, SMOi2-8, SMOi2-23, SMOi2-24, SMOi2-20,
SMOi2-26, SMOi2-11, and SMOi2-12. Also, SMOi2-6, SMOi2-7, SMOi2-2
through SMOi2-5, SMOi2-10, and SMOi2-13 are potent inhibitors.
C-terminal truncation of the second intracellular loop yields
polypeptides that were significantly more toxic to cancer cells
than the full-length loop (SMOi2-1). Both halves of the loop when
palmitoylated at the amino acids which are positioned at the end of
the loop (which end is adjacent to the membrane in the wild-type
SMO protein) are active. However, C-terminal extension of the
N-terminal half lowers the activity of the most potent 12-residue
long polypeptide (compare peptides SMOi2-8 with SMOi2-6 and
SMOi2-3).
[0112] Palmitic acid lipidation of the polypeptides appears to be
essential for the activity, since substitution of palmitate with an
acetyl residue significantly reduces the inhibitory activity
(compare SMOi2-9 and SMOi2-13). This is likely due to poor cell
penetration of the polypeptide. It appears necessary for the
palmitoylation to occur at the end of the loop (which end is
adjacent to the membrane in the wild-type SMO protein), since
positioning the palmitoyl group inside the loop generated a
significantly less active peptide (SMOi3-4 of Table 5). The growth
inhibition curves of these polypeptides either plateau at higher
concentrations or curve upward, indicating that inhibitory activity
actually decreases at higher concentrations. SMOi3-1, SMOi3-8,
SMOi3-2, SMOi3-6, and SMOi3-12 are among the most potent
polypeptides tested.
[0113] Unlike SMOi3 polypeptides, all second loop derivatives have
"normal" concentration-dependence profiles of growth inhibition
activity.
[0114] As an alternative delivery of the peptides inside the cells,
SMOi2-9 is fused to penetratin. Neither C-terminal nor N-terminal
fusion helps to restore the activity, suggesting that
palmitoylation provides more than just cell permeability. Also, the
replacement of palmitoyl residue with sequences of the
transmembrane domain of the SMO protein does not overcome the loss
of activity (SMOi2-14 and SMOi2-15). The lack of activity may be
due to the fact that these polypeptides have poor solubility.
Substitution of palmitoyl residue with slightly shorter myristoyl
resulted in 2.5-fold less potent compound (SMOi2-26). For studying
peptide localization inside the cells and characterization of the
interacting protein molecules, synthesis of cross-linkable
derivative labeled with biotin is attempted. Substitution of Trp
residue of SMOi2-8 with p-benzoyl-phenylalanine that can be UV
cross-linked to a protein ligand produces a fairly active compound
(SMOi2-18, Table 4). However, addition of maleimide-biotin coupled
through SH-group of C-terminal homocysteine (SMOi2-21) totally
abolishes the activity, thus rendering it unsuitable for receptor
identification.
[0115] The retroinverso analogues of SMOi2 polypeptides exhibit
inhibitory activity. Both SMOi2-16, which is the retroinverso
analogue of SMOi2-8, and its truncated version SMOi2-17, which is
the retroinverso analogue of SMOi2-11 (and SMOi2-12), are more
potent in inhibiting and killing melanoma cells than their all-L
parent polypeptide (FIGS. 4 and 7).
Example 5
[0116] This example demonstrates a method of inhibiting the gene
expression of Hedgehog signaling pathway proteins in cells in
accordance with an embodiment of the invention.
[0117] An analysis of expression of the genes that are known
markers of the Hedgehog signal transduction pathway, Gli-1, Gli-2,
Gli3, Ptch, Shh, Smo and NES is performed. DU145 prostate cancer
cells are exposed to SMOi3-1 (5 or 10 .mu.M), SMOi2-1 (5 or 10
.mu.M) or cyclopamine (5 .mu.M) for 48 hours. Gene expression is
assayed by quantitative PCR. For gene expression assay, DU145
prostate cancer cells were exposed to 5 .mu.M SMOi3-1 for 24 h, and
5 .mu.M and 10 .mu.M SMOi3-1 for 48 h, respectively. DU145 cells
were treated by 5 .mu.M and 10 .mu.M of SMOi2-1 for 24 h only. The
control was DU145 cells without compounds-treatment. The 5 .mu.M of
cyclopamine was always used as positive control in all
experiments.
[0118] Total cellular RNA was isolated, and further purified by
RNeasy.RTM. columns (QIAGEN, Valencia, Calif.) according to the
manufacturer's instructions. RNA quality and quantity were
determined using Agilent RNA 6000 Nano Chip (Agilent Technologies,
Inc., CA). cDNA synthesis was carried out using Random Hexamer
primer, TaqMan.RTM. Reverse Transcription Reagents kit (Applied
Biosystems, Foster, Calif.).
[0119] Taqman.RTM. Gene Expression Assay primer and probe
(FAM-labeled) sets (Applied Biosystems, Foster, Calif.) are used
for real-time quantitative PCR analysis of PTCH (Assay
ID=Hs00181117_ml), GUI (Hs00171790_ml), GLI2 (Hs00257977_ml), GLI3
(Hs00609233_ml), SMO (Hs00170665_ml), SHH (Hs00179843_ml) and NES
(Hs00707120_s1). TaqMan.RTM. Gene Expression Assay mix of primer
and probe (VIC-labeled) of 18S rRNA was used as the endogenous
control. Each sample is run in triplicate. Triplicate Ct values
were analyzed using the comparative Ct (.DELTA..DELTA.Ct) method as
described by the manufacturer (Applied Biosystems, Foster, Calif.).
The relative amount of target (2.sup.-.DELTA..DELTA.Ct) is obtained
by normalization to an endogenous reference (18s rRNA).
[0120] As shown in FIG. 5, the changes in gene expression are more
pronounced than that of SMO antagonist, cyclopamine. This is
consistent with the fact that the polypeptides have a much higher
potency than cyclopamine.
Example 6
[0121] This example demonstrates that inventive peptidomimetics in
accordance with embodiments of the invention inhibit the
proliferation of diseased cells.
[0122] Peptides SMOi2-18 and SMOi2-21 are made as essentially
described in Example 1 and tested as described in Example 2 using
SK-Mel2 cells. Cells are exposed to polypeptides or peptidomimetics
for 60 hours.
[0123] As shown in FIG. 6, peptidomimetics, SMOi2-18 and SMOi2-21,
each of which contains the synthetic amino acid, BPA, exhibit the
ability to inhibit the proliferation of SK-Mel2 cells. The
inhibitory activity is even more potent than that of the
polypeptide counterpart, SMOi2-8, which contains only
naturally-occurring amino acids.
Example 7
[0124] This example demonstrates the critical micelle concentration
of SMOi2-8.
[0125] To monitor the formation of hydrophobic nanoparticles, a
fluorescing imidazoacridone compound WMC-77
(5-{3-[4-(aminopropyl)-piperazin-1-yl]-propylamino}-2,10b-diaza-aceanthry-
len-6-one) is used (Tarasov et al., Photochem. Photobiol. 78:
313-322 (2003)). Compounds like WMC-77 tend to enhance their
intrinsic fluorescence dramatically when entering an amphiphilic
environment of biological macromolecules like DNA (Tarasova et al.,
2003, supra) or hydrophobic core of typical micelles (Tarasov et
al., Photochem. Photobiol. 70: 568-578 (1999)). Since
imidazoacridones are adsorbed on quartz from the aqueous solutions,
plastic polymethacrylate 10.times.10 mm cells from Sigma-Aldrich
(St. Louis, Mo.) are used for most measurements. Atmospheric oxygen
quenching is found to be unimportant, since similar values of
fluorescence intensity from BIAs are obtained before and after
nitrogen purging. The solutions are prepared in deionized water.
Uncorrected fluorescence emission spectra are obtained at
25.degree. C. on a Single Photon Counting Spectrofluorometer
FLUOROMAX.RTM.-2 (Horiba Jobin Yvon, Edison, N.J.). The excitation
and emission monochromator slits are adjusted to 1.5 and 3.5 nm
bandwidth, respectively. The emission spectra (increment 1 nm,
integration time 0.2 sec.) are collected at the range 450-700 nm,
using 430 nm excitation monochromator setting. The fluorometric
measurements are performed for premixed aliquots of peptide and
imidazoacridone solutions. The concentration of fluorescing agent
WMC-77 in all probes is 0.4 .mu.M.
[0126] The fluorescence data are presented in FIG. 8. The increase
of peptide/fluorophore ratio causes the permanent increase of
WMC-77 fluorescence emission intensity, leveling of at .about.2
.mu.M. The changes in the fluorescence signal are similar to those
observed during transfer of imidazoacridones from aqueous to
non-polar media such as organic solvents (Tarasov et al., 1999,
supra), to the cores of classic surfactant micelles (Tarasov et
al., 1999, supra) or upon binding to DNA (Tarasov et al., 2003,
supra). The critical micelle concentration, estimated as described
in (Tanford, The Hydrophobic Effect: Formation of Micelles and
Biological Membranes, John Wiley & Sons, New York (1980)), is
determined as 0.5-1 .mu.M of SMOi2-8.
[0127] Critical micelle concentration is tested as described above
for all other peptides of this study and is found to be around 1
.mu.M for each peptide. Micellization may be responsible for
lowering the effective concentration of free peptides in solution
and subsequent apparent reduction in potency. The majority of
peptides also precipitated out in medium at concentration higher
than 10 .mu.M.
Example 8
[0128] This example demonstrates that the peptides of the invention
exhibit different sensitivities toward different cell lines.
[0129] Cancer cells of the breast (T47D), melanoma (SK-MEL-2),
hepatoma (HepG2, PLC, JM-1), pancreas (Panc10.05, HS766T), colon
(Colo205, HCT15), and lung (A549) are cultured in medium containing
either SMOi2-12 or SMOi2-20 at a concentration of 0.001, 0.01, 0.1,
1.0, or 5 .mu.M. Cells are assayed as described in Example 2. As
shown in FIG. 9, SMOi2-12 and SMOi2-20 exhibit different
sensitivities depending on the cell treated. Cell lines exhibiting
a GI.sub.50>5 include PLC, JM-1, HS766T, Colo205, HCT15, and
A549.
Example 9
[0130] This example demonstrates the peptides of the invention have
secondary structure.
[0131] The peptides are measured by circular dichroism (CD)
spectroscopy. Peptide solutions (1 .mu.M) are prepared by
dissolving compounds in PBS containing 50 mM dodecylphophocholine
(Avanti Polar Lipids, Alabaster, Ala.). CD spectra are recorded by
an AVIV mod. 202 CD-spectrometer (Aviv Instruments, Lakewood, N.J.)
using 0.1 cm path length quartz cuvette at 22-24.degree. C. Scan
ranges are between 180 and 260 nm and the spectrum of the buffer is
subtracted from the spectrum of the compound.
[0132] The CD spectra of SMOi2-8 and SMOi2-16 (FIG. 10) demonstrate
that the peptides predominantly adopt a beta-strand conformation.
The retro-inverso peptides appear to be more structured and rigid
than the parent all-L counterparts.
Example 10
[0133] The example demonstrates alanine scanning studies of the
polypeptides of the invention.
[0134] The significance of different residues in the SMOi2-8
sequence (PalLTYAWHTSFKAL) is probed by creating a collection of
mutants of the SMOi2-8 peptides in which each mutant of the
collection has an amino acid residue substituted with Ala and every
residue of SMOi2-8 is targeted for mutation by at least one of the
mutants in the collection.
[0135] The Lys residue at the 10th position of SMOi2-8 is critical
for the activity of the SMOi2-8 peptide. Significant loss in
activity also is observed upon substitution of the Ser at position
8 of SMOi2-8. Activity increased when the Leu at position 1 was
replaced with Ala. The Phe at position 9, Tyr at position 3, and
Trp at position 5 can be substituted with Ala without significant
change in the activity. The remaining substitutions (2, 4, 6, 7,
11, and 12) result in a slight (40-60%) increase in GI.sub.50
[0136] 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.
[0137] 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.
[0138] 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
941787PRTHomo sapiensMISC_FEATUREGenbank NP_005622 1Met Ala Ala Ala
Arg Pro Ala Arg Gly Pro Glu Leu Pro Leu Leu Gly 1 5 10 15 Leu Leu
Leu Leu Leu Leu Leu Gly Asp Pro Gly Arg Gly Ala Ala Ser 20 25 30
Ser Gly Asn Ala Thr Gly Pro Gly Pro Arg Ser Ala Gly Gly Ser Ala 35
40 45 Arg Arg Ser Ala Ala Val Thr Gly Pro Pro Pro Pro Leu Ser His
Cys 50 55 60 Gly Arg Ala Ala Pro Cys Glu Pro Leu Arg Tyr Asn Val
Cys Leu Gly 65 70 75 80 Ser Val Leu Pro Tyr Gly Ala Thr Ser Thr Leu
Leu Ala Gly Asp Ser 85 90 95 Asp Ser Gln Glu Glu Ala His Gly Lys
Leu Val Leu Trp Ser Gly Leu 100 105 110 Arg Asn Ala Pro Arg Cys Trp
Ala Val Ile Gln Pro Leu Leu Cys Ala 115 120 125 Val Tyr Met Pro Lys
Cys Glu Asn Asp Arg Val Glu Leu Pro Ser Arg 130 135 140 Thr Leu Cys
Gln Ala Thr Arg Gly Pro Cys Ala Ile Val Glu Arg Glu 145 150 155 160
Arg Gly Trp Pro Asp Phe Leu Arg Cys Thr Pro Asp Arg Phe Pro Glu 165
170 175 Gly Cys Thr Asn Glu Val Gln Asn Ile Lys Phe Asn Ser Ser Gly
Gln 180 185 190 Cys Glu Val Pro Leu Val Arg Thr Asp Asn Pro Lys Ser
Trp Tyr Glu 195 200 205 Asp Val Glu Gly Cys Gly Ile Gln Cys Gln Asn
Pro Leu Phe Thr Glu 210 215 220 Ala Glu His Gln Asp Met His Ser Tyr
Ile Ala Ala Phe Gly Ala Val 225 230 235 240 Thr Gly Leu Cys Thr Leu
Phe Thr Leu Ala Thr Phe Val Ala Asp Trp 245 250 255 Arg Asn Ser Asn
Arg Tyr Pro Ala Val Ile Leu Phe Tyr Val Asn Ala 260 265 270 Cys Phe
Phe Val Gly Ser Ile Gly Trp Leu Ala Gln Phe Met Asp Gly 275 280 285
Ala Arg Arg Glu Ile Val Cys Arg Ala Asp Gly Thr Met Arg Leu Gly 290
295 300 Glu Pro Thr Ser Asn Glu Thr Leu Ser Cys Val Ile Ile Phe Val
Ile 305 310 315 320 Val Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Phe
Val Val Leu Thr 325 330 335 Tyr Ala Trp His Thr Ser Phe Lys Ala Leu
Gly Thr Thr Tyr Gln Pro 340 345 350 Leu Ser Gly Lys Thr Ser Tyr Phe
His Leu Leu Thr Trp Ser Leu Pro 355 360 365 Phe Val Leu Thr Val Ala
Ile Leu Ala Val Ala Gln Val Asp Gly Asp 370 375 380 Ser Val Ser Gly
Ile Cys Phe Val Gly Tyr Lys Asn Tyr Arg Tyr Arg 385 390 395 400 Ala
Gly Phe Val Leu Ala Pro Ile Gly Leu Val Leu Ile Val Gly Gly 405 410
415 Tyr Phe Leu Ile Arg Gly Val Met Thr Leu Phe Ser Ile Lys Ser Asn
420 425 430 His Pro Gly Leu Leu Ser Glu Lys Ala Ala Ser Lys Ile Asn
Glu Thr 435 440 445 Met Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe
Gly Phe Val Leu 450 455 460 Ile Thr Phe Ser Cys His Phe Tyr Asp Phe
Phe Asn Gln Ala Glu Trp 465 470 475 480 Glu Arg Ser Phe Arg Asp Tyr
Val Leu Cys Gln Ala Asn Val Thr Ile 485 490 495 Gly Leu Pro Thr Lys
Gln Pro Ile Pro Asp Cys Glu Ile Lys Asn Arg 500 505 510 Pro Ser Leu
Leu Val Glu Lys Ile Asn Leu Phe Ala Met Phe Gly Thr 515 520 525 Gly
Ile Ala Met Ser Thr Trp Val Trp Thr Lys Ala Thr Leu Leu Ile 530 535
540 Trp Arg Arg Thr Trp Cys Arg Leu Thr Gly Gln Ser Asp Asp Glu Pro
545 550 555 560 Lys Arg Ile Lys Lys Ser Lys Met Ile Ala Lys Ala Phe
Ser Lys Arg 565 570 575 His Glu Leu Leu Gln Asn Pro Gly Gln Glu Leu
Ser Phe Ser Met His 580 585 590 Thr Val Ser His Asp Gly Pro Val Ala
Gly Leu Ala Phe Asp Leu Asn 595 600 605 Glu Pro Ser Ala Asp Val Ser
Ser Ala Trp Ala Gln His Val Thr Lys 610 615 620 Met Val Ala Arg Arg
Gly Ala Ile Leu Pro Gln Asp Ile Ser Val Thr 625 630 635 640 Pro Val
Ala Thr Pro Val Pro Pro Glu Glu Gln Ala Asn Leu Trp Leu 645 650 655
Val Glu Ala Glu Ile Ser Pro Glu Leu Gln Lys Arg Leu Gly Arg Lys 660
665 670 Lys Lys Arg Arg Lys Arg Lys Lys Glu Val Cys Pro Leu Ala Pro
Pro 675 680 685 Pro Glu Leu His Pro Pro Ala Pro Ala Pro Ser Thr Ile
Pro Arg Leu 690 695 700 Pro Gln Leu Pro Arg Gln Lys Cys Leu Val Ala
Ala Gly Ala Trp Gly 705 710 715 720 Ala Gly Asp Ser Cys Arg Gln Gly
Ala Trp Thr Leu Val Ser Asn Pro 725 730 735 Phe Cys Pro Glu Pro Ser
Pro Pro Gln Asp Pro Phe Leu Pro Ser Ala 740 745 750 Pro Ala Pro Val
Ala Trp Ala His Gly Arg Arg Gln Gly Leu Gly Pro 755 760 765 Ile His
Ser Arg Thr Asn Leu Met Asp Thr Glu Leu Met Asp Ala Asp 770 775 780
Ser Asp Phe 785 213PRTArtificial SequenceSynthetic Polypeptide 2Thr
Phe Val Ala Asp Trp Arg Asn Ser Asn Arg Tyr Pro 1 5 10
325PRTArtificial SequenceSynthetic Polypeptide 3Leu Thr Tyr Ala Trp
His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro Leu
Ser Gly Lys Tyr Ser Tyr 20 25 431PRTArtificial SequenceSynthetic
Polypeptide 4Arg Gly Val Met Thr Leu Phe Ser Ile Lys Ser Asn His
Pro Gly Leu 1 5 10 15 Leu Ser Glu Lys Ala Ala Ser Lys Ile Asn Glu
Thr Met Leu Arg 20 25 30 56PRTArtificial SequenceSynthetic
Polypeptide 5Leu Leu Ser Glu Lys Ala 1 5 68PRTArtificial
SequenceSynthetic Polypeptide 6Leu Phe Ser Ile Lys Ser Asn His 1 5
76PRTArtificial SequenceSynthetic Polypeptide 7Gly Thr Thr Tyr Gln
Pro 1 5 89PRTArtificial SequenceSynthetic Polypeptide 8Tyr Ala Trp
His Thr Ser Phe Lys Ala 1 5 921PRTArtificial SequenceSynthetic
Polypeptide 9Arg Gly Val Met Thr Leu Phe Ser Ile Lys Ser Asn His
Pro Gly Leu 1 5 10 15 Leu Ser Glu Lys Ala 20 1026PRTArtificial
SequenceSynthetic Polypeptide 10Leu Phe Ser Ile Lys Ser Asn His Pro
Gly Leu Leu Ser Glu Lys Ala 1 5 10 15 Ala Ser Lys Ile Asn Glu Thr
Met Leu Arg 20 25 1131PRTArtificial SequenceSynthetic Polypeptide
11Arg Gly Val Met Thr Leu Phe Ser Ile Lys Ser Asn His Pro Gly Leu 1
5 10 15 Leu Ser Glu Lys Ala Ala Ser Lys Ile Asn Glu Thr Met Leu Arg
20 25 30 1216PRTArtificial SequenceSynthetic Polypeptide 12Leu Leu
Ser Glu Lys Ala Ala Ser Lys Ile Asn Glu Thr Met Leu Arg 1 5 10 15
1326PRTArtificial SequenceSynthetic Polypeptide 13Leu Phe Ser Ile
Lys Ser Asn His Pro Gly Leu Leu Ser Glu Lys Ala 1 5 10 15 Ala Ser
Lys Ile Asn Glu Thr Met Leu Arg 20 25 1418PRTArtificial
SequenceSynthetic Polypeptide 14Arg Gly Val Met Thr Leu Phe Ser Ile
Lys Ser Asn His Pro Gly Leu 1 5 10 15 Leu Ser 1513PRTArtificial
SequenceSynthetic Polypeptide 15Arg Gly Val Met Thr Leu Phe Ser Ile
Lys Ser Asn His 1 5 10 1614PRTArtificial SequenceSynthetic
Polypeptide 16Ser Glu Lys Ala Ala Ser Lys Ile Asn Glu Thr Met Leu
Arg 1 5 10 1725PRTArtificial SequenceSynthetic Polypeptide 17Leu
Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10
15 Gln Pro Leu Ser Gly Lys Thr Ser Tyr 20 25 1821PRTArtificial
SequenceSynthetic Polypeptide 18Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro Leu Ser Gly 20
1925PRTArtificial SequenceSynthetic Polypeptide 19Leu Thr Tyr Ala
Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro
Leu Ser Gly Lys Thr Ser Tyr 20 25 2023PRTArtificial
SequenceSynthetic Polypeptide 20Tyr Ala Trp His Thr Ser Phe Lys Ala
Leu Gly Thr Thr Tyr Gln Pro 1 5 10 15 Leu Ser Gly Lys Thr Ser Tyr
20 2118PRTArtificial SequenceSynthetic Polypeptide 21Leu Thr Tyr
Ala Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln
Pro 2213PRTArtificial SequenceSynthetic Polypeptide 22Gly Thr Thr
Tyr Gln Pro Leu Ser Gly Lys Thr Ser Tyr 1 5 10 2312PRTArtificial
SequenceSynthetic Polypeptide 23Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu 1 5 10 2412PRTArtificial SequenceSynthetic Polypeptide
24Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu 1 5 10
2511PRTArtificial SequenceSynthetic Polypeptide 25Thr Tyr Ala Trp
His Thr Ser Phe Lys Ala Leu 1 5 10 2611PRTArtificial
SequenceSynthetic Polypeptide 26Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala 1 5 10 2710PRTArtificial SequenceSynthetic Polypeptide
27Leu Thr Tyr Ala Trp His Thr Ser Phe Lys 1 5 10 2810PRTArtificial
SequenceSynthetic Polypeptide 28Thr Tyr Ala Trp His Thr Ser Phe Lys
Ala 1 5 10 2925PRTArtificial SequenceSynthetic Polypeptide 29Leu
Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10
15 Gln Pro Leu Ser Gly Lys Thr Ser Tyr 20 25 3012PRTArtificial
SequenceSynthetic Polypeptide 30Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu 1 5 10 3112PRTArtificial SequenceSynthetic Polypeptide
31Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu 1 5 10
3212PRTArtificial SequenceSynthetic Polypeptide 32Leu Thr Tyr Ala
Trp His Thr Ser Phe Lys Ala Leu 1 5 10 3310PRTArtificial
SequenceSynthetic Polypeptide 33Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys 1 5 10 3412PRTArtificial SequenceSynthetic Polypeptide 34Leu
Ala Lys Phe Ser Thr His Trp Ala Tyr Thr Leu 1 5 10
3511PRTArtificial SequenceSynthetic Polypeptide 35Ala Lys Phe Ser
Thr His Trp Ala Tyr Thr Leu 1 5 10 3610PRTArtificial
SequenceSynthetic Polypeptide 36Lys Phe Ser Thr His Trp Ala Tyr Thr
Leu 1 5 10 3711PRTArtificial SequenceSynthetic Polypeptide 37Ala
Lys Phe Ser Thr His Trp Ala Tyr Thr Leu 1 5 10 3823PRTArtificial
SequenceSynthetic Polypeptide 38Thr Tyr Ala Xaa His Thr Ser Phe Lys
Ala Leu Gly Thr Thr Tyr Gln 1 5 10 15 Pro Leu Ser Gly Lys Thr Ser
20 3925PRTArtificial SequenceSynthetic Polypeptide 39Leu Thr Tyr
Ala Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln
Pro Leu Ser Gly Lys Thr Ser Tyr 20 25 4021PRTArtificial
SequenceSynthetic Polypeptide 40Leu Thr Tyr Ala Xaa His Thr Ser Phe
Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro Leu Ser Gly 20
4125PRTArtificial SequenceSynthetic Polypeptide 41Leu Thr Tyr Ala
Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro
Leu Ser Gly Lys Thr Ser Tyr 20 25 4223PRTArtificial
SequenceSynthetic Polypeptide 42Tyr Ala Xaa His Thr Ser Phe Lys Ala
Leu Gly Thr Thr Tyr Gln Pro 1 5 10 15 Leu Ser Gly Lys Thr Ser Tyr
20 4318PRTArtificial SequenceSynthetic Polypeptide 43Leu Thr Tyr
Ala Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln
Pro 4412PRTArtificial SequenceSynthetic Polypeptide 44Leu Thr Tyr
Ala Xaa His Thr Ser Phe Lys Ala Leu 1 5 10 4511PRTArtificial
SequenceSynthetic Polypeptide 45Thr Tyr Ala Xaa His Thr Ser Phe Lys
Ala Leu 1 5 10 4611PRTArtificial SequenceSynthetic Polypeptide
46Leu Thr Tyr Ala Xaa His Thr Ser Phe Lys Ala 1 5 10
4710PRTArtificial SequenceSynthetic Polypeptide 47Thr Tyr Ala Xaa
His Thr Ser Phe Lys Ala 1 5 10 4810PRTArtificial SequenceSynthetic
Polypeptide 48Thr Tyr Ala Xaa His Thr Ser Phe Lys Ala 1 5 10
4912PRTArtificial SequenceSynthetic Polypeptide 49Leu Ala Lys Phe
Ser Thr His Xaa Ala Tyr Thr Leu 1 5 10 5011PRTArtificial
SequenceSynthetic Polypeptide 50Ala Lys Phe Ser Thr His Xaa Ala Tyr
Thr Leu 1 5 10 5110PRTArtificial SequenceSynthetic Polypeptide
51Lys Phe Ser Thr His Xaa Ala Tyr Thr Leu 1 5 10 5211PRTArtificial
SequenceSynthetic Polypeptide 52Ala Lys Phe Ser Thr His Xaa Ala Tyr
Thr Leu 1 5 10 5325PRTArtificial SequenceSynthetic Polypeptide
53Leu Thr Tyr Ala Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1
5 10 15 Gln Pro Leu Ser Gly Lys Thr Ser Tyr 20 25 5412PRTArtificial
SequenceSynthetic Polypeptide 54Leu Thr Tyr Ala Xaa His Thr Ser Phe
Lys Ala Leu 1 5 10 5517PRTArtificial SequenceSynthetic Polypeptide
55Val Trp Phe Val Val Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala 1
5 10 15 Leu 5616PRTArtificial SequenceSynthetic Polypeptide 56Trp
Phe Val Val Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu 1 5 10
15 5712PRTArtificial SequenceSynthetic Polypeptide 57Leu Thr Tyr
Ala Xaa His Thr Ser Phe Lys Ala Leu 1 5 10 5812PRTArtificial
SequenceSynthetic Polypeptide 58Leu Thr Tyr Ala Xaa His Thr Ser Phe
Lys Ala Leu 1 5 10 5912PRTArtificial SequenceSynthetic Polypeptide
59Leu Thr Tyr Ala Xaa His Thr Ser Phe Lys Ala Leu 1 5 10
6032PRTArtificial SequenceSynthetic Polypeptide 60Arg Gly Val Met
Thr Leu Phe Ser Ile Lys Ser Asn His Pro Gly Leu 1 5 10 15 Leu Ser
Glu Lys Ala Ala Ser Lys Ile Asn Glu Thr Met Leu Arg Xaa 20 25 30
6117PRTArtificial SequenceSynthetic Polypeptide 61Leu Leu Ser Glu
Lys Ala Ala Ser Lys Ile Asn Glu Thr Met Leu Arg 1 5 10 15 Xaa
6227PRTArtificial SequenceSynthetic Polypeptide 62Leu Phe Ser Ile
Lys Ser Asn His Pro Gly Leu Leu Ser Glu Lys Ala 1 5 10 15 Ala Ser
Lys Ile Asn Glu Thr Met Leu Arg Xaa 20 25 6315PRTArtificial
SequenceSynthetic Polypeptide 63Ser Glu Lys Ala Ala Ser Lys Ile Asn
Glu Thr Met Leu Arg Xaa 1 5 10 15 6426PRTArtificial
SequenceSynthetic Polypeptide 64Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro Leu Ser Gly Lys Thr
Ser Tyr Xaa 20 25 6524PRTArtificial SequenceSynthetic Polypeptide
65Tyr Ala Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr Gln Pro 1
5 10 15 Leu Ser Gly Lys Thr Ser Tyr Xaa 20 6614PRTArtificial
SequenceSynthetic Polypeptide 66Gly Thr Thr Tyr Gln Pro Leu Ser Gly
Lys Thr Ser Tyr Xaa 1 5 10 6713PRTArtificial SequenceSynthetic
Polypeptide 67Leu Ala Lys Phe Ser Thr His Trp Ala Tyr Thr Leu Xaa 1
5 10
6812PRTArtificial SequenceSynthetic Polypeptide 68Ala Lys Phe Ser
Thr His Trp Ala Tyr Thr Leu Xaa 1 5 10 6911PRTArtificial
SequenceSynthetic Polypeptide 69Lys Phe Ser Thr His Trp Ala Tyr Thr
Leu Xaa 1 5 10 7026PRTArtificial SequenceSynthetic Polypeptide
70Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1
5 10 15 Gln Pro Leu Ser Gly Lys Thr Ser Tyr Xaa 20 25
7126PRTArtificial SequenceSynthetic Polypeptide 71Leu Thr Tyr Ala
Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro
Leu Ser Gly Lys Thr Ser Tyr Xaa 20 25 7224PRTArtificial
SequenceSynthetic Polypeptide 72Tyr Ala Xaa His Thr Ser Phe Lys Ala
Leu Gly Thr Thr Tyr Gln Pro 1 5 10 15 Leu Ser Gly Lys Thr Ser Tyr
Xaa 20 7313PRTArtificial SequenceSynthetic Polypeptide 73Leu Ala
Lys Phe Ser Thr His Xaa Ala Tyr Thr Leu Xaa 1 5 10
7412PRTArtificial SequenceSynthetic Polypeptide 74Ala Lys Phe Ser
Thr His Xaa Ala Tyr Thr Leu Xaa 1 5 10 7511PRTArtificial
SequenceSynthetic Polypeptide 75Lys Phe Ser Thr His Xaa Ala Tyr Thr
Leu Xaa 1 5 10 7626PRTArtificial SequenceSynthetic Polypeptide
76Leu Thr Tyr Ala Xaa His Thr Ser Phe Lys Ala Leu Gly Thr Thr Tyr 1
5 10 15 Gln Pro Leu Ser Gly Lys Thr Ser Tyr Xaa 20 25
7719PRTArtificial SequenceSynthetic Polypeptide 77Ala Arg Gly Val
Met Thr Leu Phe Ser Ile Lys Ser Asn His Pro Gly 1 5 10 15 Leu Leu
Ser 7816PRTArtificial SequenceSynthetic Polypeptide 78Arg Gln Ile
Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
7913PRTArtificial SequenceSynthetic Polypeptide 79Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 809PRTArtificial
SequenceSynthetic Polypeptide 80Tyr Ala Xaa His Thr Ser Phe Lys Ala
1 5 8112PRTArtificial SequenceSynthetic Polypeptide 81Leu Thr Tyr
Ala Xaa His Thr Ser Phe Lys Ala Leu 1 5 10 8228PRTArtificial
SequenceSynthetic Polypeptide 82Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu Arg Gln Ile Lys 1 5 10 15 Ile Trp Phe Pro Asn Arg Arg
Xaa Lys Trp Lys Lys 20 25 8328PRTArtificial SequenceSynthetic
Polypeptide 83Arg Gln Ile Lys Ile Trp Phe Pro Asn Arg Arg Xaa Lys
Trp Lys Lys 1 5 10 15 Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala
Leu 20 25 8414PRTArtificial SequenceSynthetic Polypeptide 84Ala Lys
Phe Ser Thr His Trp Ala Tyr Thr Leu Xaa Lys Xaa 1 5 10
8512PRTArtificial SequenceSynthetic Polypeptide 85Leu Ala Lys Phe
Ser Thr His Trp Ala Tyr Thr Leu 1 5 10 8611PRTArtificial
SequenceSynthetic Polypeptide 86Ala Lys Phe Ser Thr His Trp Ala Tyr
Thr Leu 1 5 10 8710PRTArtificial SequenceSynthetic Polypeptide
87Lys Phe Ser Thr His Trp Ala Tyr Thr Leu 1 5 10 8825PRTArtificial
SequenceSynthetic Polypeptide 88Leu Thr Tyr Ala Trp His Thr Ser Phe
Lys Ala Leu Gly Thr Thr Tyr 1 5 10 15 Gln Pro Leu Ser Gly Lys Thr
Ser Tyr 20 25 8910PRTArtificial SequenceSynthetic Polypeptide 89Thr
Tyr Ala Trp His Thr Ser Phe Lys Ala 1 5 10 9016PRTArtificial
SequenceSynthetic Polypeptide 90Val Trp Phe Val Val Thr Tyr Ala Trp
His Thr Ser Phe Lys Ala Leu 1 5 10 15 9115PRTArtificial
SequenceSynthetic Polypeptide 91Trp Phe Val Val Thr Tyr Ala Trp His
Thr Ser Phe Lys Ala Leu 1 5 10 15 9225PRTArtificial
SequenceSynthetic Polypeptide 92Leu Ala Lys Phe Ser Thr His Trp Ala
Tyr Thr Leu Lys Leu Thr Tyr 1 5 10 15 Ala Trp His Thr Ser Phe Lys
Ala Leu 20 25 9328PRTArtificial SequenceSynthetic Polypeptide 93Asp
Ala Lys Phe Ser Thr His Trp Ala Tyr Thr Leu Lys Pro Ala Leu 1 5 10
15 Leu Thr Tyr Ala Trp His Thr Ser Phe Lys Ala Leu 20 25
9427PRTArtificial SequenceSynthetic Polypeptide 94Asp Ala Lys Phe
Ser Thr His Trp Ala Tyr Thr Leu Lys Pro Ala Leu 1 5 10 15 Leu Thr
Tyr Ala Trp His Thr Ser Phe Lys Ala 20 25
* * * * *