U.S. patent application number 11/419557 was filed with the patent office on 2006-10-26 for melanocortin metallopeptode combinatorial libraries and applications.
This patent application is currently assigned to Palatin Technologies, Inc.. Invention is credited to Hui-Zhi Cai, Shubh D. Sharma, Yiqun Shi, Wei Yang.
Application Number | 20060240481 11/419557 |
Document ID | / |
Family ID | 36440929 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060240481 |
Kind Code |
A1 |
Sharma; Shubh D. ; et
al. |
October 26, 2006 |
Melanocortin Metallopeptode Combinatorial Libraries and
Applications
Abstract
Melanocortin receptor-specific peptide combinatorial libraries
for forming metallopeptides and metallopeptide combinatorial
libraries are provided for use in biological, pharmaceutical and
related applications. The combinatorial libraries are constructed
of peptides constructed from naturally occurring amino acids,
stereoisomers and modifications of such amino acids, non-protein
amino acids, post-translationally modified amino acids,
enzymatically modified amino acids, or constructs or structures
designed to mimic amino acids, in which the peptides are
conformationally fixed on complexation of a metal ion-binding
portion thereof with a metal ion.
Inventors: |
Sharma; Shubh D.; (Cranbury,
NJ) ; Shi; Yiqun; (East Brunswick, NJ) ; Yang;
Wei; (Edison, NJ) ; Cai; Hui-Zhi; (East
Brunswick, NJ) |
Correspondence
Address: |
PALATIN TECHNOLOGIES, INC.
4-C CEDAR BROOK DRIVE
CEDAR BROOK CORPORATE CENTER
CRANBURY
NJ
08512
US
|
Assignee: |
Palatin Technologies, Inc.
Cranbury
NJ
08512
|
Family ID: |
36440929 |
Appl. No.: |
11/419557 |
Filed: |
May 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10049718 |
Feb 13, 2002 |
7049398 |
|
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PCT/US00/16396 |
Jun 15, 2000 |
|
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11419557 |
May 22, 2006 |
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60148994 |
Aug 13, 1999 |
|
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Current U.S.
Class: |
435/7.1 ; 506/18;
530/329; 530/330 |
Current CPC
Class: |
A61K 51/088 20130101;
C07K 5/1008 20130101; C07K 5/1024 20130101; C07K 5/1016 20130101;
C07K 14/665 20130101; A61K 38/00 20130101; A61K 51/086
20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/10 20060101 C40B040/10 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Grant No. R43 CA83417 awarded by the National Cancer Institute
of the National Institutes of Health of the Department of Health
and Human Services and Grant No. R43 DK55470 awarded by the
National Institute of Diabetes and Digestive and Kidney Diseases of
the National Institutes of Health of the Department of Health and
Human Services.
Claims
1. A combinatorial library targeted to melanocortin receptors of
different sequence peptide members synthesized on solid phase,
where each constituent library member comprises: (a) a peptide
sequence of three or more amino acid residues bound to solid phase
characterized by (i) a sequence of two or more amino acid residues
forming a metal ion-binding domain and including at least one amino
acid residue containing at least one sulfur atom (S) wherein the
said S is protected by an orthogonal S-protecting group, (ii) a
sequence of one or more amino acid residues at the N- or C-terminus
of the metal ion-binding domain, or at both the N- and C-terminus
of the metal ion-binding domain, and (iii) a cleavable bond
attaching the peptide sequence to solid phase; and (b) a unique
selection or sequence of amino acid residues in the peptide
sequence of at least one of the constituent members of the library;
wherein the orthogonal S-protecting group may be removed without
cleaving the peptide sequence from the solid phase.
2. The combinatorial library of claim 1 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formulas: R.sub.1-Lll-Aaa-Bbb-Ccc-R.sub.2,
R.sub.1-Bbb-Aaa-Ccc-R.sub.2, R.sub.1-Ddd-Bbb-Aaa-R.sub.3,
R.sub.4-Eee-Bbb-Ccc-R.sub.2, R.sub.1-Hhh-Aaa-Bbb-Ccc-R.sub.5, or
R.sub.1-Iii-Iii-Ccc-Jjj-Kkk-R.sub.2, wherein R.sub.1 comprises a
functionality that potentiates the intrinsic activity of the
remainder of the peptide, including but not limited to providing an
auxiliary or secondary receptor contact; Aaa is an L- or
D-configuration cationic amino acid with a positively charged side
chain; Bbb is an L- or D-configuration amino acid with an aromatic
side chain; Ccc is an amino acid that provides both a nitrogen atom
(N), from the alpha amino group, and a S, from a side chain group,
for metal ion complexation; Lll is a D-configuration amino acid
with an aromatic side chain; R.sub.2 is optionally present, and if
present, comprises an amino acid with an aromatic side chain; Ddd
is an amino acid that provides an S, from a side chain group, for
metal ion complexation; R.sub.3 is an amino acid with an aromatic
side chain that provides an N for metal ion complexation; R.sub.4
is a functionality that provides a cationic center; Eee is an
uncharged L- or D-configuration amino acid that provides an N for
metal ion complexation; R.sub.5 is an amide, substituted amide,
ester or carboxylate group, or comprises an L- or D-configuration
amino acid; Hhh is an L- or D-configuration cationic amino acid
with a positively charged side chain; Iii is an L- or
D-configuration amino acid that provides an N for metal ion
complexation; Jjj is an L- or D-configuration amino acid with an
aromatic side chain; and Kkk is an L- or D-configuration cationic
amino acid with a positively charged side chain.
3. The combinatorial library of claim 1 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formula: R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1
comprises a functionality that potentiates the intrinsic activity
of the remainder of the peptide, including but not limited to
providing an auxiliary or secondary receptor contact; Aaa is an L-
or D-configuration cationic amino acid with a positively charged
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid
wherein the aromatic ring of the aromatic side chain of Fff is
substituted with halogen, alkyl or aryl groups, an L-configuration
Phe, or an L- or D-configuration Hphe, Pgl, Trp, 1-Nal, 2-Nal,
Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl),
Tyr(BzlCl.sub.2), Tic, Tiq or Tca, or derivative, analog or homolog
thereof; Ggg is an L- or D-configuration aromatic amino acid; and
R.sub.5 is an amide, substituted amide, ester or carboxylate group,
or comprises an L- or D-configuration amino acid.
4. The combinatorial library of claim 1 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formula: R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1
comprises a functionality that potentiates the intrinsic activity
of the remainder of the peptide, including but not limited to
providing an auxiliary or secondary receptor contact; Aaa is an L-
or D-configuration cationic amino acid with a positively charged
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid;
Ggg is an L- or D-configuration aromatic amino acid wherein the
aromatic ring of the aromatic side chain of Ggg is substituted with
halogen, alkyl or aryl groups, an L-configuration Phe, or an L- or
D-configuration Hphe, Pgl, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids; and R.sub.5 is an amide, substituted
amide, ester or carboxylate group, or comprises an L- or
D-configuration amino acid.
5. The combinatorial library of claim 1 wherein the metal
ion-binding domain further comprises at least one N available for
binding to a metal ion upon removal of the orthogonal S-protecting
group.
6. The combinatorial library of claim 1 wherein the orthogonal
S-protecting group is S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl.
7. The combinatorial library of claim 1 wherein one or more
constituent library members include at least one amino acid residue
in the sequence at the N- or C-terminus of the metal ion-binding
domain containing at least one S wherein the said S is protected by
a non-orthogonal S-protecting group, whereby the orthogonal
S-protecting group may be removed without removing the
non-orthogonal S-protecting group.
8. The combinatorial library of claim 1 wherein the at least one
amino acid residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group is an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine; 3-mercapto phenylananine;
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto-3,3,-diethyl proprionic acid; 3-mercapto,3-methyl
propionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid.
9. A combinatorial library targeted to melanocortin receptors of
different sequence peptide members synthesized on solid phase and
complexed to a metal ion, where each constituent library member
comprises: (a) a peptide sequence of three or more amino acid
residues bound to solid phase characterized by (i) a sequence of
two or more amino acid residues forming a metal ion-binding domain
and including at least one amino acid residue containing at least
one S not protected by a S-protecting group, (ii) a sequence of one
or more amino acid residues at the N- or C-terminus of the metal
ion-binding domain, or at both the N- and C-terminus of the metal
ion-binding domain, and (iii) a cleavable bond attaching the
peptide sequence to solid phase; (b) a metal ion complexed to the
metal ion-binding domain; and (c) a unique selection or sequence of
amino acid residues in the peptide sequence of at least one of the
constituent members of the library.
10. The combinatorial library of claim 9 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formulas: R.sub.1-Lll-Aaa-Bbb-Ccc-R.sub.2,
R.sub.1-Bbb-Aaa-Ccc-R.sub.2, R.sub.1-Ddd-Bbb-Aaa-R.sub.3,
R.sub.4-Eee-Bbb-Ccc-R.sub.2, R.sub.1-Hhh-Aaa-Bbb-Ccc-R.sub.5, or
R.sub.1-Iii-Iii-Ccc-Jjj-Kkk-R.sub.2, wherein R.sub.1 comprises a
functionality that potentiates the intrinsic activity of the
remainder of the peptide, including but not limited to providing an
auxiliary or secondary receptor contact; Aaa is an L- or
D-configuration cationic amino acid with a positively charged side
chain; Bbb is an L- or D-configuration amino acid with an aromatic
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Lll is a D-configuration amino acid with an aromatic
side chain; R.sub.2 is optionally present, and if present,
comprises an amino acid with an aromatic side chain; Ddd is an
amino acid that provides an S, from a side chain group, for metal
ion complexation; R.sub.3 is an amino acid with an aromatic side
chain that provides an N for metal ion complexation; R.sub.4 is a
functionality that provides a cationic center; Eee is an uncharged
L- or D-configuration amino acid that provides an N for metal ion
complexation; R.sub.5 is an amide, substituted amide, ester or
carboxylate group, or comprises an L- or D-configuration amino
acid; Hhh is an L- or D-configuration cationic amino acid with a
positively charged side chain; Iii is an L- or D-configuration
amino acid that provides an N for metal ion complexation; Jjj is an
L- or D-configuration amino acid with an aromatic side chain; and
Kkk is an L- or D-configuration cationic amino acid with a
positively charged side chain.
11. The combinatorial library of claim 9 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formula: R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1
comprises a functionality that potentiates the intrinsic activity
of the remainder of the peptide, including but not limited to
providing an auxiliary or secondary receptor contact; Aaa is an L-
or D-configuration cationic amino acid with a positively charged
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid
wherein the aromatic ring of the aromatic side chain of Fff is
substituted with halogen, alkyl or aryl groups, an L-configuration
Phe or an L- or D-configuration Hphe, Pgl, Trp, 1-Nal, 2-Nal,
Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl),
Tyr(BzlCl.sub.2), Tic, Tiq or Tca, or derivative, analog or homolog
thereof; Ggg is an L- or D-configuration aromatic amino acid; and
R.sub.5 is an amide, substituted amide, ester or carboxylate group,
or comprises an L- or D-configuration amino acid.
12. The combinatorial library of claim 9 wherein the peptide
sequence of three or more amino acid residues bound to solid phase
is of the formula: R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1
comprises a functionality that potentiates the intrinsic activity
of the remainder of the peptide, including but not limited to
providing an auxiliary or secondary receptor contact; Aaa is an L-
or D-configuration cationic amino acid with a positively charged
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid;
Ggg is an L- or D-configuration aromatic amino acid wherein the
aromatic ring of the aromatic side chain of Ggg is substituted with
halogen, alkyl or aryl groups, or an L-configuration Phe, or an L-
or D-configuration Hphe, Pgl, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids; and R.sub.5 is an amide, substituted
amide, ester or carboxylate group, or comprises an L- or
D-configuration amino acid.
13. The combinatorial library of claim 9 wherein one or more
constituent library members include at least one amino acid residue
in the sequence at the N- or C-terminus of the metal ion-binding
domain containing at least one S wherein the said S is protected by
a S-protecting group.
14. The combinatorial library of claim 9 wherein the at least one
amino acid residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group is an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine; 3-mercapto phenylananine;
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto-3,3,-diethyl proprionic acid; 3-mercapto, 3-methyl
propionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid; or
2-cyclopentamethlene,2-mercaptoacetic acid.
15. The combinatorial library of claim 9 wherein the metal ion is
an ionic form of rhenium or technetium.
16. A combinatorial library targeted to melanocortin receptors of
different sequence peptide members synthesized in solution, where
each constituent library member comprises: (a) a peptide sequence
of a combination of three or more amino acid residues characterized
by (i) a sequence of two or more amino acid residues, mimics of
amino acid residues or combinations thereof forming a metal
ion-binding domain and including at least one amino acid residue or
mimic of an amino acid residue containing at least one S wherein
the said S is protected by an orthogonal S-protecting group, (ii) a
sequence of one or more amino acid residues, mimics of amino acid
residues or combinations thereof at the N- or C-terminus of the
metal ion-binding domain, or at both the N- and C-terminus of the
metal ion-binding domain; and (b) a unique selection or sequence of
amino acid residues, mimics of amino acid residues or combinations
thereof in the peptidomimetic sequence of at least one of the
constituent members of the library.
17. The combinatorial library of claim 16 wherein the peptide
sequence of three or more amino acid residues is of the formulas:
R.sub.1-Lll-Aaa-Bbb-Ccc-R.sub.2, R.sub.1-Bbb-Aaa-Ccc-R.sub.2,
R.sub.1-Ddd-Bbb-Aaa-R.sub.3, R.sub.4-Eee-Bbb-Ccc-R.sub.2,
R.sub.1-Hhh-Aaa-Bbb-Ccc-R.sub.5, or
R.sub.1-Iii-Iii-Ccc-Jjj-Kkk-R.sub.2, wherein R.sub.1 comprises a
functionality that potentiates the intrinsic activity of the
remainder of the peptide, including but not limited to providing an
auxiliary or secondary receptor contact; Aaa is an L- or
D-configuration cationic amino acid with a positively charged side
chain; Bbb is an L- or D-configuration amino acid with an aromatic
side chain; Ccc is an amino acid that provides both a N, from the
alpha amino group, and a S, from a side chain group, for metal ion
complexation; Lll is a D-configuration amino acid with an aromatic
side chain; R.sub.2 is optionally present, and if present,
comprises an amino acid with an aromatic side chain; Ddd is an
amino acid that provides an S, from a side chain group, for metal
ion complexation; R.sub.3 is an amino acid with an aromatic side
chain that provides an N for metal ion complexation; R.sub.4 is a
functionality that provides a cationic center; Eee is an uncharged
L- or D-configuration amino acid that provides an N for metal ion
complexation; R.sub.5 is an amide, substituted amide, ester or
carboxylate group, or comprises an L- or D-configuration amino
acid; Hhh is an L- or D-configuration cationic amino acid with a
positively charged side chain; Iii is an L- or D-configuration
amino acid that provides an N for metal ion complexation; Jjj is an
L- or D-configuration amino acid with an aromatic side chain; and
Kkk is an L- or D-configuration cationic amino acid with a
positively charged side chain.
18. The combinatorial library of claim 16 wherein the peptide
sequence of three or more amino acid residues is of the formula:
R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1 comprises a
functionality that potentiates the intrinsic activity of the
remainder of the peptide, including but not limited to providing an
auxiliary or secondary receptor contact; Aaa is an L- or
D-configuration cationic amino acid with a positively charged side
chain; Ccc is an amino acid that provides both a N, from the alpha
amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid
wherein the aromatic ring of the aromatic side chain of Fff is
substituted with halogen, alkyl or aryl groups, an L-configuration
Phe or an L- or D-configuration Hphe, Pgl, Trp, 1-Nal, 2-Nal,
Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl),
Tyr(BzlCl.sub.2), Tic, Tiq or Tca, or derivative, analog or homolog
thereof; Ggg is an L- or D-configuration aromatic amino acid; and
R.sub.5 is an amide, substituted amide, ester or carboxylate group,
or comprises an L- or D-configuration amino acid.
19. The combinatorial library of claim 16 wherein the peptide
sequence of three or more amino acid residues is of the formula:
R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5, wherein R.sub.1 comprises a
functionality that potentiates the intrinsic activity of the
remainder of the peptide, including but not limited to providing an
auxiliary or secondary receptor contact; Aaa is an L- or
D-configuration cationic amino acid with a positively charged side
chain; Ccc is an amino acid that provides both a N, from the alpha
amino group, and a S, from a side chain group, for metal ion
complexation; Fff is an L- or D-configuration aromatic amino acid;
Ggg is an L- or D-configuration aromatic amino acid wherein the
aromatic ring of the aromatic side chain of Ggg is substituted with
halogen, alkyl or aryl groups, an L-configuration Phe, or an L- or
D-configuration Hphe, Pgl, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids; and R.sub.5 is an amide, substituted
amide, ester or carboxylate group, or comprises an L- or
D-configuration amino acid.
20. The combinatorial library of claim 16 wherein the orthogonal
S-protecting group is S-thio-butyl, acetamidomethyl,
4-methoxytrityl, S-sulfonate or 3-nitro-2-pyridinesulfenyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/049,718, entitled Melanocortin
Metallopeptide Constructs, Combinatorial Libraries and
Applications, filed Feb. 13, 2002, and issued as U.S. Pat. No.
7,049,398 on May 23, 2006, which application is a national entry
pursuant to 37 U.S.C. .sctn. 371 of International Application No.
PCT/US00/16396, entitled Melanocortin Metallopeptide Constructs,
Combinatorial Libraries and Applications, filed Jun. 15, 2000,
which in turn claims the benefit of the filing of U.S. Provisional
Patent Application Ser. No. 60/148,994, entitled Melanocortin
Receptor-Specific Metallopeptide Constructs, Combinatorial
Libraries and Applications, filed on Aug. 13, 1999, and the
specifications thereof are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention (Technical Field)
[0004] The present invention relates to metallopeptides, metal
ion-complexed peptidomimetics, and metallo-constructs, including
metallopeptide combinatorial libraries, metal ion-complexed
peptidomimetic and peptide-like combinatorial libraries and
metallo-construct combinatorial libraries, specific for
melanocortin receptors, including methods for the use and making of
the same. The invention also relates to methods for synthesizing
and assembling such libraries, and methods for identification and
characterization of library constituents which are capable of
binding a melanocortin receptor of interest, or mediating a
melanocortin receptor-related biological activity of interest.
[0005] 2. Background Art
[0006] Note that the following discussion refers to a number of
publications by author(s) and year of publication, and that due to
recent publication dates certain publications are not to be
considered as prior art vis-a-vis the present invention. Discussion
of such publications herein is given for more complete background
and is not to be construed as an admission that such publications
are prior art for patentability determination purposes.
[0007] Melanocortin Receptors. A family of melanocortin receptor
types and subtypes has been identified, including melanocortin-1
receptors (MC1-R) expressed on normal human melanocytes and
melanoma cells, melanocortin-2 receptors (MC2-R) for ACTH
(adrenocorticotropin) expressed in cells of the adrenal gland,
melanocortin-3 and melanocortin-4 receptors (MC3-R and MC4-R)
expressed primarily in cells in the hypothalamus, mid-brain and
brainstem, and melanocortin-5 receptors (MC5-R), expressed in a
wide distribution of peripheral tissues.
[0008] Peptides specific for melanocortin receptors have been
reported to have a wide variety of biological activities, including
effects upon pigmentation and steroidogenesis, known to be mediated
by MSH (melanocyte stimulating hormone) and ACTH receptors. Several
studies have documented the presence of melanotropin receptors on
primary human melanoma cells (Tatro J B, Atkins M, Mier J W, et al.
Melanotropin receptors demonstrated in situ in human melanoma. J
Clin Invest, 85:1825-1832, 1990). Melanotropin receptors have been
reported as markers for melanotic and amelanotic human melanoma
tumors (Sharma S D, Granberry M E, Jiang J, et al. Multivalent
melanotropic peptide and fluorescent macromolecular conjugates: new
reagents for characterization of melanotropin receptors. J Clin
Invest, 85:1825-1832, 1990). Melanotropin receptors have been
reported as markers for melanotic and amelanotic human Sharma S D,
Jiang J, Hadley M E, et al. Melanotropic peptide-conjugated beads
for microscopic visualization and characterization of melanoma
melanotropin receptors. Proc Natl Acad Sci USA 93(24):13715-13720,
1996). In particular, the presence of MC1-R has been demonstrated
in human melanoma cells by an antibody to MC1-R (Xia Y, Skoog V,
Muceniece R, et al. Polyclonal antibodies against human
melanocortin MC-1 receptor: Preliminary immunohistochemical
localization of melanocortin MC1 receptor to malignant melanoma
cells. European J Pharmacol 288:277-283, 1995). MC1-R is a G
protein-coupled, 7-transmembrane receptor expressed in skin-cell
melanocytes and shares some degree of homology with related
receptors MC2-R, MC3-R, MC4-R and MC5-R. Each of these receptors
can bind various peptide analogs that contain a common melanotropic
pharmacophore, His-Phe-Arg-Trp (SEQ ID NO:1), which describes the
6-9 sequence of the alpha-melanocyte stimulating hormone
(.alpha.-MSH).
[0009] Prior to molecular characterization of the MC receptors,
.alpha.-MSH analogs were labeled with the radioisotope Indium-111
and used in melanoma imaging studies (Wraight E P, Bard D R,
Maughan T S, et al. The use of a chelating derivative of alpha
melanocyte stimulating hormone for the clinical imaging of
malignant melanoma. Brit J Radiology 65: 112-118, 1992; Bard D R,
Knight C G and Page-Thomas D P. A chelating derivative of
alpha-melanocyte stimulating hormone as a potential imaging agent
for malignant melanoma. Brit J Cancer 62:919-922, 1990; Bard D R,
Knight C G, Page-Thomas D P. Targeting of a chelating derivative of
a short chain analogue of alpha-melanocyte stimulating hormone to
Cloudman S91 melanomas. Biochem Soc Trans 18:882-883, 1990). Linear
and cyclic disulfide-containing peptides have been identified and
used for melanoma imaging and appear to be non-selective among MC
receptors (Chen J and Quinn T P. Alpha melanocyte stimulating
hormone analogues Tc-99m/Re-188 labeling and their pharmacokinetics
in malignant melanoma bearing mice. J Nucl Med 39: 222p, 1998;
Giblin M F, Wang N, Hoffman T J, et al. Design and characterization
of alpha-melanotropin peptide analogs cyclized through rhenium and
technetium metal coordination. Proc Natl Acad Sci U S A
95(22):12814-12818, 1998). In later studies, the cyclic peptide
reported by Giblin and coworkers was also found to localize in the
brain (Wang N N, Giblin M F, Hoffman T J, et al. In vivo
characterization of Tc-99m and Re-188 labeled cyclic melanotropin
peptide analogues in a murine melanoma model. J Nucl Med 39: 77p,
1998 and corresponding poster presentation at the 45th Society of
Nuclear Medicine Meeting, Toronto, June 1998). It has been recently
reported that the response of human melanocytes to UV radiation is
mediated by .alpha.-MSH induced activation of the cAMP pathway
through the MC1-R (Im S, Moro O, Peng F, et al. Activation of the
cyclic AMP pathway by alpha-melanotropin mediates the response of
human melanocytes to ultraviolet B radiation. Cancer Res 58: 47-54,
1998).
[0010] MC4-R is also a G protein-coupled, 7-transmembrane receptor,
but is believed to be expressed primarily in the brain.
Inactivation of this receptor by gene targeting has been reported
to result in mice with the maturity-onset obesity syndrome that is
associated with hyperphagia, hyperinsulinemia, and hyperglycemia
(Huszar D, Lynch C A, Fairchild-Huntress V, et al. Targeted
disruption of the melanocortin-4 receptor results in obesity in
mice. Cell 88:131-141, 1997). MC4-R is a molecular target for
therapeutic intervention in energy homeostasis.
[0011] Alpha-MSH has been described as a potent anti-inflammatory
agent in all major forms of inflammation (Star R A, Rajora N, Huang
J, Stock R C, Catania A, and Lipton J M: Evidence of autocrine
modulation of macrophage nitric oxide synthase by alpha-melanocyte
stimulating hormone. Proc Natl Acad Sci U S A 92:8016-8020, 1995;
Getting S J, and Perretti M: MC3-R as a novel target for
antiinflammatory therapy. Drug News and Perspectives 13:19-27,
2000). Implication of both MC1-R and MC3-R receptors in
anti-inflammatory processes has been stressed. In particular, the
activation of these MC receptors by melanocortin receptor agonists
has been reported to inhibit the expression of nitric oxide
synthase and subsequent nitric oxide production.
[0012] Significant work has been done in determining the structure
of melanocortin receptors, including both the nucleic acid
sequences encoding for the receptors and the amino acid sequences
constituting the receptors. See, for example, International Patent
Applications No. PCT/US98/12098 and PCT/US99/16862 and U.S. Pat.
No. 5,994,087. A large number of ligands specific for melanocortin
receptors, both agonists and antagonists, have also been developed.
See, for example, International Patent Applications No.
PCT/US98/03298 (iodo group-containing melanocortin
receptor-specific linear peptide), PCT/GB99/01388 (MC1-R specific
linear peptides), PCT/GB99/01195 (MC3-R, MC4-R and MC5-R specific
cyclic peptides), PCT/US99/04111 (MC1-R specific peptide
antagonists for melanoma therapy), PCT/US99/09216 (isoquinoline
compounds as melanocortin receptor ligands), PCT/US99/13252
(spiropiperdine derivatives as melanocortin receptor agonists), and
U.S. Pat. No. 6,054,556 (cyclic lactam peptides as MC1-R, MC3-R,
MC4-R and MC5-R antagonists). In addition, a large number of
patents teach various methods of screening and determining
melanocortin receptor-specific compounds, as for example
International Patent Applications No. PCT/US97/15565,
PCT/US98/12098 and PCT/US99/16862 and U.S. Pat. Nos. 5,932,779 and
5,994,087.
[0013] In general, compounds specific for MC1-R are believed to be
useful for treatment of melanoma, including use as radiotherapeutic
or drug delivery agent, and as diagnostic imaging agents,
particularly when labeled with a diagnostic radionuclide. Compounds
specific for MC3-R, MC4-R or MC5-R are believed to be useful in
regulation of energy homeostasis, including use as agents for
attenuating food intake and body weight gain, for use in treatment
of anorexia, as a weight gain aid, for treatment of obesity, and
other treatment of other food intake and metabolism-related
purposes. Compounds specific for MC3-R and MC4-R, among other
melanocortin receptors, can be used as agents for treatment of
sexual dysfunction, including male erectile dysfunction. Compounds
specific for MC3-R and MC4-R, among other melanocortin receptors,
can be used to regulate blood pressure, heart rate and other
neurophysiologic parameters. Other melanocortin receptor peptides
can be used as tanning agents, to increase melanin production, such
as peptides that are MCR-1 agonists. Compounds specific for MCR-1
and MCR-3 may be useful in regulation of inflammatory
processes.
[0014] There remains a significant need for ligands with high
specificity for discrete melanocortin receptors, as well as ligands
or compounds that are either agonists or antagonists of specific
melanocortin receptors. High affinity peptide ligands of
melanocortin receptors can be used to exploit varied physiological
responses associated with the melanocortin receptors, either as
agonists or antagonists. In addition, melanocortin receptors have
an effect on the activity of various cytokines, and high affinity
peptide ligands of melanocortin receptors can be used to regulate
cytokine activity.
[0015] Peptide Libraries and Combinatorial Chemistry. Libraries of
peptides and other small molecules, with enormous pools of
structurally diverse molecules, are well suited for pharmaceutical
lead generation and lead optimization. Libraries of a variety of
molecular species have been described in literature and screened
for drug discovery, including peptides, peptoids, peptidomimetics,
oligonucleotides, benzodiazepines, and other libraries of small
organic molecules. Various approaches have been used to construct
libraries of structurally diverse chemical compounds, including
chemical synthesis and genetic engineering methods. Chemically
synthesized libraries have been synthesized by general solution
chemical means and by solid-phase methods. The prior art on
designing, synthesizing, screening, and evaluation of peptide-based
libraries has been reviewed in numerous articles.
[0016] Spatially Addressable Parallel Synthesis of Solid Phase
Bound Libraries. Various strategies for chemical construction of a
library of peptides or other small molecules are well established.
One strategy involves spatially separate synthesis of compounds in
parallel on solid phase or on a solid surface in a predetermined
fashion so that the location of one compound or a subset of
compounds on the solid surface is known. Other methods, such as
light-directed spatially addressable parallel chemical synthesis
techniques, based upon use of photolithographic techniques in
peptide synthesis on a solid surface, such as a borosilicate glass
microscope slide, provide libraries containing more than 100,000
spatially separated compounds. However, synthesis of libraries that
are structurally more diverse than simple peptides requires the
development of orthogonal photolabile protecting groups that can be
cleaved at different wavelengths of light. In addition, the solid
surface bearing these libraries also has been reported to cause a
pronounced effect on binding affinities in library screening
assays.
[0017] Pooling and Split Synthesis Strategies. Large libraries of
compounds can be assembled by a pooling strategy that employs
equimolar mixtures of reactants in each synthetic step or by
adjusting the relative concentration of various reactants in the
mixture according to their reactivities in each of the coupling
reactions. In one approach equimolar mixtures of compounds are
obtained by splitting the resin in equal portions, each of which is
separately reacted with each of the various monomeric reagents. The
resin is mixed, processed for the next coupling, and again split
into equal portions for separate reaction with individual reagents.
The process is repeated as required to obtain a library of desired
oligomeric length and size. This approach is the basis of the
"one-bead one-peptide" strategy which employs amino acid sequencing
to ascertain the primary structure of the peptide on a hit bead in
a bioassay. Automated systems have been developed for carrying out
split synthesis of these libraries with rather more efficiency. A
common artifact occasionally seen with all these resin bound
libraries is altered target-specific affinity by some solid phase
bound compounds in bioassays, which can result in totally
misleading results.
[0018] Another strategy involves construction of soluble libraries.
This strategy involves a deconvolution process of iterative
re-synthesis and bioassaying until all the initially randomized
amino acid positions are defined. Several modifications to this
strategy have been developed, including co-synthesis of two
libraries containing orthogonal pools, which eliminates the need of
iterative re-synthesis and evaluation. A major limitation of the
soluble library approach is its applicability to high affinity
systems. The abundance of each compound in solution can be
influenced by the total number of compounds in a library that can
affect the biological activity. For this reason, a highly active
compound in any pool may not in fact be the most potent molecule.
Lack of reasonable solubilities of certain members in a library may
further influence this phenomenon.
[0019] Among the various classes of libraries of small molecules,
peptide libraries remain the most versatile because of the
structural diversity offered by the use of naturally occurring
amino acids, incorporation of a variety of "designer" amino acids,
and the high efficiency and ease with which peptide synthesis can
be accomplished. In addition, another level of structural diversity
in peptide-based libraries has been added by post-synthesis
modification of the libraries. These modifications include
permethylation, acylation, functionalization of the side chain
functionality, and reductive amination of the N-terminus.
SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)
[0020] In one preferred embodiment, the invention provides a
combinatorial library targeted to melanocortin receptors of
different sequence peptide members synthesized on solid phase,
where each constituent library member comprises:
[0021] (a) a peptide sequence of three or more amino acid residues
bound to solid phase characterized by (i) a sequence of two or more
amino acid residues forming a metal ion-binding domain and
including at least one amino acid residue containing at least one S
wherein the said S is protected by an orthogonal S-protecting
group, (ii) a sequence of one or more amino acid residues at the N-
or C-terminus of the metal ion-binding domain, or at both the N-
and C-terminus of the metal ion-binding domain, and (iii) a
cleavable bond attaching the peptide sequence to solid phase;
and
[0022] (b) a unique selection or sequence of amino acid residues in
the peptide sequence of at least one of the constituent members of
the library; [0023] wherein the orthogonal S-protecting group may
be removed without cleaving the peptide sequence from the solid
phase.
[0024] In another preferred embodiment, the invention provides a
combinatorial library targeted to melanocortin receptors of
different sequence peptidomimetic members synthesized on solid
phase, where each constituent library member comprises:
[0025] (a) a peptidomimetic sequence of a combination of three or
more amino acid residues and mimics of amino acid residues bound to
solid phase characterized by (i) a sequence of two or more amino
acid residues, mimics of amino acid residues or combinations
thereof forming a metal ion-binding domain and including at least
one amino acid residue or mimic of an amino acid residue containing
at least one S wherein the said S is protected by an orthogonal
S-protecting group, (ii) a sequence of one or more amino acid
residues, mimics of amino acid residues or combinations thereof at
the N- or C-terminus of the metal ion-binding domain, or at both
the N- and C-terminus of the metal ion-binding domain, and (iii) a
cleavable bond attaching the peptidomimetic sequence to solid
phase; and
[0026] (b) a unique selection or sequence of amino acid residues,
mimics of amino acid residues or combinations thereof in the
peptidomimetic sequence of at least one of the constituent members
of the library; [0027] wherein the orthogonal S-protecting group
may be removed without cleaving the peptidomimetic sequence from
the solid phase.
[0028] In another preferred embodiment, the invention provides a
combinatorial library targeted to melanocortin receptors of
different sequence peptide or peptidomimetic members synthesized in
solution, where each constituent library member comprises:
[0029] (a) a peptidomimetic sequence of a combination of three or
more amino acid residues and mimics of amino acid residues
characterized by (i) a sequence of two or more amino acid residues,
mimics of amino acid residues or combinations thereof forming a
metal ion-binding domain and including at least one amino acid
residue or mimic of an amino acid residue containing at least one S
wherein the said S is protected by an orthogonal S-protecting
group, (ii) a sequence of one or more amino acid residues, mimics
of amino acid residues or combinations thereof at the N- or
C-terminus of the metal ion-binding domain, or at both the N- and
C-terminus of the metal ion-binding domain; and
[0030] (b) a unique selection or sequence of amino acid residues,
mimics of amino acid residues or combinations thereof in the
peptidomimetic sequence of at least one of the constituent members
of the library.
[0031] The libraries of this invention include compositions of the
formulas: R.sub.1-Lll-Aaa-Bbb-Ccc-R.sub.2,
R.sub.1-Bbb-Aaa-Ccc-R.sub.2, R.sub.1-Ddd-Bbb-Aaa-R.sub.3,
R.sub.4-Eee-Bbb-Ccc-R.sub.2, R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5,
R.sub.1-Hhh-Aaa-Bbb-Ccc-R.sub.5, and
R.sub.1-Iii-Iii-Ccc-Jjj-Kkk-R.sub.2,
[0032] wherein [0033] R.sub.1 is any functionality that potentiates
the intrinsic activity of the remainder of the molecule, including
but not limited to providing an auxiliary or secondary receptor
contact. Any of a variety of amino acids and non-peptide groups may
be employed, including an amino acid chain from one to about four
neutral or charged L- or D-configuration amino acid residues. If
R.sub.1 is a non-peptide group, it may be a linear or branched
alkyl, aryl, alkene, alkenyl or aralkyl chain; [0034] Aaa is an L-
or D-configuration cationic amino acid with a positively charged
side chain. Preferred amino acids include L-configuration Lys, Arg,
Orn, Dpr or Dbu, and derivatives, analogs or homologs thereof,
including both natural and synthetic amino acids. Aaa provides an N
(nitrogen atom) for metal ion complexation; [0035] Bbb is an L- or
D-configuration amino acid with an aromatic side chain. Preferred
amino acids include D-configuration Phe, Phe(4'Cl), Phe(3',4'
Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl), Hphe, Pgl,
Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz),
Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and derivatives, analogs
or homologs thereof. The aromatic ring in Bbb may be functionalized
with halogen, alkyl or aryl groups. Bbb provides an N for metal ion
complexation; [0036] Ccc is an amino acid that provides both an N,
from the alpha amino group, and an S (sulfur atom), from a side
chain group, for metal ion complexation. Preferred amino acids
include L- or D-configuration Cys, Pen and Hcys; [0037] Lll is a
D-configuration amino acid with an aromatic side chain. Preferred
amino acids include D-configuration Phe, Phe(4'Cl), Phe(3',4'
Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl), Hphe, Pgl,
Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz),
Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and derivatives, analogs
or homologs thereof. The aromatic ring in Lll may be functionalized
with halogen, alkyl or aryl groups. Lll does not provide an N for
metal ion complexation; [0038] R.sub.2 is an amino acid with an
aromatic side chain. Preferred amino acids include L- or
D-configuration Phe, Trp, Phe(4'Cl), Phe(3',4' Di-Cl),
Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl), Hphe, Pgl, Trp,
1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz), Thr(Bzl),
Cys(Bzl) or Tyr(BzlCl.sub.2), and derivatives, analogs or homologs
thereof, including both natural and synthetic amino acids. The
C-terminus may be free or amidated. R.sub.2 may also be the
corresponding des-carboxyl amino acid of any of the foregoing.
Alternatively, R.sub.2 may be eliminated; [0039] Ddd is an amino
acid that provides an S, from a side chain group, for metal ion
complexation. Preferred amino acids include L- or D-configuration
Cys, Pen and Hcys; [0040] R.sub.3 is an amino acid with an aromatic
side chain that provides an N for metal ion complexation. Preferred
amino acids include L- or D-configuration Phe, Trp, Phe(4'Cl),
Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl),
Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br),
Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and derivatives,
analogs or homologs thereof, including both natural and synthetic
amino acids. The C-terminus may be free or amidated. R.sub.3 may
also be the corresponding des-carboxylamino acid of any of the
foregoing; [0041] R.sub.4 is a functionality that provides a
cationic center. Preferred amino acids include L- or
D-configuration Lys, Arg, Orn, Dpr or Dbu, and derivatives, analogs
or homologs thereof, including both natural and synthetic amino
acids. The N-terminus of the amino acid may be functionalized with
any of a variety of neutral amino acid and non-peptide groups,
including linear or branched alkyl, aryl, alkene, alkenyl or
aralkyl chains; [0042] Eee is an uncharged L- or D-configuration
amino acid that provides an N for metal ion complexation. Preferred
amino acids include Gly and L-configuration Ala, Nle, Leu, Val, Phe
or Trp, and derivatives, analogs or homologs thereof, including
both natural and synthetic amino acids. In a preferred embodiment,
Eee isn an amino acid with an aliphatic side chain; [0043] Fff is
an L- or D-configuration aromatic amino acid. Preferred amino acids
include D-configuration Phe, Phe(4'Cl), Phe(3',4' Di-Cl),
Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl), Hphe, Pgl, Trp,
1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz), Thr(Bzl),
Cys(Bzl), Tyr(BzlCl.sub.2), Tic, Tiq or Tca, and derivatives,
analogs or homologs thereof, including both natural and synthetic
amino acids. The aromatic ring in Fff may be substituted with
halogen, alkyl or aryl groups. Fff does not provide an N for metal
ion complexation; [0044] Ggg is an L- or D-configuration aromatic
amino acid. Preferred amino acids include L-configuration Phe,
Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids. The aromatic ring in Ggg may be
substituted with halogen, alkyl or aryl groups. Ggg provides an N
for metal ion complexation; [0045] R.sub.5 is preferably an amide,
substituted amide, ester or carboxylate group. R.sub.5 may also be
and L- or D-configuration amino acid or amino acid amide, including
an aromatic, aliphatic, neutral or charged amino acid; [0046] Hhh
is an L- or D-configuration cationic amino acid with a positively
charged side chain. Preferred amino acids include L-configuration
Lys, Arg, Orn, Dpr or Dbu, and derivatives, analogs or homologs
thereof, including both natural and synthetic amino acids. Hhh does
not provide an N for metal ion complexation; [0047] Iii is an L- or
D-configuration amino acid that provides an N for metal ion
complexation. Preferred amino acids includes Ala, Gly, Nle, Val.
Leu, Ile, His, Lys, or Arg, and derivatives, analogs or homologs
thereof, including both natural and synthetic amino acids; [0048]
Jjj is an L- or D-configuration amino acid with an aromatic side
chain. Preferred amino acids include D-configuration Phe,
Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof. The aromatic ring in Jjj
may be functionalized with halogens, alkyl or aryl groups. Jjj does
not provide an N for metal ion complexation; and [0049] Kkk is an
L- or D-configuration cationic amino acid with a positively charged
side chain. Preferred amino acids include L-configuration Lys, Arg,
Orn, Dpr or Dbu, and derivatives, analogs or homologs thereof,
including both natural and synthetic amino acids. Aaa does not
provide an N for metal ion complexation.
[0050] In the compositions of this invention, the metal ion-binding
domain can be complexed with a metal ion, and such compositions are
included within the invention. The invention further includes
compositions wherein the composition is substantially more specific
for one or more melanocortin receptors when the metal ion-binding
domain is complexed with a metal ion than is the composition when
the metal ion-binding amino acid sequence is not complexed with a
metal ion.
[0051] The combinatorial libraries of this invention include
libraries wherein the metal ion-binding domain further comprises at
least one N available for binding to a metal ion upon removal of
the orthogonal S-protecting group. The combinatorial libraries
include compositions wherein the metal ion-binding domain comprises
three residues forming an N.sub.3S.sub.1 ligand.
[0052] In the combinatorial libraries, the orthogonal S-protecting
group is S-thio-butyl, acetamidomethyl, 4-methoxytrityl,
S-sulfonate or 3-nitro-2-pyridinesulfenyl. The orthogonal
S-protecting group may be removed from constituent library members
thereof without otherwise altering the constituent library members
or any amino acid side chain protecting group therein. In the
combinatorial libraries, structural diversity may occurs in the
metal ion-binding domain or outside the metal ion-binding
domain.
[0053] In the combinatorial libraries, one or more constituent
library members may include at least one amino acid residue or
mimic of an amino acid residue in the sequence at the N- or
C-terminus of the metal ion-binding domain containing at least one
S wherein the said S is protected by a non-orthogonal S-protecting
group, whereby the orthogonal S-protecting group may be removed
without removing the non-orthogonal S-protecting group.
[0054] For combinatorial libraries limited to amino acids, the
amino acid residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group can be an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine. For combinatorial libraries
including amino acid residues and mimics of amino acid residues,
the residue containing at least one S wherein the said S is
protected by an orthogonal S-protecting group can be an L- or
D-3-mercapto amino acid, including but not limited to L- or
D-cysteine or L- or D-penicillamine; 3-mercapto phenylananine;
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto-3,3,-diethyl proprionic acid; 3-mercapto, 3-methyl
propionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethylene,3-mercaptopropionic acid; or
2-cyclopentamethylene,2-mercaptoacetic acid.
[0055] It is an object of this invention to devise, demonstrate and
illustrate the preparation and use of highly specific
conformationally restricted peptides, peptoids, related
pseudopeptides, peptidomimetics and metallo-constructs formed by
complexing sequences thereof to a desired metal ion so that the
topography of the side chains in the resulting complex is a
biologically active three-dimensional structure specific for
melanocortin receptors.
[0056] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors and which have a
higher level of stability and are less susceptible to proteolysis
than either the uncomplexed peptide, or other peptides known in the
art.
[0057] Another object of this invention is to provide peptide-metal
ion complexes that are specific for different subsets of
melanocortin receptors, such as specific only for MC1-R or for
MC4-R.
[0058] Another object of this invention is to provide peptide-metal
ion complexes which are specific for melanocortin receptors and
which are agonists or antagonists.
[0059] Another object of this invention is to provide for peptide
analogs which are not conformationally restricted in the absence of
a metal ion, whereby the uncomplexed peptide analog is either
inactive or demonstrates low potency, but which is conformationally
restricted on complexation with a metal ion and specific for
melanocortin receptors with high potency.
[0060] Another object of this invention is to utilize metal
complexation in a peptide specific for melanocortin receptors to
cause specific regional conformational restrictions in the peptide
so that the peptide conformation at the metal binding site is
conformationally fixed on metal complexation.
[0061] Another object of this invention is to complex a peptide to
a metal ion, whereby the resulting metallopeptide is specific for
melanocortin receptors, and exhibits a preferred in vivo
biodistribution profile, rate and mode of clearance,
bioavailability and pharmacokinetics in mammals.
[0062] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors utilizing stable
non-radioactive metal ions, for use in therapeutic treatment of
disease, including as an agent to modify energy metabolism and
feeding behavior, such as for treatment of pathologic obesity and
related conditions.
[0063] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors utilizing metal
ions which are radionuclides for use in diagnostic methods,
including imaging and staging of melanoma tumors and melanoma tumor
metastases.
[0064] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors utilizing stable
non-radioactive metal ions, for use as a prevention agent against
ultra-violet radiation-induced DNA damages, including
sunlight-induced DNA damage in the skin.
[0065] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors utilizing stable
non-radioactive metal ions, for use as a tanning agent, including
but not limited to therapeutic use as a tanning agent.
[0066] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors utilizing stable
non-radioactive metal ions, for use as anti-inflammatory
agents.
[0067] Another object of this invention is to complex peptides with
radiometal ions, including but not limted to technetium-99m, for
use in diagnostic imaging, so that the resulting peptide-metal ion
complex is substantially more specific for melanocortin receptors
than the uncomplexed peptide molecule, and the resulting
radiolabeled species is essentially carrier-free in terms of
specificity for melanocortin receptors.
[0068] Another object of this invention is to complex peptides with
radiometal ions, including radioisotopes of rhenium such as
rhenium-186 and rhenium-188, for use in targeted radiotherapy, such
as for treatment of melanoma.
[0069] Another object of this invention is to provide peptide-metal
ion complexes specific for melanocortin receptors that can transit
the gut-blood barrier, without significant enzymatic or peptidase
degradation, and may be adapted for oral administration.
[0070] Another object of this invention is to provide libraries of
conformationally constrained peptide-metal ion complexes directed
to melanocortin receptors.
[0071] Another object of this invention is to provide combinatorial
peptide libraries of peptide-metal ion complexes specific for
melanocortin receptors, wherein the peptides include a metal
ion-binding domain, such that a specific conformational restriction
is obtained upon labeling the peptides with a metal ion.
[0072] Another object of this invention is to provide combinatorial
peptide libraries of peptide-metal ion complexes specific for
melanocortin receptors, wherein the amino acids comprising the
peptides may be naturally occurring amino acids, isomers and
modifications of such amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically modified
amino acids, constructs or structures designed to mimic amino
acids, and the like, so that the library includes pseudopeptides
and peptidomimetics.
[0073] Another object of this invention is to provide
metallopeptide libraries specific for one or more melanocortin
receptors, wherein the metallopeptides include a metal ion-binding
domain, such that a determined conformational restriction is
obtained upon labeling the peptides with a metal ion, and the
metallopeptides further include distinct, unique and different
amino acid sequences.
[0074] Another object of this invention is to provide both soluble
and solid phase metallopeptide libraries specific for one or more
melanocortin receptors, wherein the metallopeptides include a metal
ion-binding domain.
[0075] Another object of this invention is to provide methods for
synthesis of peptides specific for melanocortin receptors wherein
the peptide contains a reactive SH group forming a part of a metal
ion-binding domain, whereby the reactive SH group is protected
during synthesis, and is deprotected only upon complexing the
peptides with a metal ion.
[0076] Another object of this invention is to provide combinatorial
metallopeptide libraries specific for melanocortin receptors
wherein the peptides forming the library contain a reverse turn
structure as a consequence of metal ion complexation.
[0077] Another object of this invention is to provide a method for
rapid and efficient complexation of a pool of diverse peptides
specific for melanocortin receptors with a metal ion, including a
rhenium metal ion.
[0078] Another object of this invention to provide libraries of
conformationally constrained peptide-metal ion complexes as
surrogates for reverse turn structures, such as beta turns and
gamma turns commonly found in naturally occurring peptides and
proteins specific for melanocortin receptors. The turns formed as a
consequence of metal ion complexation are more stable than the
naturally occurring turn structures, which are stabilized only by
weaker interactions such as van der Waals=interactions and hydrogen
bonds.
[0079] Another object of this invention is to provide combinatorial
metallopeptide libraries wherein each of the peptides forming the
library contain a reverse turn structure as a consequence of metal
ion complexation.
[0080] Another object of this invention is to provide a method for
the identification of specific metallopeptides through internal
signatures resulting from use of metal ions with two or more
isotopic peaks, such as through use of rhenium containing two
isotopes in fixed relative abundance that differ in mass by 2
units.
[0081] Other objects, advantages and novel features, and the
further scope of applicability of the present invention, will be
set forth in part in the detailed description to follow, taken in
conjunction with the accompanying drawings, and in part will become
apparent to those skilled in the art upon examination of the
following, or may be learned by practice of this invention. The
objects and advantages of this invention may be realized and
attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate several embodiments of
the present invention and, together with the description, serve to
explain the principles of the invention. The drawings are only for
the purpose of illustrating a preferred embodiment of the invention
and are not to be construed as limiting the invention. In the
drawings:
[0083] FIG. 1 is a molecular structure for Template 1.
[0084] FIG. 2 is a molecular structure for Template 2.
[0085] FIG. 3 is a molecular structure for Template 3.
[0086] FIG. 4 is a molecular structure for Template 4.
[0087] FIG. 5 is a molecular structure for Template 5.
[0088] FIG. 6 is a molecular structure for Template 6.
[0089] FIG. 7 is a molecular structure for Template 7.
[0090] FIG. 8 is a flow chart of a split pool and combination
synthesis method according to Example 2.
[0091] FIG. 9 is a mass spectrum of a library pool of 25
metallopeptides synthesized according to Example 2.
[0092] FIG. 10 is a mass spectrum of a library pool of 4
metallopeptides synthesized according to Example 6.
[0093] FIGS. 11A-11E are reversed phased HPLC profiles of a library
pool of 4 metallopeptides synthesized according to Example 6.
DESCRIPTION OF THE PREFERRED EMBODIMENTS (BEST MODES FOR CARRYING
OUT THE INVENTION)
[0094] Definitions. Certain terms as used throughout the
specification and claims are defined as follows:
[0095] The terms "bind," "binding," "complex," and "complexing," as
used throughout the specification and claims, are generally
intended to cover all types of physical and chemical binding,
reactions, complexing, attraction, chelating and the like.
[0096] The "peptides" of this invention can be a)
naturally-occurring, b) produced by chemical synthesis, c) produced
by recombinant DNA technology, d) produced by biochemical or
enzymatic fragmentation of larger molecules, e) produced by methods
resulting from a combination of methods a through d listed above,
or f) produced by any other means for producing peptides.
[0097] By employing chemical synthesis, a preferred means of
production, it is possible to introduce various amino acids which
do not naturally occur along the chain, modify the N- or
C-terminus, and the like, thereby providing for improved stability
and formulation, resistance to protease degradation, and the
like.
[0098] The term "peptide" as used throughout the specification and
claims is intended to include any structure comprised of two or
more amino acids, including chemical modifications and derivatives
of amino acids. For the most part, the peptides of this invention
comprise fewer than 100 amino acids, and preferably fewer than 60
amino acids, and most preferably ranging from about 2 to 20 amino
acids. The amino acids forming all or a part of a peptide may be
naturally occurring amino acids, stereoisomers and modifications of
such amino acids, non-protein amino acids, post-translationally
modified amino acids, enzymatically modified amino acids,
constructs or structures designed to mimic amino acids, and the
like, so that the term "peptide" includes pseudopeptides and
peptidomimetics, including structures which have a non-peptidic
backbone. The term "peptide" also includes dimers or multimers of
peptides. A "manufactured" peptide includes a peptide produced by
chemical synthesis, recombinant DNA technology, biochemical or
enzymatic fragmentation of larger molecules, combinations of the
foregoing or, in general, made by any other method.
[0099] The "amino acids" used in this invention, and the term as
used in the specification and claims, include the known naturally
occurring protein amino acids, which are referred to by both their
common three letter abbreviation and single letter abbreviation.
See generally Synthetic Peptides: A User's Guide, GA Grant, editor,
W.H. Freeman & Co., New York, 1992, the teachings of which are
incorporated herein by reference, including the text and table set
forth at pages 11 through 24. As set forth above, the term "amino
acid" also includes stereoisomers and modifications of naturally
occurring protein amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically
synthesized amino acids, derivatized amino acids, constructs or
structures designed to mimic amino acids, and the like. Modified
and unusual amino acids are described generally in Synthetic
Peptides: A User's Guide, cited above; Hruby V J, Al-obeidi F and
Kazmierski W: Biochem J 268:249-262, 1990; and Toniolo C: Int J
Peptide Protein Res 35:287-300, 1990; the teachings of all of which
are incorporated herein by reference. In addition, the following
abbreviations have the meanings giving: TABLE-US-00001 Abu
gamma-amino butyric acid 2-Abz 2-amino benzoic acid 3-Abz 3-amino
benzoic acid 4-Abz 4-amino benzoic acid Achc
1-amino-cyclohexane-1-carboxylic acid Acpc
1-amino-cyclopropane-1-carboxylic acid 12-Ado 12-amino dodecanoic
acid 7-Ahept 7-amino heptanoic acid Aic 2-aminoindane-2-carboxylic
acid 6-Ahx 6-amino hexanoic acid 8-Aoc 8-amino octanoic acid
Arg(Tos) N.sup.G-para-tosyl-arginine Asp(anilino)
beta-anilino-aspartic acid Asp(3-Cl-anilino)
beta-(3-chloro-anilino)-aspartic acid Asp(3,5-diCl-anilino)
beta-(3,5-dichloro anilino)-aspartic acid D/L Atc
(D,L)-2-aminotetralin-2-carboxylic acid 11-Aun 11-amino undecanoic
acid AVA 5-amino valeric acid Bip biphenylalanine Bz Benzoyl Cha
Cyclohexylalanine Chg Cyclohexylglycine Dip 3,3-Diphenylalanine Et-
Ethyl GAA epsilon-guanidino acetic acid GBzA 4-guanidino benzoic
acid B-Gpa 3-guanidino propionic acid GVA(Cl)
beta-chloro-epsilon-guanidino valeric acid Hphe Homophenylalanine
Inp isonipecotic acid Lys(Z) N-epsilon-benzyloxycarbonyl-lysine Me-
Methyl Nal 1 3-(1-naphthyl)alanine Nal 2 3-(2-naphthyl)alanine
(N-Bzl)Nal 2 N-benzyl-3-(2-naphthyl) alanine (N-PhEt)Nal 2
N(2-phenylethyl)-3-(2-naphthyl) alanine Phg Phenylglycine pF-Phe
para-fluoro-phenylalanine Phe(4-Br) 4-bromo-phenylalanine
Phe(4-CF.sub.3) 4-trifluoromethyl-phenylalanine Phe(4-Cl)
4-chloro-phenylalanine Phe(2-Cl) 2 chloro-phenylalanine
Phe(2,4-diCl) 2,4,-dichloro-phenylalanine Phe(3,4-diCl)
3,4,-dichloro-phenylalanine Phe(3,4-diF)
3,4,-difluoro-phenylalanine Phe(4-I) 4-iodo-phenylalanine
Phe(3,4-di-OMe) 3,4,-dimethoxy-phenylalanine Phe(4-Me)
4-methyl-phenylalanine Phe(4-NO.sub.2) 4-nitro-phenylalanine Pip
Pipecolic acid Qal(2') beta-(2-quinolyl)-alanine Sal
3-styrylalanine TFA trifluoroacetyl Tic
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Tle
tert-butylalanine Tyr(Bzl) O-benzyl-tyrosine Tyr(BzlDiCl 2,6)
O-(2,6 dichloro)benzyl-tyrosine
[0100] A single amino acid, including stereoisomers and
modifications of naturally occurring protein amino acids,
non-protein amino acids, post-translationally modified amino acids,
enzymatically synthesized amino acids, derivatized amino acids,
constructs or structures designed to mimic amino acids, and the
like, including all of the foregoing, is sometimes referred to
herein as a "residue."
[0101] The library constructs of this invention also include a
metal ion, which may be an ionic form of any element in metallic
form, including but not limited to metals and metalloids. The metal
ion may, but need not, be radioactive, paramagnetic or
superparamagnetic. The metal ion can be of any oxidation state of
any metal, including oxidation states of vanadium (V), manganese
(Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn),
gallium (Ga), arsenic (As), selenium (Se), yttrium (Y), molybdenum
(Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium
(Pd), silver (Ag), cadmium (Cd), indium (In), tin (Sn), tungsten
(W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold
(Au), mercury (Hg), thallium (TI), lead (Pb), bismuth (Bi),
polonium (Po), astatine (At), samarium (Sm), europium (Eu), and
gadolinium (Gd). The metal ion can also be a radionuclide of any of
the foregoing, including In, Au, Ag, Hg, Tc, Re, Sn, At, Y and Cu.
A preferred metal ion with a tetradentate coordination sphere is
Re. For radiopharmaceutical applications, or applications wherein a
radioisotope is desirable for screening, an alpha-, gamma- or
beta-emitting radionuclide may be employed.
[0102] The coordination sphere of various common metal ions, in
general, is tetradentate to hexadentate. In one embodiment
according to this invention, an amino acid or amino acid mimetic
sequence is included within each library member such that it
contains the desired number of groups (4 to 6 in most cases) for
complexing with the metal. The molecule is designed so that, upon
complexing with a metal, it forms a mimic of a reverse turn
structure about the site of metal complexation. A metal with
coordination number 4, 5 or 6, and complexing respectively with an
amino acid sequence forming a tetra, penta, or hexadentate ligand,
will fold and constrain the ligand. The amino acid or amino acid
mimetic sequence forming a ligand is defined as the metal
ion-binding domain ("MBD") of the peptide or peptidomimetic. A
highly flexible molecule like a peptide, in other words, is folded
to form a kind of reverse turn upon its complexation with a metal.
This resulting turn is a highly constrained structure in the
conformational sense.
[0103] The biological-binding domain ("BBD") of the peptide or
peptidomimetic is defined in the specification and claims as a
sequence of one or more amino acids which constitute a biologically
active sequence, exhibiting binding to a melanocotin-associated
receptor, including MC1-R, MC2-R, MC3-R, MC4-R and MC5-R, thereby
constituting the peptide as a member of a specific binding pair.
The BBD also includes any sequence, which may be consecutive amino
acids or mimetics (sychnological) or non-consecutive amino acids or
mimetics (rhegnylogical) which forms a melanocortin-associated
ligand, which ligand is capable of forming a specific interaction
with its acceptor or receptor. The term "receptor" is intended to
include both acceptors and receptors. The receptor may be a
biological receptor. The sequence or BBD may transmit a signal to
the cells, tissues or other materials associated with the
biological receptor after binding, but such is not required. The
BBD may be either an agonist or antagonist, or a mixed
agonist-antagonist. A peptide or peptidomimetic complexed to a
metal ion with such a BBD constitutes a member of a "specific
binding pair," which specific binding pair is made up of at least
two different molecules, where one molecule has an area on the
surface or in a cavity which specifically binds to a particular
spatial and polar organization of the other molecule. Frequently,
the members of a specific binding pair are referred to as ligand
and receptor or anti-ligand.
[0104] The BBD is further defined to include the portion of a
construct, wherein the construct is a peptidomimetic, peptide-like,
or metallo-construct molecule, which upon binding of the construct
with a metal ion, is biologically active, exhibiting binding to a
melanocortin receptor found on cells, tissues, organs and other
biological materials. The BBD may, in this instance, be
sychnological or rhegnylogical, and generally has the attributes
and functions of a BBD of a peptide. The BBD may be coextensive
with all or a portion of the MBD, so that the same amino acids or
other residues which constitute the MBD also constitute all or a
part of the BBD. In some instances, one or amino acids of the MBD
will form a part of the BBD, and one or more additional amino
acids, which are not part of the MBD, form the remainder of the
BBD.
[0105] Conformational constraint refers to the stability and
preferred conformation of the three-dimensional shape assumed by a
peptide or other construct. Conformational constraints include
local constraints, involving restricting the conformational
mobility of a single residue in a peptide; regional constraints,
involving restricting the conformational mobility of a group of
residues, which residues may form some secondary structural unit;
and global constraints, involving the entire peptide structure. See
generally Synthetic Peptides: A User's Guide, cited above.
[0106] The primary structure of a peptide is its amino acid
sequence. The secondary structure deals with the conformation of
the peptide backbone and the folding up of the segments of the
peptide into regular structures such as .alpha.-helices,
.beta.-sheets, turns and the like. Thus, the three-dimensional
shape assumed by a peptide is directly related to its secondary
structure. See generally Synthetic Peptides: A User's Guide, cited
above, including the text, figures and tables set forth at pages
24-33, 39-41 and 58-67. A global structure refers to a peptide
structure which exhibits a preference for adopting a
conformationally constrained three-dimensional shape.
[0107] The product resulting from the methods set forth herein can
be used for both medical applications and animal husbandry or
veterinary applications. Typically, the product is used in humans,
but may also be used in other mammals. The term "patient" is
intended to denote a mammalian individual, and is so used
throughout the specification and in the claims. The primary
applications of this invention involve human patients, but this
invention may be applied to laboratory, farm, zoo, wildlife, pet,
sport or other animals. The products of this invention may
optionally employ radionuclide ions, which may be used for
diagnostic imaging purposes or for radiotherapeutic purposes.
[0108] Peptide and Metallo-Construct Molecule Libraries and
Combinatorial Chemistries. Using the methods of this invention,
libraries of peptides and peptidomimetics are designed wherein each
constituent library member includes an MBD sequence necessary for
providing a coordination site for complexation with a metal, it
being understood that such sequence may differ among the
constituent members of the library. Upon complexing the MBD with a
metal, a specific structure results, forming a mimic of a reverse
turn structure. The specific stereochemical features of this
complex are due to the stereochemistry of the coordination sphere
of the complexing metal ion. Thus the preferred geometry of the
coordination sphere of the metal dictates and defines the nature
and extent of conformational restriction.
[0109] Libraries of this invention contain constituents which are
either locally or globally constrained structures. Libraries may
include molecules with either local conformation restrictions or
global conformation restrictions, or some combination thereof. This
aspect of the invention includes a variety of methods of synthesis,
screening and structural elucidation of positive hits in screening
systems. The importance of these aspects is well known to those
skilled in the art and will also become evident from the following
description and examples.
[0110] In general, most of the metals that may prove useful in this
invention have a coordination number of 4 to 6, and rarely as high
as 8, which implies that the putative MBD must be made of residues
with reactive groups located in a stereocompatible manner so as to
establish a bond with a metal ion of given geometry and
coordination sphere. Coordinating groups in the peptide chain
include nitrogen atoms of amine, amide, imidazole, or guanidino
functionalities; sulfur atoms of thiols or disulfides; and oxygen
atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities.
In addition, the peptide chain or individual amino acids can be
chemically altered to include a coordinating group, such as oxime,
hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or
morpholino groups. For a metal with a coordination number of 4, a
tetrapeptide amino acid sequence may be employed (such as
Gly-Gly-Gly-Gly (SEQ ID NO:2)), or a tripeptide amino acid sequence
in which at least one of the amino acids has a side chain with a
coordinating group can similarly be employed (such as Gly-Gly-Cys).
The side chain can have a nitrogen, oxygen or sulfur-based
coordination group. Thus, an amino acid sequence can provide an
N.sub.4, N.sub.3S, N.sub.2S.sub.2, NS.sub.3, N.sub.2SO or similar
ligand, yielding tetradentate coordination of a metal ion utilizing
nitrogen, sulfur and oxygen atoms.
[0111] In another embodiment of the invention, the MBD includes one
or more amino acid residues and one or more derivatized amino acids
or spacer sequences, with the derivatized amino acid or spacer
sequence having a nitrogen, sulfur or oxygen atom available for
complexing with the various oxidation states of the metal. Examples
of derivatized amino acids include amide, primary alkyl or aryl
amide, 1,2,3,4-tetrahydroisoquinoline-2-carboxylic acid and its
corresponding 7-hydroxy derivative, N-carboxymethylated amino
acids, 2'-mercapto-Trp, N.sup..beta.-(2
mercaptoethane)-.alpha.,.beta.-diaminopropionic acid and similar
higher homologs of other homologous amino acids, N.sup..beta.-(2
aminoethane)-.alpha.,.beta.-diaminopropionic acid and similar
higher homologs of other homologous amino acids,
N.sup..beta.-(picolinoyl)-.alpha.,.beta.-diaminopropionic acid and
similar higher homologs of other homologous amino acids,
.beta.-(picolylamide)-Asp and similar homologs of other homologous
amino acids,
N.sup..beta.-(2-amino-benzoyl)-.alpha.,.beta.-diaminopropionic acid
and similar higher homologs of other homologous amino acids,
.beta.-(2-amidomethylpyridine)-Asp and similar homologs of other
homologous amino acids, N-benzyloxycarbonyl amino acid, N-tert
butyloxycarbonyl amino acid, N-fluorenylmethyloxycarbonyl amino
acid and other similar urethane-protected amino acid derivatives,
and other derivatized or synthetic amino acids relating to any of
the foregoing. Examples of spacer sequences which may be employed
in this invention include 2-mercaptoethylamine, succinic acid,
glutaric acid, 2-mercaptosuccinic acid, ethylenediamine,
diethylenetriamine, triethylenetetraamine, tetraethylenepentaamine,
glycol, polyethylene glycol, thioglycolic acid, mercaptopropionic
acid, pyridine-2-carboxylate, picolylamine, 2-mercaptoaniline,
2-aminobenzoic acid, and 2-aminomethylpyridine. In general, any
sequence which may be linked, directly or indirectly, to one or two
amino acids so as to form a continuous sequence, and which has a
nitrogen, sulfur or oxygen atom available for complexing with the
valences of the metal ion, may be employed as an element of the
MBD.
[0112] S-Protected Thiol Group Compounds in Metallo-Libraries. A
free thiol (SH) group is preferred for complexation of most metal
ions to the peptides and peptidomimetics of this invention, and in
many cases an SH group is necessary in order to form a stable
exchange-inert complex with a metal. Peptides and other organic
molecules with free SH groups, however, are easily oxidized in air
and in solution, and can often form a disulfide-linked dimer. If
more than one free SH group is present in a molecule, oxidation may
lead to a complex polymer. Similarly, if a mixture of different
peptides or organic molecules with free SH groups are prepared,
oxidation generally leads to a complex mixture of polymers of
unknown composition. This is of serious concern in preparing
libraries of metallopeptides or other organic molecules where one
or more SH group is intended for use in metal complexation.
[0113] A variety of SH protecting groups have been employed for a
variety of purposes, including radiopharmaceutical manufacture and
formulation. For example, in its protected form
S-Benzoyl-mercaptoacetyl-glycyl-glycyl-glycine (Bz-MAG.sub.3) has
been used to complex Tc-99m (.sup.99mTc) under conditions where the
S-Bz group splits during .sup.99mTc complexation. The use of S-Bz
protection, however, is not compatible with the methods of peptide
synthesis.
[0114] In order to construct metallopeptide libraries of this
invention which incorporate an SH group, if mixed pool synthesis is
employed the peptides must be S-protected derivatives. The SH
protecting group is chosen such that (a) the synthesis of peptide
derivatives with S-protecting group is compatible with methods of
solution and solid phase peptide synthesis, so that the
S-protecting group is stable during synthetic procedures, and (b)
the S-protecting group can be deprotected in situ, without cleavage
from the resin in the case of solid phase synthesis, during the
metal complexation step. Many prior art methods, such as
Bz-MAG.sub.3, meet at most only one of the two criteria specified
above (Bz-MAG.sub.3 meets only criterion (a) above).
[0115] Use of orthogonally S-protected thiol groups permits
synthesis of metallo-compounds in a single pot. A mixture of
compounds, each compound containing an orthogonal S-protected group
("OSPG"), is used for complexation with a metal ion, and it is only
during metal ion complexation that the S-protected group is
deprotected, and accordingly polymerization and cross-linking is
avoided. This procedure thus provides homogenous libraries of
metallo-compounds.
[0116] One OSPG meeting the criteria specified above, and which can
be used in this invention, employs an S.sup.tBu (S-thio-butyl or
S-t-butyl) group to protect the SH group. The S.sup.tBu group is
stable under both the acidic and basic conditions typically
employed in peptide synthesis. Further, the S.sup.tBu group may be
cleaved by reduction using a suitable phosphine reagent, which
reduction step may be employed immediately prior to or in
conjunction with complexation of a metal ion to the peptide. Such
OSPG cleavage does not cleave the peptide from the resin, or
otherwise alter the structure of the peptide.
[0117] Another OSPG meeting the criteria specified above and
suitable for this invention employs an S-Acm (S-acetamidomethyl)
group to protect the SH group. The Acm group is also stable under
the acid and base conditions usually employed during peptide
synthesis. The S-Acm group may be removed by treatment of
S-Acm-protected peptide or peptide resin with mercury (II) acetate
or silver (I) tertrafluoroborate, which liberates the thiol peptide
in its mercury or silver ion-complexed state. Free thiol-containing
peptide can then be recovered by treating the mercury or silver ion
and thiol complexed salts with an excess of a thiol-containing
reagent, such as beta-mercaptoethanol or dithiothreitol. The
resulting peptide is then used for metal complexation.
Alternatively, the mercury or silver ion and thiol complexed
peptide may be directly treated with a metal ion complexing reagent
to form the desired metallopeptide.
[0118] Other examples of OSPGs for metallopeptides include
4-methoxytrityl (Mmt), 3-nitro-2-pyridinesulfenyl (Npys) and
S-sulfonate (SO.sub.3H). Mmt is selectively removed upon treatment
with 1% TFA in dichloromethane. Npys and S-sulfonate are
selectively removed by treatment with a thiol-containing reagent
such as beta-mercaptoethanol or dithiothreitol or a phosphine
reagent such as tributyl phosphine. The Npys group (R. G. Simmonds
R G et al: Int J Peptide Protein Res, 43:363, 1994) is compatible
with Boc chemistry for peptide synthesis and the S-sulfonate
(Maugras I et al: Int J Peptide Protein Res, 45:152, 1995) is
compatible with both Fmoc and Boc chemistries. Similar OSPGs
derived from homologous series of S-alkyl, or S-aryl, or S-aralkyl
may also be used in this invention. A primary characterization of
the OSPG is that its use results in the formation of a disulfide
(S--S) bond utilizing one sulfur atom each from the
thiol-containing amino acid and the protecting group. In addition,
the resulting disulfide (S--S) bond is cleavable by the use of any
of a variety of disulfide cleaving agents, including but not
limited to phosphine- and thiol-containing reagents.
[0119] The method employing S.sup.tBu protected SH groups, or other
OSPGs, may be employed for the generation of either solid phase or
soluble libraries. For solid phase libraries, peptides may be
synthesized by use of conventional Fmoc chemistry. In the case of
conventional Fmoc chemistry, Fmoc-L-Cys-(S.sup.tBu) is coupled to
an appropriate resin, via one or more intermediate amino acids, and
additional amino acids are thereafter coupled to the
L-Cys-(S.sup.tBu) residue. S.sup.tBu may be employed with either L-
or D-Cys, and any of a variety of other amino acids, including
designer or unnatural amino acids and mimics thereof, characterized
by an SH group available for binding to a metal ion, including, but
not limited to, 3-mercapto phenylananine and other related
3-mercapto amino acids such as 3-mercapto valine (penicillamine);
2-mercaptoacetic acid; 3-mercaptopropionic acid;
2-mercaptopropionic acid; 3-mercapto-3,3,-dimethyl propionic acid;
3-mercapto,3-methyl propionic acid; 3-mercapto-3,3,-diethyl
proprionic acid; 2-mercapto,2-methyl acetic acid;
3-cyclopentamethlene,3-mercaptopropionic acid;
2-cyclopentamethlene,2-mercaptoacetic acid and related amino acids.
In all these cases, S-protection can be by S.sup.tBu, S-Acm, Mmt,
Npys, S-sulfonate and related groups, as described above.
[0120] Metal Ion Complexation to MBD. The complexation of metal
ions to the sequences in a library, and specifically to the MBD, is
achieved by mixing the sequences with the metal ion. This is
conveniently done in solution, with the solution including an
appropriate buffer. In one approach, the metal ion is, when mixed
with the peptide or peptidomimetic constituents, already in the
oxidation state most preferred for complexing to the MBD. Some
metal ions are complexed in their most stable oxidation state, such
as calcium (II), potassium (I), indium (III), manganese (II),
copper (II), zinc (II) and other metals. In other instances, the
metal must be reduced to a lower oxidation state in order to be
complexed to the MBD. This is true of ferrous, ferric, stannous,
stannic, technetiumoxo[V], pertechnetate, rheniumoxo[V], perrhenate
and other similar metal ions. Reduction may be performed prior to
mixing with the sequences, simultaneously with mixing with the
sequences, or subsequent to mixing with the sequences. Any means of
reduction of metal ions to the desired oxidation state known to the
art may be employed.
[0121] For tetradentate coordination with a metal ion, rhenium is a
preferred ion. Solid phase resin bound peptide or peptidomimetic
sequences may be labeled with rhenium ion by treatment with the
rhenium transfer agent ReOCl.sub.3(PPh.sub.3).sub.2 in the presence
of 1,8-Diazabicyclo[5,4,0] undec-7-ene as a base. The sequences may
then be cleaved from the resin. Alternatively, peptide or
peptidomimetic sequences in a soluble library may similarly be
labeled by treatment with the rhenium transfer agent
ReOCl.sub.3(PPh.sub.3).sub.2 in the presence of
1,8-Diazabicyclo[5,4,0] undec-7-ene as a base. Metal complexation
in the presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) as a
base can conveniently be accomplished at ambient room
temperature.
[0122] In an alternative method of metal complexation a mild base,
such as sodium acetate, can be used. In this case the
thiol-containing sequence, either in solution or bound to solid
phase, is taken in a suitable solvent, such as DMF, NMP, MeOH, DCM
or a mixture thereof, and heated to 60-70.degree. C. with the
rhenium transfer agent ReOCl.sub.3(PPh.sub.3).sub.2 in the presence
of sodium acetate for 15 minutes. Similarly, other bases such as
triethylamine, ammonium hydroxide and so on, may be employed.
According to this invention, MeOH is a preferred choice of solvent
for rhenium complexation in the case of S-deprotected peptides in
solution. The solvent choice for S-deprotected peptides still
attached to the solid phase is guided mainly by considerations of
superior solvation (swelling) of the solid phase. DMF and NMP may
be employed. Various mixtures of these solvents, also in
combination with MeOH, and DCM, CHCl.sub.3 and so on, may also be
employed to yield optimized complexation results.
[0123] In one embodiment of this invention, an S.sup.tBu protected
peptide is treated in situ with rhenium transfer agent in the
presence of DBU and tributylphosphine to effect S-deprotection and
rhenium complexation in one pot. Alternately, complexation of
rhenium to the S.sup.tBu protected peptide in the presence of
rhenium perrhenate may be accomplished by treatment with
Sn[II]Cl.sub.2. This reagent effects S-deprotection as well as
conversion of ReO.sub.4 state to ReO state in situ to cause
complexation of the rhenium to the S-deprotected peptide. A
preferred procedure in this invention is the use of S.sup.tBu
protected peptide with S-deprotection by treatment with
tributylphosphine, and metal complexation of the resulting peptide
utilizing ReOCl.sub.3(PPh.sub.3).sub.2 in the presence of DBU at
room temperature.
[0124] In the libraries of this invention, the MBD forms a reverse
turn structure upon complexation with a metal ion, with the library
constructed such that side chains of amino acids within the MBD are
varied, and similarly amino acids not forming a part of the MBD are
also varied. Various compounds in a library of metallopeptides can
be obtained by varying the sequence of amino acids in a set of
peptides that are all optimized to form a complex of nearly similar
geometry when coordinated with a metal ion. This optimization can
be obtained, for example, by appropriate positioning of amino acids
having high affinity to complex a metal ion. Examples of naturally
occurring amino acids with high affinity for metal complexation
include Cys and His. A library of such peptides, therefore, would
have at least one of these amino acids that is suitably placed in
the sequence, with this amino acid being common to all the
molecules in the library, with this amino acid thus
non-randomized.
[0125] A conceptual, generalized view of a solid phase library of
metallopeptides that is constructed using local conformational
restriction is: ##STR1## where M is a metal ion, A.sub.1 and
A.sub.2 are amino acid side chains forming parts of the reverse
turn structure which is the BBD, and "Peptide Chain" denotes one or
more amino acids. A similar library can also be constructed in
which the components are soluble, and thus not bound to a
resin.
[0126] Another embodiment of this invention provides for
construction of a library with global conformational restriction.
In this embodiment, the MBD can be held constant, and a randomized
or selected series of sequences of amino acids or mimetics varied
to form the library. This type of library encompasses
metallopeptides in which a MBD is an isosteric replacement for a
disulfide, lactam, lactone, thioether or thioester moiety in cyclic
peptides. In these constructs a set MBD is introduced between two
pre-selected ends of a linear peptide or peptidomimetic that
contains the randomized or selected series of sequences of amino
acids or mimetics under investigation. The general structure of a
metallopeptide library of this type is: ##STR2##
Peptide Chain (Containing Biological Function
Domain).about.Resin
[0127] where M is a metal ion and A.sub.1 and A.sub.2 are
structural elements that may provide additional stability to metal
complexation, or may modulate biological activity, such as
determining the organ of clearance, or altering biodistribution
patterns or pharmacokinetics. The "Peptide Chain" sequence may be
randomly varied, thereby resulting in a random library, or may be
directed in a predetermined fashion, based upon known
characteristics of the target molecule.
[0128] One illustration of a globally-constrained metallopeptide
library is a library of peptides wherein all the individual members
of the library include a metal ion-binding domain and the library
is directed specifically towards a family of melanocortin
receptors. The general formula of this library of peptides, before
complexation to a metal ion, is:
[0129] A.sub.1-Aa.sub.1-Aa.sub.2-Aa.sub.3-A.sub.2.about.Resin
[0130] where X is a fixed MBD including a plurality of amino acids,
so that all of the valences of the metal ion are satisfied upon
complexation of the metal ion with X, A.sub.1 and A.sub.2 each
comprise from 0 to about 20 amino acids, and Aa.sub.1, Aa.sub.2 and
Aa.sub.3 each comprise one or more amino acids connected to X
through an amide, thioether, thioester, ester, carbamate, or
urethane bond, wherein each of Aa.sub.1, Aa.sub.2 and Aa.sub.3 is
varied. In this example, the MBD may include an OSPG. Other thiols
in the sequence may optionally include S-protecting groups that are
not orthogonal, such that the OSPG may be removed without removal
of other S-protecting groups in the sequence.
[0131] For solid phase libraries the peptide constructs are
attached to a resin, and the resin is omitted for soluble
libraries. The functional equivalent of each these peptide
libraries may also be obtained through the development of a library
of non-amino acid building blocks so as to result in structural
mimics of these peptides. The peptide bonds may be replaced by
pseudopeptide bonds, such as thioamides, thioethers, substituted
amines, carbanate, urethane, aliphatic moieties, and functionally
similar constructs.
[0132] A peptide library is first assembled according to the
sequence specification and degeneration, as described above, by
well-known methods of peptide synthesis. These libraries can be
synthesized as discreet, spatially addressable compounds in
parallel synthesis, using split synthesis approaches, or by
deconvolution techniques of soluble libraries. Using similar
methods, a pseudopeptide, peptidomimetic or non-peptide library can
be obtained. The non-peptide libraries may also optionally
incorporate one of various tagging approaches that are well known
to those skilled in the art. Both solid-phase and soluble libraries
can be obtained in this manner. The entire library is then reacted
with an appropriate metal-complexing agent to obtain the
corresponding metal-coordinated library, comprising a similar class
of predetermined structures. For example, to complex a peptide
library with rheniumoxo metal ion, the peptide library can be
treated with Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the presence of
sodium acetate. This procedure results in quantitative complexation
of ReO with the peptide. In order to complex Zn, Co, Mn, Fe or Cu
ions, the peptide library is treated with chloride or other
suitable salts of these metal ions to yield the library of
corresponding metal ions. Essentially, a variety of metal ions can
be used to construct different metallopeptide libraries. One
limiting factor in selection of the appropriate metal ion is the
relative stability of a particular metal-peptide complex, related
in large part to the metal-peptide binding constant or constants.
It is well known in the art that some metal-peptide constructs are
stable only within specified pH or other special conditions, or are
easily oxidized in air. Some peptide-metal ion complexes, such as
those with ReO, are stable in pure form and can be isolated and
stored under normal storage conditions for a long period of
time.
[0133] A metallopeptide library constructed according to this
invention can be screened to identify one or more receptor-binding
or pharmacologically-active melanocortin receptor-specific
candidates by various techniques that have been reported in the
prior art. Both soluble and solid phase libraries may be directly
employed in these assays. These techniques include direct target
binding approaches as described by Lam and coworkers (Lam K S et
al: Nature 354:82-84, 1991; Lam K S et al: Nature 360:768, 1992),
deconvolution and iterative re-synthesis approaches (Houghten R A
et al: Proc Natl Acad Sci USA 82:5131-5135, 1985; Berg et al: J Am
Chem Soc 111:8024-8026, 1989; Dooley C T et al: Science
266:2019-2022, 1994; Blondelle S E: Antimicrob Agents Chemother
38:2280-2286, 1994; Panilla C: Biopolymers 37:221-240, 1995),
approaches using orthogonal pools of two co-synthesized libraries
according to Tartar and coworkers (Deprez B et al: J Am Chem Soc
117: 5405-5406, 1995), positional scanning methods devised by
Houghton and coworkers that eliminate iterative re-synthesis
(Dooley C T et al: Life Sci 52:1509-1517, 1993; Pinilla C et al:
Biotechniques 13:901-905, 1992; Pinilla C et al: Drug Dev Res
33:133-145, 1992), and a combination of the positional scanning
method with split synthesis methods (Erb E et al: Proc Natl Acad
Sci USA, 91:11422-11426, 1994).
[0134] Among these techniques, the deconvolution and iterative
resynthesis approach, the approach involving orthogonal pools of
two co-synthesized libraries, and the positional scanning method
may be directly applied to soluble metallopeptide libraries to
elucidate the structure of a "hit," or peptide identified as a
receptor-binding or pharmacologically-active candidate in the
screening process. For solid phase libraries, other than spatially
addressable parallel synthesis libraries, the structure of hits can
be directly determined by various strategies well known to those
skilled in the art. These include direct mass spectrometric
analysis of compounds covalently bound to solid phase matrix of
particles by the use of matrix-assisted laser desorption/ionization
(MALDI) techniques (Siuzdak G et al: Bioorg Med Chem Lett 6:979,
1996; Brown B B et al: Molecular Diversity 1:4-12, 1995). The
technique of creating a series of partially end-capped compounds at
each of the synthetic steps during library assembly also helps in
unambiguous identification by mass spectrometry (Youngquist R S et
al: J Am Chem Soc, 117:3900-3906, 1995; Youngquist R S et al: Rapid
Commun Mass Spectr 8:77-81, 1994). In addition to these analytical
techniques, various encoding strategies that have been devised for
structure elucidation in organic molecule-based libraries,
including non-peptide and non-nucleotide libraries, may be
utilized. Various encoding strategies, such as DNA encoding,
peptide encoding, haloaromatic tag encoding, and encoding based on
radiofrequency transponders, are now well known in the art and can
be used directly in combination with metallopeptide libraries.
These tagging strategies require the incorporation of the tags
during the course of synthesis of libraries, which can be
accomplished during the construction of metallopeptide libraries,
since metal complexation is a final, post-synthesis step.
[0135] Structural Diversity of Melanocortin Receptor-Specific
Library Members. Examples of some of the molecular templates which
may be employed in this invention are shown below for tetradentate
metal ion complexation. In general, these molecular templates
define groups of metallopeptides of this invention which, by
substitution as provided, give rise to libraries of metallopeptides
for use in determining melanocortin receptor-specific compounds,
which may be either agonist or antagonist compounds. The templates
are provided without the metal ion, it being understood that the
compounds exhibit enhanced specificity for melanocortin receptors
only upon metal ion complexation. R.sub.1-Lll-Aaa-Bbb-Ccc-R.sub.2
Template 1 and R.sub.1-Bbb-Aaa-Ccc-R.sub.2 Template 2
[0136] Where R.sub.1 is any functionality that potentiates the
intrinsic activity of the remainder of the molecule, including but
not limited to providing an auxiliary or secondary receptor
contact. Any of a variety of amino acids and non-peptide groups may
be employed, including an amino acid chain from one to about four
neutral or charged L- or D-configuration amino acid residues. If
R.sub.1 is a non-peptide group, it may be a linear or branched
alkyl, aryl, alkene, alkenyl or aralkyl chain.
[0137] Where Aaa is an L- or D-configuration cationic amino acid
with a positively charged side chain. Preferred amino acids include
L-configuration Lys, Arg, Orn, Dpr or Dbu, and derivatives, analogs
or homologs thereof, including both natural and synthetic amino
acids. Aaa provides an N (nitrogen atom) for metal ion
complexation.
[0138] Where Bbb is an L- or D-configuration amino acid with an
aromatic side chain. Preferred amino acids include D-configuration
Phe, Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof. The aromatic ring in Bbb
may be functionalized with halogen, alkyl or aryl groups. Bbb
provides an N for metal ion complexation.
[0139] Where Ccc is an amino acid that provides both an N, from the
alpha amino group, and an S (sulfur atom), from a side chain group,
for metal ion complexation. Preferred amino acids include L- or
D-configuration Cys, Pen and Hcys.
[0140] Where Lll is a D-configuration amino acid with an aromatic
side chain. Preferred amino acids include D-configuration Phe,
Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof. The aromatic ring in Lll
may be functionalized with halogen, alkyl or aryl groups. Lll does
not provide an N for metal ion complexation.
[0141] Where R.sub.2 is an amino acid with an aromatic side chain.
Preferred amino acids include L- or D-configuration Phe, Trp,
Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids. The C-terminus may be free or amidated.
R.sub.2 may also be the corresponding des-carboxylamino acid of any
of the foregoing. Alternatively, R.sub.2 may be eliminated.
[0142] FIG. 1 depicts the structure of Template 1, and FIG. 2
depicts the structure of Template 2, in both cases showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3 .mu.l metal ion bond.
R.sub.1-Ddd-Bbb-Aaa-R.sub.3 Template 3
[0143] Where R.sub.1, Bbb and Aaa are as described above.
[0144] Where Ddd is an amino acid that provides an S, from a side
chain group, for metal ion complexation. Preferred amino acids
include L- or D-configuration Cys, Pen and Hcys.
[0145] Where R.sub.3 is an amino acid with an aromatic side chain
that provides an N for metal ion complexation. Preferred amino
acids include L- or D-configuration Phe, Trp, Phe(4'Cl), Phe(3',4'
Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl), Hphe, Pgl,
Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br), Lys(Bz),
Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and derivatives, analogs or
homologs thereof, including both natural and synthetic amino acids.
The C-terminus may be free or amidated. R.sub.3 may also be the
corresponding des-carboxylamino acid of any of the foregoing.
[0146] FIG. 3 depicts the structure of Template 3, showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3S.sub.1 metal ion bond.
R.sub.4-Eee-Bbb-Ccc-R.sub.2 Template 4
[0147] Where R.sub.2, Bbb and Ccc are as described above.
[0148] Where R.sub.4 is a functionality that provides a cationic
center. Preferred amino acids include L- or D-configuration Lys,
Arg, Orn, Dpr or Dbu, and derivatives, analogs or homologs thereof,
including both natural and synthetic amino acids. The N-terminus of
the amino acid may be functionalized with any of a variety of
neutral amino acid and non-peptide groups, including linear or
branched alkyl, aryl, alkene, alkenyl or aralkyl chains.
[0149] Where Eee is an uncharged L- or D-configuration amino acid
that provides an N for metal ion complexation. Preferred amino
acids include Gly and L-configuration Ala, Nle, Leu, Val, Phe or
Trp, and derivatives, analogs or homologs thereof, including both
natural and synthetic amino acids. In a preferred embodiment, Eee
isn an amino acid with an aliphatic side chain.
[0150] FIG. 4 depicts the structure of Template 4, showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3S.sub.1 metal ion bond.
R.sub.1-Fff-Aaa-Ggg-Ccc-R.sub.5 Template 5
[0151] Where R.sub.1, Aaa and Ccc are as described above.
[0152] Where Fff is an L- or D-configuration aromatic amino acid.
Preferred amino acids include D-configuration Phe, Phe(4'Cl),
Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl),
Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br),
Lys(Bz), Thr(Bzl), Cys(Bzl), Tyr(BzlCl.sub.2), Tic, Tiq or Tca, and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids. The aromatic ring in Fff may be
substituted with halogen, alkyl or aryl groups. Fff does not
provide an N for metal ion complexation.
[0153] Where Ggg is an L- or D-configuration aromatic amino acid.
Preferred amino acids include L-configuration Phe, Phe(4'Cl),
Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl), Phe(4'-Phenyl),
Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z), Lys(Z-2'Br),
Lys(Bz), Thr(Bzl), Cys(Bzl) or Tyr(BzlCl.sub.2), and derivatives,
analogs or homologs thereof, including both natural and synthetic
amino acids. The aromatic ring in Ggg may be substituted with
halogen, alkyl or aryl groups. Ggg provides an N for metal ion
complexation.
[0154] Where R.sub.5 is preferably an amide, substituted amide,
ester or carboxylate group. R.sub.5 may also be and L- or
D-configuration amino acid or amino acid amide, including an
aromatic, aliphatic, neutral or charged amino acid.
[0155] FIG. 5 depicts the structure of Template 5, showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3S.sub.1 metal ion bond.
R.sub.1-Hhh-Aaa-Bbb-Ccc-R.sub.5 Template 6
[0156] Where R.sub.1, Aaa, Bbb, Ccc and R.sub.2 are as described
above.
[0157] Where Hhh is an L- or D-configuration cationic amino acid
with a positively charged side chain. Preferred amino acids include
L-configuration Lys, Arg, Orn, Dpr or Dbu, and derivatives, analogs
or homologs thereof, including both natural and synthetic amino
acids. Hhh does not provide an N for metal ion complexation.
[0158] FIG. 6 depicts the structure of Template 6, showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3S.sub.1 metal ion bond.
R.sub.1-Iii-Iii-Ccc-Jjj-Kkk-R.sub.2 Template 7
[0159] Where R.sub.1, Ccc and R.sub.2 are as described above.
[0160] Where Iii is an L- or D-configuration amino acid that
provides an N for metal ion complexation. Preferred amino acids
includes Ala, Gly, Nle, Val, Leu, Ile, His, Lys, or Arg, and
derivatives, analogs or homologs thereof, including both natural
and synthetic amino acids.
[0161] Where Jjj is an L- or D-configuration amino acid with an
aromatic side chain. Preferred amino acids include D-configuration
Phe, Phe(4'Cl), Phe(3',4' Di-Cl), Phe(4'-nitro), Phe(4'-methyl),
Phe(4'-Phenyl), Hphe, Pgl, Trp, 1-Nal, 2-Nal, Ser(Bzl), Lys(Z),
Lys(Z-2'Br), Lys(Bz), Thr(Bzl), Cys(Bzl), or Tyr(BzlCl.sub.2), and
derivatives, analogs or homologs thereof. The aromatic ring in Jjj
may be functionalized with halogens, alkyl or aryl groups. Jjj does
not provide an N for metal ion complexation.
[0162] Where Kkk is an L- or D-configuration cationic amino acid
with a positively charged side chain. Preferred amino acids include
L-configuration Lys, Arg, Orn, Dpr or Dbu, and derivatives, analogs
or homologs thereof, including both natural and synthetic amino
acids. Aaa does not provide an N for metal ion complexation.
[0163] FIG. 7 depicts the structure of Template 7, showing
coordination with a tetradenate coordination sphere metal ion,
resulting in an N.sub.3S.sub.1 metal ion bond.
[0164] The foregoing templates may be employed with tetradentate
coordination sphere metal ions, such as various forms of technetium
and rhenium. Corresponding templates may be constructed for use
with metal ions of other coordination spheres.
[0165] Representative Peptides of this Invention. Representative
peptides of this invention were made using library and synthesis
methods described herein, and selected peptides were tested using a
binding assay. Table 1 sets forth peptides of this invention, and
the results of competitive inhibition binding assays. The peptides
were synthesized using conventional peptide synthesis methods, and
were complexed with rhenium using the methods described herein.
[0166] The competitive inhibition binding assay was conducted using
membranes prepared from hMC4-R and B-16 mouse melanoma cells
(containing MC1-R) using 0.4 nM .sup.125I-NDP-.alpha.-MSH (New
England Nuclear, Boston, Mass., USA) in 50 mM HEPES buffer
containing 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, and 5 mM KCl, at pH
7.2. The assay tube also contained a chosen concentration of the
test peptide of this invention, complexed to a rhenium metal ion as
indicated, for determining its efficacy in inhibiting the binding
of .sup.125I-NDP-.alpha.-MSH to its receptor. Non-specific binding
was measured by complete inhibition of binding of
.sup.125I-NDP-.alpha.-MSH in the assay with the presence of 1 .mu.M
.alpha.-MSH. Incubation was for 90 minutes at room temperature,
after which the assay mixture was filtered and the membranes washed
three times with ice cold buffer. The filter was dried and counted
in a gamma counter for remaining radioactivity bound to the
membranes. 100% specific binding was defined as the difference in
radioactivity (cpm) bound to cell membranes in the absence and
presence of 1 .mu.M .alpha.-MSH. The cpm obtained in presence of
test compounds were normalized with respect to 100% specific
binding to determine the percent inhibition of
.sup.125I-NDP-.alpha.-MSH binding. Each assay was conducted in
triplicate and the actual mean valves are described in Table 1.
Negative numbers in Table 1 under "% Inhibition" result from
experimental variance, and are indicative of no inhibition;
similarly, values over 100% also result from experimental variance,
and are indicative of complete inhibition. TABLE-US-00002 TABLE 1
Melanocortin Receptor Screening Results: Receptor Binding Assay
Ion/ Conc. % Inhibition Linear Cut off MC1-R ID peptide Sequence
Structure (.mu.M) MC4-R (B-16) PL-808 ReO[V]
Ac-L-His-L-Trp-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 3) 1 0 -3 PL-809
ReO[V] Ac-L-His-L-Hphe-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 4) 1 0 -4
PL-810 ReO[V] Ac-L-His-L-Nal 2-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 5)
1 -1 -2 PL-811 ReO[V] Ac-L-His-L-Phg-L-Cys-L-Trp-NH.sub.2 (SEQ ID
NO: 6) 1 0 -5 PL-812 Linear Ac-L-His-L-Phe-L-Cys-L-Trp-NH.sub.2
(SEQ ID NO: 7) 10 29 54 PL-813 Linear
Ac-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 8 91 PL-814 ReO[V]
Ac-L-His-L-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 7) 1 0 -4 PL-815
ReO[V] Ac-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 10 7 93 PL-816
ReO[V] Ac-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 28 70 PL-836 ReO[V]
Ac-L-His-L-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 10 26 78 PL-837 ReO[V]
Ac-L-His-D-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 10 0 54 PL-838 ReO[V]
Ac-L-His-L-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10 0 35 NO: 8)
PL-839 ReO[V] Ac-L-His-L-Phe-L-Trp-L-Cys-NH.sub.2 (SEQ ID NO: 9) 10
9 12 PL-840 ReO[V] Ac-L-His-L-Phe-D-Trp-L-Cys-NH.sub.2 10 10 8
PL-841 ReO[V] Ac-L-His-D-Phe-D-Trp-L-Cys-NH.sub.2 10 0 19 PL-842
ReO[V] Ac-L-His-D-Phe-L-Trp-L-Cys-NH.sub.2 10 2 4 PL-843 ReO[V]
Ac-D-Phe-Gly-L-Cys-L-Trp-NH.sub.2 10 1 10 PL-844 ReO[V]
Ac-L-Phe-Gly-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 10) 10 2 10 PL-845
ReO[V] Ac-D-Phe-L-His-Gly-L-Cys-L-Trp-NH.sub.2 10 0 5 PL-846 ReO[V]
Ac-L-Phe-D-His-Gly-L-Cys-L-Trp-NH.sub.2 10 0 2 PL-847 ReO[V]
Ac-L-Phe-L-His-Gly-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10 6 -6 NO: 11)
PL-848 ReO[V] Ac-D-Phe-D-His-Gly-L-Cys-L-Trp-NH.sub.2 10 10 19
PL-989 ReO[V] Bz-D-Tyr-L-Nal-L-Cys-L-Phe-NH.sub.2 10 11 35 PL-997
ReO[V] Bz-D-Nal-L-Tyr-L-Cys-L-Phe-NH.sub.2 10 36 6 PL-1073 ReO[V]
Ac-L-Ala-L-Tle-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 12) 1 13 -6 PL-1089
ReO[V] Ac-L-Ala-L-pF-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10 10 0 NO:
13) PL-1090 ReO[V] Ac-L-Ala-L-Tyr(3',5' di-I,
4'-Ac)-L-Cys-L-Trp-NH.sub.2 1 10 2 (SEQ ID NO: 14) PL-1091 ReO[V]
BAla-Gly-L-Cys(Bzl)-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1 13 -1 NO: 15)
PL-1092 ReO[V] BAla-Gly-L-Lys(TFA)-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1 4
4 NO: 16) PL-1093 ReO[V] BAla-Gly-L-Phe(2,4-di
Cl)-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1 2 2 NO: 17) PL-1094 ReO[V]
BAla-Gly-L-Phe(2-Cl)-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10 19 57 NO: 18)
PL-1095 ReO[V] BAla-Gly-L-Lys(Z)-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO:
19) 10 8 3 PL-1096 ReO[V] BAla-Gly-L-Leu-L-Cys-L-Trp-NH.sub.2 (SEQ
ID NO: 20) 10 12 6 PL-1102 ReO[V] BAla-Gly-D-Nal
2-L-Cys-L-Trp-NH.sub.2 10 60 9 PL-1103 ReO[V]
BAla-Gly-D-Phg-L-Cys-L-Trp-NH.sub.2 10 12 -3 PL-1109 ReO[V]
L-Lys-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 37 73 PL-1110 ReO[V]
L-Lys-L-His-L-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 21) 10 36 11
PL-1111 ReO[V] L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 0 0 PL-1112
ReO[V] L-His-L-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 7) 10 8 0
PL-1113 ReO[V] BAla-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 31 82
PL-1114 ReO[V] BAla-L-His-L-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO:
22) 10 10 2 PL-1140 ReO[V] Ac-D-Phe-Arg-L-Cys-L-Phe-NH.sub.2 10 12
62 PL-1141 ReO[V] Ac-D-Phe-Arg-L-Cys-L-Trp-NH.sub.2 10 11 57
PL-1144 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Cys-L-Phe-NH.sub.2 1 14 99
PL-1145 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 17 96
PL-1146 ReO[V] Ac-D-Phe-Gly-L-Cys-L-Phe-NH.sub.2 10 9 39 PL-1147
ReO[V] Ac-D-Pip-Gly-L-Cys-L-Phe-NH.sub.2 10 12 40 PL-1156 ReO[V]
Ac-D-Phe-L-Tle-L-Cys-L-Trp-NH.sub.2 10 14 -19 PL-1157 ReO[V]
Ac-L-Nle-L-Arg-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 92 53 PL-1158
ReO[V] BAla-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 62 0 PL-1159
ReO[V] BAla-L-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 63 67 PL-1160
ReO[V] BAla-L-Phe-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 66 1 PL-1161
ReO[V] BAla-L-Nal 2-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 64 3 PL-1162
ReO[V] Heptanoyl-L-Arg-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 79 64
PL-1163 ReO[V] BAla-D-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 51 4
PL-1164 ReO[V] BAla-D-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 42 2
PL-1165 ReO[V] BAla-D-Phe-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 43 7
PL-1166 ReO[V] BAla-D-Nal 2-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 44 5
PL-1167 ReO[V] Abu-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 31 -1
PL-1168 ReO[V] 6-Axh-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 41 -2
PL-1169 ReO[V] 8-Aoc-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 41 -3
PL-1170 ReO[V] 11-Aun-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 27 9
PL-1171 ReO[V] 12-Ado-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 29 -5
PL-1172 ReO[V] Ac-L-Arg-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 58 -7
PL-1173 ReO[V] Ac-L-Lys-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 41 -2
PL-1174 ReO[V] Ac-L-Orn-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 53 -2
PL-1175 ReO[V] Ac-L-Nle-L-Arg-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10
96 88 PL-1176 ReO[V] 7-Ahept-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 40
-8 PL-1177 ReO[V] B-Gpa-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 48 -6
PL-1178 ReO[V] Ac-L-Nle-L-Ala-L-His-D-HPhe-L-Arg-L-Cys-L-Trp- 1 4
51 NH.sub.2 PL-1179 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-His-L-Arg-L-Cys-L-Trp-NH.sub.2 1 21 -5
PL-1180 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Trp-L-Arg-L-Cys-L-Trp-NH.sub.2 1 24 86
PL-1181 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Tyr-L-Arg-L-Cys-L-Trp-NH.sub.2 1 27 86
PL-1183 ReO[V] Ac-L-Nle-L-Ala-L-Arg-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 82
96 NH.sub.2 PL-1184 ReO[V] Ac-L-Nle-L-Ala-L-Tyr-D-Nal
2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 58 36 PL-1185 ReO[V]
Ac-L-Nle-L-Ala-L-Phe-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 35 52 NH.sub.2
PL-1186 ReO[V] Ac-L-Nle-L-Ala-L-Trp-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 35
17 NH.sub.2 PL-1187 ReO[V] Ac-L-Ala-L-His-D-Nal
2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 48 58 PL-1188 ReO[V]
Ac-L-Nle-L-Ala-D-Trp-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 38 -12 NH.sub.2
PL-1189 ReO[V] Ac-L-Nle-L-Ala-D-Phe-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 36
-15 NH.sub.2 PL-1190 ReO[V] Ac-L-Nle-L-Ala-D-His-D-Nal
2-L-Arg-L-Cys-L-Trp- 1 31 17 NH.sub.2 PL-1191 ReO[V]
Ac-L-Nle-L-Ala-D-Arg-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 41 38 NH.sub.2
PL-1192 ReO[V] Ac-L-Nle-L-Ala-D-Tyr-D-Nal 2-L-Arg-L-Cys-L-Tyr- 1 26
-5 NH.sub.2 PL-1193 ReO[V] Ac-L-Glu-L-Ala-D-Nal
2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 29 51 PL-1194 ReO[V]
HOOC--(CH.sub.2).sub.3--CO-L-His-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 -3 23
NH.sub.2 PL-1195 ReO[V] Ac-L-Ala-L-Glu-L-His-D-Nal
2-L-Arg-L-Cys-L-Trp- 1 9 45 NH.sub.2 PL-1196 ReO[V]
Ac-L-Nle-L-Glu-L-His-D-Nal 2-L-Arg-L-Cys-L-Trp- 1 31 89 NH.sub.2
PL-1197 ReO[V] Ac-L-Glu-L-His-D-Nal 2-L-Arg-L-Cys-L-Trp-NH.sub.2 1
9 44 PL-1198 ReO[V] Ac-L-Glu-L-His-D-Nal 2-L-Cys-L-Arg-NH.sub.2 1 4
-5 PL-1199 ReO[V] Ac-L-Glu-L-His-L-Cys-D-Nal 2-L-Arg-NH.sub.2 1 1
-11 PL-1200 ReO[V] Ac-L-Glu-L-His-D-Nal
2-L-Cys-L-Arg-L-Trp-NH.sub.2 1 10 8 PL-1201 ReO[V]
Ac-L-Glu-L-His-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1 65 -7 PL-1202
ReO[V] Ac-L-Nle-L-Ala-D-Trp-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 17
10 PL-1203 ReO[V] Ac-L-Nle-L-Ala-D-Nal 2-D-Nal 2-L-Arg-L-Cys-L-Trp-
1 35 4 NH.sub.2 PL-1204 ReO[V] Ac-L-Nle-L-Ala-D-Nal 1-D-Nal
2-L-Arg-L-Cys-L-Trp- 1 32 0 NH.sub.2 PL-1205 ReO[V]
Ac-L-Nle-L-Ala-Gly-D-Nal 2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 54 60
PL-1206 ReO[V] Heptanoyl-D-Trp-D-Nal 2-L-Arg-L-Cys-L-Trp-NH.sub.2 1
17 0 PL-1207 ReO[V] Ac-L-Ala-L-His-D-Nal 2-L-Cys-D-Nal
2-L-Arg-L-Trp- 1 84 26 NH.sub.2 PL-1209 ReO[V] Ac-L-Nal
2-L-His-D-Nal 2-L-Cys-D-Nal 2-L-Arg-L- 1 77 1 Trp-NH.sub.2 PL-1210
ReO[V] Ac-D-Nal 2-L-His-D-Nal 2-L-Cys-D-Nal 2-L-Arg-L- 1 71 0
Trp-NH.sub.2 PL-1211 ReO[V] Heptanoyl-L-Glu-L-His-D-Nal
2-L-Cys-D-Nal 2-L-Arg- 1 67 26 L-Trp-NH.sub.2 PL-1212 ReO[V]
Ac-L-Glu-D-Trp-D-Nal 2-L-Cys-D-Nal 2-L-Arg-L-Trp- 1 80 0 NH.sub.2
PL-1213 ReO[V] Ac-D-Trp-D-Arg-L-Nal 2-D-Nal 2-L-Cys-D-Nal 2-L- 1 94
34 Arg-L-Trp-NH.sub.2 PL-1214 ReO[V] Heptanoyl-L-Arg-Gly-D-Nal
2-D-Nal 2-L-Cys-D-Nal 1 78 47 2-L-Arg-L-Trp-NH.sub.2 PL-1215 ReO[V]
Gly-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 31 20 PL-1216 ReO[V]
L-Lys-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 47 0 PL-1217 ReO[V]
L-Lys(Z)-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 52 24 PL-1218 ReO[V]
BAla-L-Arg(Tos)-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 57 1 PL-1220 ReO[V]
BAla-L-Tle-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 69 30 PL-1221 ReO[V]
BAla-L-Tyr(BzlDiCl 2,6)-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 35 37
PL-1222 ReO[V] BAla-Gly-D-Phe-L-CysL-Trp-NH.sub.2 10 27 9 PL-1223
ReO[V] Ac-L-Nle-L-Arg-D-Phe-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 64 79
PL-1225 ReO[V] GBzA-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 12 0
PL-1226 ReO[V] AVA-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 19 0 PL-1227
ReO[V] 2-Abz-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 5 0 PL-1228 ReO[V]
BAla-L-His-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 11 64 PL-1229 ReO[V]
BAla-L-Nle-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 32 21 PL-1230 ReO[V]
GAA-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 32 20 PL-1231 ReO[V]
GVA(Cl)-Gly-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 32 28 PL-1232 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 90 49 PL-1233
ReO[V] BAla-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 94 100 PL-1234
ReO[V] BAla-D-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 83 84 PL-1235
ReO[V] Heptanoyl-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 96 100
PL-1236 ReO[V] Heptanoyl-D-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 54
70 PL-1237 ReO[V] BAla-Gly-D-Lys(Z)-L-Cys-L-Trp-NH.sub.2 10 26 86
PL-1238 ReO[V] BAla-Gly-D-Tyr(Bzl)-L-Cys-L-Trp-NH.sub.2 10 40 40
PL-1239 ReO[V] BAla-Gly-D-Phe(DiF 3,4)-L-Cys-L-Trp-NH.sub.2 10 20
18 PL-1240 ReO[V] BAla-Gly-D-Val-L-Cys-L-Trp-NH.sub.2 10 13 0
PL-1241 ReO[V] BAla-Gly-D-Nal 1-L-Cys-L-Trp-NH.sub.2 10 34 23
PL-1242 ReO[V] D-Nal 2-Gly-L-Arg-L-Cys-L-Trp-NH.sub.2 10 61 80
PL-1243 ReO[V] D-Nal 2-Gly-D-Arg-L-Cys-L-Trp-NH.sub.2 10 62 88
PL-1244 ReO[V] D-Phe-Gly-L-Arg-L-Cys-L-Trp-NH.sub.2 10 46 69
PL-1245 ReO[V] D-Phe-Gly-D-Arg-L-Cys-L-Trp-NH.sub.2 10 50 74
PL-1248 ReO[V] Ac-L-Nle-D-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10
69 63 PL-1249 ReO[V] BAla-L-Nle-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2
10 64 40 PL-1250 ReO[V] BAla-D-Nle-L-Ala-D-Nal
2-L-Cys-L-Trp-NH.sub.2 1 79 10 PL-1251 ReO[V]
Ac-L-Nle-L-Arg-L-Phe-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 82 77 PL-1252
ReO[V] D-Lys(Z)-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 44 34 PL-1253
ReO[V] L-Ala-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 54 27 PL-1254
ReO[V] L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 40 43 PL-1255 ReO[V]
Bz-L-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 85 14 PL-1256 ReO[V]
L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 60 57 PL-1257 ReO[V]
HOOC(CH.sub.2).sub.2--CO-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 51
43 PL-1258 ReO[V] BAla-L-Nle-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 66 57
PL-1259 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Cys-NH.sub.2 1 31
82 PL-1260 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Nal 2-L-Cys-NH.sub.2 1 74
49 PL-1261 ReO[V] Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2
1 30 101 PL-1262 ReO[V] Heptanoyl-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2
1 20 44 PL-1263 ReO[V]
Ac-L-Arg(Tos)-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 26 33 PL-1264
ReO[V] Ac-L-Nle-L-Glu-L-His-D-Nal 2-L-Arg-L-Trp-L-Cys- 0.1 54 45
NH.sub.2 PL-1265 ReO[V]
Ac-L-Glu-L-His-Gly-L-Arg-L-Trp-L-Cys-NH.sub.2 (SEQ ID 1 67 34 NO:
23) PL-1266 ReO[V] Ac-L-Nle-L-Ala-D-Trp-D-Nal 2-L-Arg-L-Trp-L-Cys-
1 87 30 NH.sub.2 PL-1267 ReO[V] Admentoyl-Gly-D-Nal
2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 55 36 PL-1268 ReO[V]
Bz-L-His-Gly-D--Nal 2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 97 57 PL-1269
ReO[V] Bz-L-His-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1 99 25 PL-1270
Linear Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 57
101 PL-1271 ReO[V] Ac-D-Trp-D-Arg-L-Nal 2-L-Cys-D-His-L-Nal
2-NH.sub.2 1 32 35 PL-1272 Linear
Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 36 100 PL-1273
ReO[V] Ac-L-Nle-L-Arg-D-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 53 78
PL-1274 ReO[V] Ac-L-Nle-L-Arg-D-Phe-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10
45 75 PL-1275 ReO[V] Des-aminoTyr-L-Lys-L-Ala-D-Nal
2-L-Cys-L-Trp-NH.sub.2 1 17 -4 PL-1276 ReO[V]
Heptanoyl-L-Lys-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 64 4 PL-1277
ReO[V] 3-Pyridine propionyl-L-Lys-L-Ala-D-Nal 2-L-Cys-L- 1 38 -17
Trp-NH.sub.2
PL-1278 ReO[V] EtOOC--(CH.sub.2).sub.4--CO-L-Lys-L-Ala-D-Nal
2-L-Cys-L-Trp- 1 32 -13 NH.sub.2 PL-1279 ReO[V]
(s)-2-OH-isocaproyl--L-Lys-L-Phe-D-Nal 2-L-Cys-L- 1 55 -10
Trp-NH.sub.2 PL-1280 ReO[V] 4-MePhenoxyacetyl--L-Lys-L-Ala-D-Nal
2-L-Cys-L- 1 49 -11 Trp-NH.sub.2 PL-1281 ReO[V]
Heptanoyl-L-Lys-L-Ala-D-Phe-L-Cys-L-Trp-NH.sub.2 1 29 60 PL-1282
ReO[V] Heptanoyl-L-Lys-L-Ala-D-Phe-D-Cys-L-Trp-NH.sub.2 1 14 -30
PL-1283 ReO[V] Des-aminoPhe-L-Lys-L-Ala-D-Nal 2-L-Cys-L-Trp- 1 45
-20 NH.sub.2 PL-1284 ReO[V] BAla-L-Lys(Ac)-L-Ala-D-Nal
2-L-Cys-L-Trp-NH.sub.2 1 27 -26 PL-1285 ReO[V]
Ac-L-Nle-L-Ala-D-Trp-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 32 54
PL-1286 ReO[V] Ac-L-Nle-L-Ala-D-Nal 2-D-Phe-L-Arg-L-Trp-L-Cys- 1 34
7 NH.sub.2 PL-1287 ReO[V]
Ac-L-Nle-L-Ala-D-His-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 40 62
PL-1288 ReO[V]
EtOOC--(CH.sub.2).sub.4--CO-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 16
-3 PL-1289 ReO[V] 4-n-Heptanoyl-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1
31 -7 PL-1290 ReO[V] Des-aminoTyr-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2
1 24 -22 PL-1291 ReO[V]
Me2-CH--CH(L-OH)--CO-D-Phe-L-Arg-L-Cys-L-Trp- 1 29 1 NH.sub.2
PL-1292 ReO[V] Ac-D-Ala-L-HisL-Cys-D-Nal 2-L-Arg-Tryptamide 1 87 25
PL-1293 ReO[V] Ac-D-Ala-L-HisL-Cys-D-Nal 2-L-Arg-Tryptamide 1 96 21
PL-1294 ReO[V] Bz-L-His-L-Cys-D-Phe-L-Arg-L-Trp-NH.sub.2 1 65 62
PL-1295 ReO[V] Bz-L-Ala-L-Cys-D-Phe-L-Arg-L-Trp-NH.sub.2 1 72 62
PL-1296 ReO[V] Bz-L-Nal 2-L-Cys-D-Phe-L-Arg-L-Trp-NH.sub.2 1 51 51
PL-1297 ReO[V] Des-aminoPhe-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1 91
31 PL-1298 ReO[V] Heptanoyl-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1 95
43 PL-1299 ReO[V] Heptanoyl-BAla-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID
1 19 1 NO: 24) PL-1300 ReO[V]
Heptanoyl-L-Ala-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1 22 62 NO: 25)
PL-1301 ReO[V] L-Lys(Z)-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 72 49
PL-1302 ReO[V] Heptanoyl-L-His-D-Phe-L-Arg-D-Cys-L-Trp-NH.sub.2 1
24 77 PL-1303 ReO[V] Heptanoyl-L-His-D-Nal
2-L-Arg-D-CysL-Trp-NH.sub.2 1 60 39 PL-1304 ReO[V]
D-Lys(Z)-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 42 -10 PL-1305 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 91 14 PL-1306
ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe-L-Cys-L-Trp-NH.sub.2 1 36 56
PL-1309 ReO[V] Heptanoyl-L-Cys-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1
10 9 NO: 26) PL-1310 ReO[V] Des-aminoTyr-L-Lys-L-Ala-D-Nal
2-L-Cys-L-Trp-NH.sub.2 10 78 100 PL-1311 ReO[V]
Heptanoyl-L-Lys-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 61 40 PL-1312
ReO[V] 3-Pyridine propionyl-L-Lys-L-Ala-D-Nal 2-L-Cys-L- 10 71 43
Trp-NH.sub.2 PL-1313 ReO[V]
Heptanoyl-L-Ala-L-Trp-D-Arg-L-Cys-L-Phe-NH.sub.2 1 6 16 PL-1314
ReO[V] C.sub.11H.sub.23--CO-L-Trp-D-Arg-L-Cys-L-Phe-NH.sub.2 10 85
53 PL-1315 ReO[V] Ac-L-Arg-L-Trp-L-Nle-L-Cys-L-Phe-NH.sub.2 (SEQ ID
10 70 69 NO: 27) PL-1316 ReO[V]
Ac-L-Arg-D-Trp-L-Nle-L-Cys-L-Phe-NH.sub.2 10 82 91 PL-1317 ReO[V]
Ac-L-Trp-L-Nle-D-Phe-L-Cys-L-Arg-NH.sub.2 10 79 92 PL-1318 ReO[V]
C.sub.6H.sub.5--(CH.sub.2).sub.2--CO-L-Nle-D-Trp-L-Cys-L-Arg-NH.sub.2
10 82 82 PL-1319 ReO[V] Ac-L-Trp-D-Arg-L-Phe-L-Cys-L-Nle-NH.sub.2
10 61 89 PL-1320 ReO[V]
C.sub.6H.sub.5--(CH.sub.2).sub.2--CO-L-Arg-L-Trp-L-Cys-L-Nle-NH.sub.2
(SEQ 10 94 96 ID NO: 28) PL-1321 ReO[V]
Bz-L-Arg-L-Ala-D-Phe-L-Cys-L-Phe-NH.sub.2 10 79 93 PL-1322 ReO[V]
Bz-L-Arg-L-Ala-D-Trp-L-Cys-L-Phe-NH.sub.2 10 66 81 PL-1323 ReO[V]
Bz-L-Arg-L-Ala-D-Phe-L-Cys-L-Nle-NH.sub.2 10 77 99 PL-1324 ReO[V]
Des-aminoPhe-L-Lys-L-Arg-D-Phe-L-Cys-L-Nle-NH.sub.2 10 75 93
PL-1325 ReO[V] Des-aminoPhe-L-Cys-D-Phe-L-Arg-L-Phe-NH.sub.2 10 78
94 PL-1326 ReO[V] Heptanoyl-D-Phe-L-Arg-D-Trp-L-Cys-NH.sub.2 10 62
68 PL-1327 ReO[V] Heptanoyl-L-Phe-L-Arg-D-Trp-L-Cys-NH.sub.2 10 87
93 PL-1328 ReO[V] Ac-D-Nal 2-L-Ala-L-Arg-L-Cys-L-Trp-NH.sub.2 10 78
83 PL-1329 ReO[V] Ac-L-Nle-L-Ala-D-Trp-D-Nal 2-L-Arg-D-Trp-L-Cys- 1
88 30 NH.sub.2 PL-1330 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Nal
2-L-Cys-D-Trp-NH.sub.2 1 94 53 PL-1331 ReO[V]
Des-aminoPhe-L-Cys-D-Phe-L-Arg-L-Nle-NH.sub.2 10 74 82 PL-1332
ReO[V] Des-aminoPhe-L-Cys-D-Nal 2-L-Arg-Gly-L-Phe-NH.sub.2 10 98 80
PL-1333 ReO[V] Des-aminoPhe-L-Cys-D-Nal 2-L-Arg-Gly-L-Trp-NH.sub.2
10 95 77 PL-1334 ReO[V]
C.sub.6H.sub.5--(CH.sub.2).sub.2--CO-L-Nle-L-Trp-L-Cys-L-Arg-NH.sub.2
(SEQ 1 12 26 ID NO: 29) PL-1335 ReO[V] Heptanoyl-L-HPhe-D-Nal
2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 98 36 PL-1340 ReO[V] D-(N-Bzl)Nal
2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 99 34 PL-1341 ReO[V] D-(N-PhEt)Nal
2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 100 46 PL-1342 ReO[V]
Ac-L-Nle-L-Arg-D-His-D-Phe-L-Cys-L-Trp-NH.sub.2 1 37 89 PL-1343
ReO[V] Ac-L-Nle-L-Arg-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 50 86
PL-1344 ReO[V] Heptanoyl-L-Arg-L-Phe-L-His-L-Cys-L-Trp-NH.sub.2
(SEQ 10 81 89 ID NO: 30) PL-1345 ReO[V]
Heptanoyl-L-Arg-L-Phe-D-His-L-Cys-L-Trp-NH.sub.2 10 80 95 PL-1345
ReO[V] Heptanoyl-L-Arg-L-Phe-D-His-L-Cys-L-Trp-NH.sub.2 1 52 38
PL-1346 ReO[V]
Ph(CH.sub.2).sub.2--CO-L-His-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10
77 86 NO: 31) PL-1347 ReO[V]
Ph(CH.sub.2).sub.2--CO-D-His-L-Arg-L-Cys-L-Trp-NH.sub.2 10 70 64
PL-1348 ReO[V] Ac-L-Arg-L-His-L-Phe-L-Cys-L-Trp-NH.sub.2 (SEQ ID 10
51 57 NO: 32) PL-1349 ReO[V]
Ac-L-Arg-D-His-L-Phe-L-Cys-L-Trp-NH.sub.2 10 46 60 PL-1350 ReO[V]
Ac-L-Arg-D-His-D-Phe-L-Cys-L-Trp-NH.sub.2 10 42 70 PL-1351 ReO[V]
Ac-L-Arg-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 1 6 68 PL-1358 ReO[V]
Ac-L-Nle-L-Arg-L-Trp-D-Phe-L-Cys-L-His-NH.sub.2 10 66 98 PL-1359
ReO[V] Ac-L-Nle-L-Arg-D-Trp-D-Phe-L-Cys-L-His-NH.sub.2 10 62 90
PL-1360 ReO[V] Ac-L-Arg-L-Trp-L-Phe-L-Cys-L-His-NH.sub.2 (SEQ ID 10
62 57 NO: 33) PL-1362 ReO[V]
Ac-L-Arg-L-Trp-D-Phe-L-Cys-L-His-NH.sub.2 10 59 74 PL-1363 ReO[V]
Ac-L-Arg-D-Trp-D-Phe-L-Cys-L-His-NH.sub.2 10 74 92 PL-1364 ReO[V]
Ac-L-Trp-L-Phe-L-His-L-Cys-L-Arg-NH.sub.2 (SEQ ID 10 72 74 NO: 34)
PL-1365 ReO[V] Ac-L-Trp-L-Phe-D-His-L-Cys-L-Arg-NH.sub.2 10 64 71
PL-1366 ReO[V] Ac-L-His-L-Phe-L-Trp-L-Cys-L-Arg-NH.sub.2 (SEQ ID 10
64 78 NO: 35) PL-1367 ReO[V]
Ac-L-His-L-Phe-D-Trp-L-Cys-L-Arg-NH.sub.2 10 73 95 PL-1370 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-CF.sub.3)-L-Cys-L-Trp- 1 87 51
NH.sub.2 PL-1371 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe(3,4
di-OMe)-L-Cys-L- 1 44 35 Trp-NH.sub.2 PL-1372 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Me)-L-Cys-L-Trp-NH.sub.2 1 87 82
PL-1373 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe(3,4 diCl)-L-Cys-L-Trp- 1
92 54 NH.sub.2 PL-1374 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 89 83
PL-1375 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe(3,4 diF)-L-Cys-L-Trp- 1
54 78 NH.sub.2 PL-1376 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Val-L-Cys-L-Trp-NH.sub.2 1 31 33 PL-1385
ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Leu-L-Cys-L-Trp-NH.sub.2 1 45 34
PL-1386 ReO[V]
HOOC--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp- 0.1 1 71
NH.sub.2 PL-1387 Linear
HOOC--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp- 0.1 1 76
NH.sub.2 PL-1388 ReO[V]
NH.sub.2--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2
0.1 -10 80 PL-1389 Linear
NH.sub.2--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2
0.1 7 87 PL-1390 ReO[V] L-Lys(Z)-D-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2
10 63 88 PL-1391 ReO[V] Ac-L-Nle-L-Arg-D-His-D-Nal
2-L-Cys-L-Trp-NH.sub.2 10 72 103 PL-1391 ReO[V]
Ac-L-Nle-L-Arg-D-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 25 58 PL-1392
ReO[V] B Ala-D-Nle-D-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 80 84
PL-1393 ReO[V] Bz-L-Arg-D-His-D-Nal 2-L-Cys-L-Trp-NH.sub.2 10 65 86
PL-1394 ReO[V] Ac-L-His-L-Arg-L-Trp-L-Cys-L-Phe-NH.sub.2 (SEQ ID 10
59 84 NO: 36) PL-1394 ReO[V]
Ac-L-His-L-Arg-L-Trp-L-Cys-L-Phe-NH.sub.2 (SEQ ID 1 0 27 NO: 36)
PL-1395 ReO[V] Ac-L-His-D-Arg-L-Trp-L-Cys-L-Phe-NH.sub.2 1 5 10
PL-1395 ReO[V] Ac-L-His-D-Arg-L-Trp-L-Cys-L-Phe-NH.sub.2 10 78 74
PL-1396 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Nal
2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 70 96 PL-1405 ReO[V]
Heptanoyl-L-Phe-L-His-L-Cys-L-Trp-NH.sub.2 (SEQ ID 1 27 20 NO: 37)
PL-1406 ReO[V] Heptanoyl-L-Arg-L-Phe-L-His-L-Cys-L-Trp-NH.sub.2
(SEQ 1 42 32 ID NO: 30) PL-1412 ReO[V]
L-Phe-L-His-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 38) 1 54 43 PL-1413
ReO[V] Heptanoyl-D-Nal 2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 86 51 PL-1414
ReO[V] Heptanoyl-Psi D-Nal 2-L-Arg-L-Trp-L-Cys-NH.sub.2 1 53 26
PL-1415 ReO[V] Heptanoyl-D-Nal 2-Psi-L-Arg-L-Trp-L-Cys-NH.sub.2 1 0
25 PL-1416 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 35 85 (SEQ
ID NO: 39) PL-1417 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 1 11 20
PL-1418 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 1 24 80
PL-1419 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Nal
2-L-Arg-L-Cys-L-Trp-NH.sub.2 1 51 70 PL-1420 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Nal 2-D-Arg-L-Cys-L-Trp-NH.sub.2 1 52 43
PL-1421 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Nal 2-D-Arg-L-Cys-L-Trp- 1 59
81 NH.sub.2 PL-1422 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-His-NH.sub.2 1 63 69
PL-1423 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Phe(4- 1 71
78 NO.sub.2)--NH.sub.2 PL-1424 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Phe-NH.sub.2 1 66 85
PL-1425 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Glu-NH.sub.2 1 6 39
PL-1426 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Gln-NH.sub.2 1 16 71
PL-1427 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 82 90
PL-1428 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-His-L-Cys-L-Trp-NH.sub.2 1 45
56 PL-1429 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Me)-L-Cys-L-Trp-NH.sub.2 1 84 80
PL-1430 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-HomoPhe-L-Cys-L-Trp-NH.sub.2
1 40 26 PL-1431 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phg-L-Cys-L-Trp-NH.sub.2 1 41 20 PL-1432
ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Trp-L-Cys-L-Trp-NH.sub.2 1 47 38
PL-1433 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Cha-L-Cys-L-Trp-NH.sub.2 1 38
14 PL-1434 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Chg-L-Cys-L-Trp-NH.sub.2 1
53 33 PL-1435 ReO[V]
Ac-L-Nle-L-Arg-L-His-L-Ala-D-Phe(4-Cl)-L-Cys-L- 1 59 44
Trp-NH.sub.2 PL-1436 ReO[V]
Ac-L-Nle-L-His-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L- 1 58 51
Trp-NH.sub.2 PL-1438 ReO[V]
Ac-L-Nle-L-Arg-L-His-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 91 89
PL-1439 ReO[V]
Ac-L-Nle-L-Arg-D-His-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 43 92
PL-1440 ReO[V]
Ac-L-Nle-L-Arg-L-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 94 79
PL-1441 ReO[V]
Ac-L-Nle-L-Arg-D-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 51 71
PL-1442 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-F)-L-Cys-L-Trp-NH.sub.2
1 85 93 PL-1443 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-I)-L-Cys-L-Trp-NH.sub.2 1 99 72
PL-1444 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Tyr-L-Cys-L-Trp-NH.sub.2 1 52
68 PL-1445 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-NO.sub.2)-L-Cys-L-Trp- 1 83 63
NH.sub.2 PL-1446 ReO[V] L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 50 14
PL-1447 ReO[V] Heptanoyl-L-Ala-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 44 12
PL-1448 ReO[V] L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 62 26 PL-1449
ReO[V] Heptanoyl-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 80 45 PL-1450
ReO[V] Ac-L-Nle-L-Ala-L-His-L-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 66
73 (SEQ ID NO: 40) PL-1451 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Br)-L-Cys-L-Trp-NH.sub.2 1 92 77
PL-1452 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(4-Br)-L-Cys-L-Trp-NH.sub.2 1 97 86
PL-1457 ReO[V] Ac-L-Ala-L-His-L-Cys-D-Bip-L-Arg-L-Trp-NH.sub.2 1 79
26 PL-1458 ReO[V] Ac-L-Ala-L-His-L-Cys-D-Phe-L-Arg-L-Trp-NH.sub.2 1
61 48 PL-1459 ReO[V]
Ac-L-Ala-L-His-L-Cys-D-Asp(3Cl-anilino)-L-Arg-L- 1 31 68
Trp-NH.sub.2 PL-1460 ReO[V]
Ac-L-Ala-L-His-L-Cys-D-Asp(3,5diCl-anilino)-L-Arg- 1 40 47
L-Trp-NH.sub.2 PL-1461 ReO[V]
Ac-L-Ala-L-His-L-Cys-D-Asp(anilino)-L-Arg-L-Trp- 1 60 43 NH.sub.2
PL-1462 ReO[V] Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-L-Nal 2-NH.sub.2 1
39 99 PL-1463 ReO[V] Heptanoyl-L-His-D-Phe-L-Arg-D-Cys-L-Nal
2-NH.sub.2 1 57 93 PL-1464 ReO[V]
Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-D-Nal 2-NH.sub.2 1 80 94 PL-1465
ReO[V] Heptanoyl-L-His-D-Phe-L-Arg-D-Cys-D-Nal 2-NH.sub.2 1 48 84
PL-1466 ReO[V] Heptanoyl-L-His-D-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 1
37 67 PL-1467 ReO[V]
Heptanoyl-L-His-D-Phe-D-Arg-D-Cys-L-Trp-NH.sub.2 1 47 69 PL-1478
ReO[V] Ac-L-Arg-L-Phe-L-Phe-L-Cys-L-Ser-NH.sub.2 (SEQ ID 1 21 28
NO: 41) PL-1480 ReO[V] Ac-L-Arg-D-Phe-L-Phe-L-Cys-L-Ser-NH.sub.2 1
9 21 PL-1481 ReO[V] Ac-L-Arg-D-Phg-L-Phe-L-Cys-L-Ser-NH.sub.2 1 15
19
PL-1483 ReO[V] Ac-L-Arg-L-Phe-L-Nal 2-L-Cys-L-Ser-NH.sub.2 1 31 56
PL-1484 ReO[V] Ac-L-Arg-D-Phe-L-Nal 2-L-Cys-L-Ser-NH.sub.2 1 37 72
PL-1485 ReO[V] Ac-L-Arg-D-Phg-L-Nal 2-L-Cys-L-Ser-NH.sub.2 1 41 60
PL-1486 ReO[V] Ac-D-Ala-L-His-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1
90 26 PL-1488 Linear Ac-D-Ala-L-His-L-Cys-D-Nal
2-L-Arg-L-Trp-NH.sub.2 1 85 35 PL-1489 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Nal 2-L-Arg-L-Trp-L-Cys- 1 99 30 NH.sub.2
PL-1490 Linear Heptanoyl-L-Ser(Bzl)-D-Nal 2-L-Arg-L-Trp-L-Cys- 1 86
48 NH.sub.2 PL-1491 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Nal
2-L-Arg-L-Phe-L-Cys- 1 76 66 NH.sub.2 PL-1492 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Nal 2-L-Arg-D-Phe-L-Cys- 1 90 63 NH.sub.2
PL-1493 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Nal 2-L-Arg-D-Trp-L-Cys- 1
102 26 NH.sub.2 PL-1494 ReO[V]
Ac-L-Lys-L-Cys-D-Phe-L-Trp-L-Nle-NH.sub.2 1 30 27 PL-1496 ReO[V]
Ac-L-Asp-L-Lys-L-Pro-L-Pro-L-Arg-L-Ala-D-Nal 2-L- 1 59 43
Cys-L-Trp-NH.sub.2 PL-1497 ReO[V]
L-Asp-L-Lys-L-Pro-L-Pro-L-Arg-L-Ala-D-Nal 2-L-Cys- 1 71 47
L-Trp-NH.sub.2 PL-1498 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Nal
2-L-Cys-L-Trp-Ahx-L- 1 86 59 Lys-L-Asp-NH.sub.2 PL-1499 ReO[V]
Heptanoyl-L-Lys-D-Phe-L-Trp-L-Cys-NH.sub.2 1 20 23 PL-1500 ReO[V]
Heptanoyl-D-Phe-L-Trp-L-Cys-L-Lys-NH.sub.2 1 33 25 PL-1501 ReO[V]
Heptanoyl-L-His-D-Trp-L-Cys-L-Trp-NH.sub.2 1 44 78 PL-1502 ReO[V]
Heptanoyl-L-His-D-Trp-Gly-L-Cys-L-Lys-NH.sub.2 1 41 29 PL-1503
ReO[V] Heptanoyl-L-Phe-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 41 52
PL-1504 ReO[V] Ac-L-Arg-L-Phe-L-Nal 2-L-Asn-L-Cys-L-Phe-NH.sub.2 1
21 36 (SEQ ID NO: 42) PL-1505 ReO[V] Ac-L-Arg-L-Phe-L-Nal
2-L-Asn-L-Cys-L-Phe-NH.sub.2 1 29 45 (SEQ ID NO: 42) PL-1506 ReO[V]
Ac-L-Arg-D-Phe-L-Nal 2-L-Asn-L-Cys-L-Phe-NH.sub.2 1 22 30 PL-1507
ReO[V] Ac-L-Arg-L-Phe-L-Phe-L-Asn-L-Cys-L-Phe-NH.sub.2 1 22 20 (SEQ
ID NO: 43) PL-1508 ReO[V]
Ac-L-Arg-D-Phe-L-Phe-L-Asn-L-Cys-L-Phe-NH.sub.2 1 31 20 PL-1509
ReO[V] Ac-L-Arg-L-Phe-L-Cys-L-Phe-L-Asn-L-Ala-L-Phe- 1 30 19
NH.sub.2 (SEQ ID NO: 44) PL-1510 ReO[V]
Ac-L-Arg-L-Phe-L-Phe-L-Cys-L-Asn-L-Ala-L-Phe- 1 31 21 NH.sub.2 (SEQ
ID NO: 45) PL-1511 ReO[V]
Ac-L-Arg-L-Phe-L-Phe-L-Asn-L-Cys-L-Ala-L-Phe- 1 26 21 NH.sub.2 (SEQ
ID NO: 46) PL-1512 ReO[V]
Ac-L-Arg-L-Phe-L-Phe-L-Asn-L-Ala-L-Cys-L-Phe- 1 24 20 NH.sub.2 (SEQ
ID NO: 47) PL-1513 ReO[V]
Ac-L-Arg-L-Phe-L-Phe-L-Asn-L-Phe-L-Cys-NH.sub.2 1 18 23 (SEQ ID NO:
48) PL-1514 ReO[V] Ac-L-Arg-L-Phe-L-Phe-L-Asn-L-Phe-L-Cys-NH.sub.2
1 30 28 (SEQ ID NO: 48) PL-1515 ReO[V]
Heptanoyl-L-His-D-Phe-L-Lys-L-Cys-L-Glu 1 34 21 PL-1518 ReO[V]
Heptanoyl-L-Cys-D-Phe-L-Trp-L-Lys-NH.sub.2 1 25 8 PL-1519 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp-L-Pro-L- 1 60 22
Pro-L-Lys-L-Asp-NH.sub.2 PL-1522 ReO[V]
Ac-L-Nle-L-Arg-L-His-D-Phe(4-Br)-L-Cys-L-Trp-NH.sub.2 1 91 87
PL-1523 ReO[V] Ac-L-Nle-L-Arg-L-Ala-L-His-D-Phe(4-Br)-L-Cys-L- 1 59
69 Trp-NH.sub.2 PL-1524 ReO[V]
Ac-L-Nle-L-Arg-L-His-L-Ala-D-Phe(4-Br)-L-Cys-L- 1 44 36
Trp-NH.sub.2 PL-1525 ReO[V]
Ac-L-Nle-L-Arg-L-Phe(4-Br)-L-Ala-L-His-L-Cys-L- 1 41 57
Trp-NH.sub.2 PL-1526 ReO[V]
Ac-L-Nle-L-Arg-L-Trp-L-Ala-D-Phe(4-Br)-L-Cys-L- 1 34 63
His-NH.sub.2 PL-1581 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 98 91
PL-1582 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Cys-L-Arg-L-Trp-NH.sub.2 1 66 93
PL-1583 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Cys-D-Phe-L-Arg-L-Trp-NH.sub.2 1 75 84
PL-1584 ReO[V]
Ac-L-Nle-L-His-L-Cys-L-His-D-Phe-L-Arg-L-Trp-NH.sub.2 1 71 96
PL-1585 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Phe-L-Cys-L-Arg-L-Trp-NH.sub.2 1 22 45
PL-1587 ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1
98 96 PL-1592 ReO[V]
Ac-L-Nle-L-Ala-L-Arg-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 1 7 19
PL-1593 ReO[V]
Ac-L-Nle-L-Ala-D-Arg-L-His-D-Phe-L-Cys-L-Trp-NH.sub.2 1 16 71
PL-1594 ReO[V]
Ac-L-Nle-L-Ala-L-His-D/L-Atc-L-Arg-L-Cys-L-Trp-NH.sub.2 1 24 100
PL-1595 ReO[V] Ac-L-Nle-L-Ala-L-His-Aic-L-Arg-L-Cys-L-Trp-NH.sub.2
1 3 60 (SEQ ID NO: 49) PL-1597 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D/L-Atc-L-Cys-L-Trp-NH.sub.2 1 11 68 PL-1598
ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Qal(2')-L-Cys-L-Trp-NH.sub.2 1 9 22
PL-1605 ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-2-L-Cys-L-Trp-NH.sub.2 1 100
100 PL-1606 ReO[V] Ac-L-Nle-L-Arg-L-Ala-Aic-L-Cys-L-Trp-NH.sub.2
(SEQ ID 1 63 44 NO: 50) PL-1607 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Qal(2')-L-Arg-L-Cys-L-Trp- 1 52 100 NH.sub.2
PL-1621 ReO[V] Ac-L-Nle-L-Ala-L-His-Achc-L-Arg-L-Cys-L-Trp-NH.sub.2
1 34 36 (SEQ ID NO: 51) PL-1623 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Sal-L-Arg-L-Cys-L-Trp-NH.sub.2 1 55 92
PL-1624 ReO[V] Ac-L-Nle-L-Arg-L-Ala-D-Sal-L-Cys-L-Trp-NH.sub.2 1 48
25 PL-1626 ReO[V] Ac-L-Nle-L-Arg-L-Trp-D-Nal 2-L-Cys-L-Trp-NH.sub.2
1 54 66 PL-1633 ReO[V] Ac-L-Nle-D-Arg-L-Arg-D-Nal
2-L-Cys-L-Trp-NH.sub.2 1 87 86 PL-1633 ReO[V]
Ac-L-Nle-D-Arg-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 87 91 PL-1634
ReO[V] Ac-L-Nle-L-Arg-D-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 50 42
PL-1635 ReO[V] Ac-L-Nle-L-Arg-L-Ala-Acpc-L-Cys-L-Trp-NH.sub.2 (SEQ
1 43 14 ID NO: 52) PL-1636 ReO[V]
Ac-L-Nle-L-Ala-L-His-Acpc-L-Arg-L-Cys-L-Trp-NH.sub.2 1 38 20 (SEQ
ID NO: 53) PL-1638 ReO[V]
Ac-L-Nle-L-Arg-L-Arg-D-Qal(2')-L-Cys-L-Trp-NH.sub.2 1 48 67 PL-1649
ReO[V] Ac-L-His-Gly-Gly-L-Cys-L-Trp-NH.sub.2 (SEQ ID NO: 54) 10 62
19 PL-1650 ReO[V] Ac-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 10 66
52 PL-1651 ReO[V] Ac-L-His-D-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 10 58
95 PL-1652 ReO[V] Ac-L-His-L-Phe-D-Arg-L-Cys-L-Trp-NH.sub.2 10 40
11 PL-1655 ReO[V] Ac-L-His-L-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 (SEQ ID
10 51 45 NO: 8) PL-1658 ReO[V]
Ac-L-Nle-L-Arg-L-Arg-D-Phe(3,4-diCl)-L-Cys-L-Trp- 1 100 99 NH.sub.2
PL-1658 ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-Phe(3,4-diCl)-L-Cys-L-Trp- 1
97 93 NH.sub.2 PL-1659 ReO[V]
Ac-L-Nle-L-Lys-L-Lys-D-L-Cys-L-Trp-NH.sub.2 1 91 58 PL-1660 ReO[V]
Ac-L-Nle-L-Arg-L-Arg-D-Phe-L-Cys-L-Trp-NH.sub.2 1 67 100 PL-1661
ReO[V] Ac-L-Nle-L-Cit-L-Cit-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 58 27
PL-1662 ReO[V] Ac-L-Nle-L-Ala-L-Arg-L-Arg-D-Nal 2-L-Cys-L-Trp- 1 93
69 NH.sub.2 PL-1663 ReO[V]
Ac-L-Nle-L-Arg-L-Arg-D-Trp-L-Cys-L-Trp-NH.sub.2 1 85 81 PL-1664
ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-Nal 2-L-Cys-NH.sub.2 1 100 96 PL-1665
ReO[V] Ac-L-Arg-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 93 94 PL-1666
ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-Phe(p-I)-L-Cys-L-Trp-NH.sub.2 1 101
101 PL-1667 ReO[V] Heptanoyl-L-Arg-L-Arg-D-Nal
2-L-Cys-L-Trp-NH.sub.2 1 97 92 PL-1684 ReO[V]
Ac-L-Val-L-Pro-L-Arg-L-Ala-D-Nal 2-L-Cys-L-Trp- 1 36 26 NH.sub.2
PL-1685 ReO[V] Ac-L-Nle-L-Arg-BAla-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1
83 54 PL-1686 ReO[V] Ac-L-Nle-D-Arg-D-Arg-L-Nal
2-L-Cys-L-Trp-NH.sub.2 1 24 23 PL-1690 ReO[V]
Ac-L-Nle-D-Arg-L-Arg-D-Nal 2-L-Cys-L-Trp-NH.sub.2 1 94 71 PL-1691
Linear Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-L-Trp-L-Lys- 1 90 62
NH.sub.2 PL-1692 Linear
NH.sub.2--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp-L- 1
79 60 Lys-NH.sub.2 PL-1694 ReO[V]
Heptanoyl-L-His-D-Phe-L-Arg-L-Cys-L-Trp-L-Lys- 1 43 61 NH.sub.2
PL-1695 ReO[V]
NH.sub.2--(CH.sub.2).sub.5--CO-L-His-D-Phe-L-Arg-L-Cys-L-Trp-L- 1
44 87 Lys-NH.sub.2 PL-1702 ReO[V] Ac-L-His-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 83 62 PL-1703 ReO[V] Ac-L-Trp-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 95 73 PL-1704 ReO[V] Ac-L-Phe-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 98 75 PL-1705 ReO[V] Ac-L-Lys-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 90 53 PL-1706 ReO[V] Ac-L-Ser-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 89 60 PL-1707 ReO[V] Ac-L-Glu-L-Arg-L-Arg-D-Nal
2-L-Cys-NH.sub.2 1 46 36 PL-1708 ReO[V] Ac-L-Arg-L-His-L-Cys-D-Nal
2-L-Arg-L-Trp-NH.sub.2 1 99 34 PL-1709 ReO[V]
Ac-D-Ala-L-His-L-Cys-L-Arg-D-Nal 2-L-Arg-L-Trp- 1 100 98 NH.sub.2
PL-1710 ReO[V] Ac-D-Ala-L-Arg-L-Cys-D-Nal 2-L-Arg-L-Trp-NH.sub.2 1
99 25 PL-1718 ReO[V]
Ac-L-Nle-L-Arg-L-Ala-D-Phe(3-Cl)-L-Cys-L-Trp-NH.sub.2 1 59 35
PL-1722 ReO[V] Ac-L-Trp-L-Arg-L-Arg-D-Phe-L-Cys-NH.sub.2 1 29 38
PL-1723 ReO[V] Ac-L-Nle-L-Arg-L-Arg-D-Nal 2-L-Cys-OH 1 85 66
PL-1726 ReO[V] Ac-L-Nle-L-Arg-L-Hphe-D-Phe(4-Cl)-L-Cys-NH.sub.2 1
70 42 PL-1727 ReO[V] Ac-L-Nle-L-Arg-L-Pal
2'-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 84 62 PL-1728 ReO[V]
Ac-L-Nle-L-Arg-L-Phe-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 73 53 PL-1730
ReO[V] Ac-L-Nle-L-Arg-L-Nal 1-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 35 39
PL-1731 ReO[V] Ac-L-Nle-L-Arg-L-Nal 2-D-Phe(4-Cl)-L-Cys-NH.sub.2 1
63 38 PL-1732 ReO[V]
Ac-L-Nle-L-Arg-L-Trp-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 74 53 PL-1733
ReO[V] L-Tic-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 8 14 PL-1734 ReO[V]
L-Tic-D-Phe(4-Cl)-L-Trp-L-Cys-NH.sub.2 1 7 6 PL-1735 ReO[V]
L-Tic-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 13 12 PL-1736 ReO[V]
Ac-D-Ala-L-His-L-Cys-D-(N-Bzl)Phe-L-Arg-L-Trp-NH.sub.2 1 3 6
PL-1737 ReO[V]
Ac-D-Ala-L-His-L-Cys-L-(N-Bzl)Phe-L-Arg-L-Trp-NH.sub.2 1 3 48
PL-1738 ReO[V] Ac-D-Ala-L-His-L-Cys-D-(N-Bzl)Nal 2-L-Arg-L-Trp- 1
23 13 NH.sub.2 PL-1751 ReO[V]
Ac-L-His-L-(N-2'naphthalene)Phe-L-Arg-L-Trp-L- 1 70 78 Cys-NH.sub.2
(SEQ ID NO: 55) PL-1752 ReO[V]
Ac-D-Ala-L-His-L-Cys-L-(N-2'naphthalene)Phe-L- 1 5 29
Arg-L-Trp-NH.sub.2 PL-1753 ReO[V]
Ac-D-Ala-L-His-L-Cys-D-(N-2'naphthalene)Phe-L- 1 22 50
Arg-L-Trp-NH.sub.2 PL-1754 ReO[V]
D-Tic-D-Phe(4-Cl)-L-Trp-L-Cys-NH.sub.2 1 7 4 PL-1755 ReO[V]
Ac-L-Arg-L-Lys-L-Phe-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 40 48
PL-1756 ReO[V]
Ac-L-Nle-L-Lys-L-Phe-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 63 64
PL-1757 ReO[V]
Ac-L-Arg-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 45 38
PL-1758 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 94 78
PL-1759 ReO[V]
Ac-L-Arg-L-Phe-L-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 62 61
PL-1760 ReO[V]
Ac-L-Nle-L-Phe-L-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 72 84
PL-1761 ReO[V]
Ac-L-Arg-L-Leu-L-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 16 51
PL-1762 ReO[V]
Ac-L-Nle-L-Leu-L-Lys-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 69 82
PL-1774 ReO[V]
Ac-L-Nle-L-Lys-L-Val-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 83 79
PL-1775 ReO[V]
Ac-L-Nle-L-Lys-L-Ile-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 78 57
PL-1776 ReO[V]
Ac-L-Nle-L-Lys-L-Nle-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 76 33
PL-1777 ReO[V]
Ac-L-Nle-L-Lys-L-Thr-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 79 86
PL-1778 ReO[V]
Ac-L-Nle-L-Lys-L-Tle-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 89 60
PL-1779 ReO[V]
Ac-L-Nle-L-Lys-L-Chg-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 85 71
PL-1780 ReO[V]
Ac-L-Nle-L-Lys-L-Cha-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 77 34
PL-1781 ReO[V]
Ac-L-Nle-L-Lys-L-Trp-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 69 66
PL-1782 ReO[V] Ac-L-Nle-L-Lys-L-Hphe-D-Phe(4-Cl)-L-Cys-L-Trp- 1 67
14 NH.sub.2 PL-1783 ReO[V]
Ac-L-Nle-L-Lys-L-Lys(Z)-D-Phe(4-Cl)-L-Cys-L-Trp- 1 72 87 NH.sub.2
PL-1785 ReO[V] Ac-D-Ala-L-His-L-Cys-D-Tic-L-Arg-L-Trp-NH.sub.2 1 16
51 PL-1787 ReO[V]
Ac-D-Ala-L-His-L-Cys-D-3,3,Dip-L-Arg-L-Trp-NH.sub.2 1 31 49 PL-1788
ReO[V] Ac-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 78 62
PL-1789 ReO[V]
Ac-L-Pro-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 87 63
PL-1790 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-L- 1 99
93 Trp-NH.sub.2 PL-1791 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-L- 1 98 87
Leu-NH.sub.2 PL-1792 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-L- 1 100 96
Lys-NH.sub.2 PL-1793 ReO[V]
Ac-L-Pro-L-His-D-Phe-L-Arg-D-Trp-L-Cys-NH.sub.2 1 39 41 PL-1794
ReO[V] Ac-L-Pro-L-His-D-Phe-L-Arg-D-Trp-L-Cys-D-Trp-NH.sub.2 1 20
-7 PL-1795 ReO[V] Ac-D-Tic-D-Phe-L-Arg-D-Trp-L-Cys-NH.sub.2 1 85 51
PL-1796 ReO[V] Ac-D(3,3)Bpa-D-Phe-L-Arg-D-Trp-L-Cys-NH.sub.2 1 14
-7 PL-1797 ReO[V] Ac-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1
42 49 PL-1798 ReO[V] Heptanoyl-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Trp-
1 56 68 NH.sub.2 PL-1799 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-NH.sub.2 1 69 70 PL-1800
ReO[V] Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-D-Cys-L-Trp-NH.sub.2 1 35
54 PL-1801 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Tic-NH.sub.2 1 64 89
PL-1802 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Tyr-NH.sub.2 1 16 72
PL-1803 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Leu-NH.sub.2 1 23 55
PL-1804 ReO[V] Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Tyr(Bzl)- 1
47 70 NH.sub.2 PL-1805 ReO[V]
Ac-L-Nle-L-Lys-L-Leu-D-Phe(4-Cl)-L-Cys-L-Phe(3- 1 67 74 Cl)--NH2
PL-1806 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1 13.0 66.0
PL-1807 ReO[V]
Ac-L-Nle-L-Glu-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 1
38.0 96.0 PL-1808 ReO[V]
Ac-L-His-D-Phe-L-Arg-L-Trp-L-Cys-L-Lys-L-Pro-L- 1 75.0 94.0
Val-NH.sub.2 PL-1809 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Phe-L-Arg-L-Trp-L- 1 77.0 88.0
Asp-L-Arg-L-Cys-L-Phe-NH.sub.2 (SEQ ID NO: 56) PL-1810 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Phe-L-Arg-L-Trp-L- 1 88.0 91.0
Asp-L-Cys-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 57) PL-1811 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Phe-L-Arg-L-Trp-L- 1 92.0 95.0
Cys-L-Asp-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 58) PL-1812 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Phe-L-Arg-L-Cys-L- 1 98.0 98.0
Trp-L-Asp-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 59) PL-1813 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Phe-L-Cys-L-Arg-L- 1 36.0 67.0
Trp-L-Asp-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 60) PL-1814 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-His-L-Cys-L-Phe-L-Arg-L- 1 26.0 62.0
Trp-L-Asp-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 61) PL-1815 ReO[V]
L-Tyr-L-Val-L-Nle-Gly-L-Cys-L-His-Phe-L-Arg-L-Trp- 1 36.0 60.0
L-Asp-L-Arg-L-Phe-NH.sub.2 (SEQ ID NO: 62) PL-1816 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-D-Trp-L- 1 83.0 46.0
Cys-NH.sub.2 PL-1817 ReO[V]
Bz-L-His-Gly-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 68.0 45.0
PL-1818 ReO[V] Ac-L-Nle-L-Ala-D-Trp-D-Phe(2-Cl)-L-Arg-D-Trp-L- 1
36.0 15.0 Cys-NH.sub.2 PL-1819 ReO[V]
Ac-L-Ala-L-His-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 67.0 76.0
PL-1820 ReO[V] Bz-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 54.0
17.0 PL-1821 ReO[V] Lys(Z)-L-Ala-D-Phe-L-Cys-L-Trp-NH.sub.2 1 18 43
PL-1822 ReO[V] Lys(Z)-L-Ala-D-Phe(2-Cl)-L-Cys-L-Trp-NH.sub.2 1 15
24 PL-1823 ReO[V] Lys(Z)-L-Ala-D-Phe(3-Cl)-L-Cys-L-Trp-NH.sub.2 1
35 11 PL-1824 ReO[V] Lys(Z)-L-Ala-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2
1 69 34 PL-1825 ReO[V]
Lys(Z)-L-Ala-D-Phe-(3,4-diCl)-L-Cys-L-Trp-NH.sub.2 1 58 15 PL-1828
ReO[V] Heptanoyl-L-Tyr-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 29 29
NH.sub.2 PL-1829 ReO[V]
Heptanoyl-L-Trp-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 18 10 NH.sub.2
PL-1830 ReO[V] Heptanoyl-L-Nal 2-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 5
11 NH.sub.2 PL-1831 ReO[V]
Heptanoyl-L-Bip-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 -2 13 NH.sub.2
PL-1832 ReO[V] Heptanoyl-L-Phe(3,4-diCl)-D-Phe(2-Cl)-L-Arg-D-Trp- 1
25 12 L-Cys-NH.sub.2 PL-1833 ReO[V]
Heptanoyl-L-Tle-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 59 49
PL-1834 ReO[V] Heptanoyl-L-Cha-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 16
46 NH.sub.2 PL-1835 ReO[V]
Heptanoyl-L-Phe(p-NO.sub.2)-D-Phe(2-Cl)-L-Arg-D-Trp-L- 1 20 15
Cys-NH.sub.2 PL-1836 ReO[V]
Heptanoyl-L-HPhe-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 82 50 NH.sub.2
PL-1837 ReO[V]
Heptanoyl-L-Tic-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 42 24
PL-1838 ReO[V] D-Tic-L-Arg-L-Trp-L-Cys-NH.sub.2 1 54 74 PL-1839
ReO[V] Ac-D-Tic-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 11 35 PL-1840
ReO[V] Ac-L-Tic-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 34 24 PL-1841
ReO[V] Ac-L-Pro-L-His-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 34 61
PL-1842 ReO[V]
Ac-L-Nle-L-Ala-L-His-L-Arg-L-Phe-L-Trp-L-Cys-NH.sub.2 1 2 27 (SEQ
ID NO: 63) PL-1843 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Arg-L-Phe-L-Trp-L-Cys-NH.sub.2 1 -8 16
PL-1844 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-D-Cys-L- 1 83
98 Trp-NH.sub.2 PL-1845 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-L-Cys-D- 1 96 99
Trp-NH.sub.2 PL-1846 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Cys-L-Trp-NH.sub.2 0.1 4 85
PL-1849 ReO[V] C.sub.6H.sub.5--CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2
1 12 39 PL-1850 ReO[V]
C.sub.6H.sub.5--CH.dbd.CH--CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2 1 -2
24 PL-1851 ReO[V] Pyridine-3-CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2 1
-5 26 PL-1852 ReO[V] Tetralin-2-CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2
1 0 15 PL-1853 ReO[V]
Naphthalene-1-CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2 1 9 27 PL-1854
ReO[V] Naphthalene-2-CO-L-Lys-D-Phe-L-Cys-L-Trp-NH.sub.2 1 -6 24
PL-1855 ReO[V] Lys(Z)-Gly-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 0 32
PL-1856 ReO[V] Lys(Z)-L-Val-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1 31
53 PL-1857 ReO[V] Lys(Z)-L-Nle-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2 1
32 40 PL-1858 ReO[V] Lys(Z)-L-Leu-D-Phe(4-Cl)-L-Cys-L-Trp-NH.sub.2
1 31 36 PL-1859 ReO[V] Ac-L-Phe-L-Phe-L-Cys-L-Tic-L-Lys-NH.sub.2
(SEQ ID 1 -8 9 NO: 64) PL-1860 ReO[V]
Ac-L-Phe-L-Phe-L-Cys-L-Inp-L-Lys-NH.sub.2 (SEQ ID 1 0 6 NO: 65)
PL-1861 ReO[V] Ac-L-Phe-L-Phe-L-Cys-4-Abz-L-Lys-NH.sub.2 (SEQ ID 1
-14 0 NO: 66) PL-1862 ReO[V]
Ac-L-Phe-L-Phe-L-Cys-3-Abz-L-Lys-NH.sub.2 (SEQ ID 1 -7 17 NO: 67)
PL-1863 ReO[V] Ac-L-Phe-L-Phe-L-Cys-2-Abz-L-Lys-NH.sub.2 (SEQ ID 1
6 19 NO: 68) PL-1864 ReO[V]
Ac-L-Phe-D-Trp-L-Cys-2-Abz-L-Lys-NH.sub.2 1 -7 17 PL-1865 ReO[V]
Ac-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 40 13
PL-1866 ReO[V] Bz-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2
1 30 16 PL-1867 ReO[V]
Heptanoyl-L-Asn-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 60 52 NH.sub.2
PL-1868 ReO[V] Heptanoyl-L-Asp-D-Phe(2-Cl)-L-Arg-D-Trp-L-Cys- 1 -3
5 NH.sub.2 PL-1869 ReO[V]
Heptanoyl-L-Lys(NH-Bz)-D-Phe(2-Cl)-L-Arg-D-Trp-L- 1 42 25
Cys-NH.sub.2 PL-1870 ReO[V]
Heptanoyl-D-B-Hphe(4-F)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 11 12 PL-1871
ReO[V] Heptanoyl-D-B-Hphe(2-Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 1 3 10
PL-1872 ReO[V]
Ac-D-Ala-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 79 27
PL-1873 ReO[V] Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-D-Cys-Trp-NH.sub.2
1 31 92.6 PL-1874 ReO[V]
Ac-L-Nle-L-Ala-L-His-D-Phe-L-Arg-L-Trp-D-Cys-D- 1 90 98
Trp-NH.sub.2 PL-1875 ReO[V]
1-Naphthalene-acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L- 1 77 34
Cys-Trp-NH.sub.2 PL-1876 ReO[V]
2-Naphthalene-acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L- 1 52 8
Cys-Trp-NH.sub.2 PL-1877 ReO[V] 3-Bromophenyl
acetyl-L-Lys-L-Ala-D-Phe(4-I)-L-Cys- 1 92 31 Trp-NH.sub.2 PL-1878
ReO[V] 4-Bromophenyl acetyl-L-Lys-L-Ala-D-Phe(p-I)-L-Cys- 1 75 53
Trp-NH.sub.2 PL-1879 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(3-Cl)-L-Arg-D-Trp-L- 1 86 28
Cys-NH.sub.2 PL-1880 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(3,4-Cl.sub.2)-L-Arg-D-Trp-L- 1 96 18
Cys-NH.sub.2 PL-1881 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-HPhe-L-Arg-D-Trp-L-Cys- 1 -6 16 NH.sub.2
PL-1882 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Tic-L-Arg-D-Trp-L-Cys-NH.sub.2 1 -14 11
PL-1883 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(4-Cl)-L-Arg-D-Trp-L- 1 97
49 Cys-NH.sub.2 PL-1884 ReO[V]
Ac-D-Phe-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 64 32
PL-1885 ReO[V]
Ac-D-Nle-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 84 68
PL-1886 ReO[V] Ac-D-HPhe-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 73
62 NH.sub.2 PL-1887 ReO[V]
Ac-D-Phe-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 50 24 NH.sub.2
PL-1888 ReO[V]
Ac-D-Ala-L-Nle-L-Cys-D-Phe(2-Cl)$$-L-Arg-L-Trp-NH.sub.2 1 90 40
PL-1889 ReO[V]
Ac-L-Nle-L-His-L-Cys-D-Phe(2-Cl)$$-L-Arg-L-Trp-NH.sub.2 1 49 58
PL-1890 ReO[V] Heptanoyl-D-Ala-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L- 1
24 31 Trp-NH.sub.2 PL-1891 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-BHphe(2-Cl)-L-Arg-D-Trp-L- 1 -7 10
Cys-NH.sub.2 PL-1892 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-BHphe(2-F)-L-Arg-D-Trp-L- 1 6 10
Cys-NH.sub.2 PL-1893 ReO[V]
Ac-D-Phg-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 78 52
PL-1894 ReO[V]
Ac-D-Ala-L-Phg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 81 37
PL-1895 ReO[V]
Ac-D-Ala-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 63 19
PL-1896 ReO[V] Ac-D-Nle-L-His-L-Cys-D-(NMe)Phe-L-Arg-L-Trp-NH.sub.2
1 -1 56 PL-1897 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Tiq-L-Arg-D-Trp-L-Cys-NH.sub.2 1 -24 12
PL-1899 ReO[V]
C.sub.6H.sub.5(CH.sub.2).sub.3-CO-L-Ser(Bzl)-D-(NMe)-Phe-L-Arg-D- 1
0 19 Trp-L-Cys-NH.sub.2 PL-1900 ReO[V] 3-Bromophenyl
acetyl-L-Lys-L-Ala-D-Trp-L-Cys- 1 47 10 L-Trp-NH.sub.2 PL-1901
ReO[V] 3-Bromophenyl acetyl-L-Lys-L-Ala-D-Phe-L-Cys-L- 1 51 26
Trp-NH.sub.2 PL-1902 ReO[V] 3-Bromophenyl
acetyl-L-Lys-L-Ala-D-Phe(4-Me)-L- 1 93 17 Cys-L-Trp-NH.sub.2
PL-1903 ReO[V] 3-Bromophenyl acetyl-L-Lys-L-Ala-D-Phe(3-Cl)-L- 1 71
5 Cys-L-Trp-NH.sub.2 PL-1904 ReO[V] 3-Bromophenyl
acetyl-L-Lys-L-Ala-D-HPhe-L-Cys-L- 1 11 2 Trp-NH.sub.2 PL-1905
ReO[V] 2-Chlorophenyl acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L- 1 86 31
Cys-L-Trp-NH.sub.2 PL-1906 ReO[V] 4-Chlorophenyl
acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L- 1 91 68 Cys-L-Trp-NH.sub.2
PL-1907 ReO[V] 4-Methylphenyl acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L- 1
69 44 Cys-L-Trp-NH.sub.2 PL-1908 ReO[V] Indonyl
acetyl-L-Lys-L-Ala-D-Phe(4-Cl)-L-Cys-L-Trp- 1 33 8 NH.sub.2 PL-1909
ReO[V] 3-Bromophenyl acetyl-L-Arg-L-Ala-D-Phe(4-Cl)-L- 1 95 32
Cys-L-Trp-NH.sub.2 PL-1910 ReO[V]
Heptanoyl-L-Dpr(Bz)-D-Phe(2-Cl)-L-Arg-D-Trp-L- 1 23 42 Cys-NH.sub.2
PL-1911 ReO[V] Heptanoyl-L-Dpr(2'-Naphthalene acetyl)-D-Phe(2- 1 -6
11 Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 PL-1912 ReO[V]
Heptanoyl-L-Dpr(1'-Adamantane carbonyl)-D-Phe(2- 1 -3 2
Cl)-L-Arg-D-Trp-L-Cys-NH.sub.2 PL-1913 ReO[V]
Heptanoyl-L-Dpr(4'-MePhenyl acetyl)-D-Phe(2-Cl)-L- 1 22 35
Arg-D-Trp-L-Cys-NH.sub.2 PL-1914 ReO[V] Heptanoyl-L-Dpr(3'-BrPhenyl
acetyl)-D-Phe(2-Cl)-L- 1 20 53 Arg-D-Trp-L-Cys-NH.sub.2 PL-1915
ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)$$-L-Arg-L-Trp-L- 1 94 72
Cys-NH.sub.2 PL-1916 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-L-His-L- 1 9 44 Cys-NH.sub.2
PL-1917 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-L-Nal 2'-L- 1
94 48 Cys-NH.sub.2 PL-1918 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-L-Bip-L- 1 10 21
Cys-NH.sub.2 PL-1919 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-L-Pal 3'-L- 1 17 47
Cys-NH.sub.2 PL-1920 ReO[V] D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 52
65 PL-1921 ReO[V] Ac-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 20 25
PL-1922 ReO[V] Ac-L-Nle-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 25 28
PL-1923 ReO[V] Ac-L-Nle-L-Ala-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 68
70 PL-1924 ReO[V] Ac-L-Pro-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 44 33
PL-1925 ReO[V] Heptanoyl-D-Phe-L-Arg-L-Trp-L-Cys-NH.sub.2 1 6 18
PL-1926 ReO[V] Bz-L-Arg-L-Trp-L-Cys-NH.sub.2 (SEQ ID NO: 69) 1 7 25
PL-1927 ReO[V] Phenyl acetyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 8 28
PL-1928 ReO[V] 3-Phenyl-propanoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 8 32
PL-1929 ReO[V] 4-Phenyl-butanoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 2 18
PL-1930 ReO[V] t-Cinnamoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 -20 9
PL-1931 ReO[V] 1-Naphthyl-acetyl-L-Arg-L-Trp-L-Cys-NH.sub.2 10 92
47 PL-1932 ReO[V] 2-Naphthyl-acetyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 1
16 PL-1933 ReO[V] 1-Naphthoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 0 14
PL-1934 ReO[V] 2-Naphthoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 6 34
PL-1935 ReO[V] Heptanoyl-L-Arg-L-Trp-L-Cys-NH.sub.2 1 8 39 PL-1936
ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(4-F)-L-Arg-L-Trp-L-Cys- 1 81 71
NH.sub.2 PL-1937 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(penta-F)-L-Arg-L-Trp-L- 1 91 65
Cys-NH.sub.2 PL-1938 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Pal(2)-L-Arg-L-Trp-L-Cys- 1 16 16 NH.sub.2
PL-1939 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(2-Br)-L-Arg-L-Trp-L- 1 91
73 Cys-NH.sub.2 PL-1940 ReO[V]
Ac-D-Ala-L-Nle-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 89 26
PL-1941 ReO[V] Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Lys-L-Nal 2-L- 1
90 25 Cys-NH.sub.2 PL-1942 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(2-Cl)-L-Arg-L-Nal 1-L- 1 53 16
Cys-NH.sub.2 PL-1943 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(penta-F)-L-Arg-L-Nal 2- 1 93 64
L-Cys-NH.sub.2 PL-1944 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(penta-F)-L-Lys-L-Nal 2- 1 80 40
L-Cys-NH.sub.2 PL-1945 ReO[V]
Heptanoyl-L-Hyp(Bzl)-D-Phe(2-Cl)-L-Arg-L-Nal 2-L- 1 94 30
Cys-NH.sub.2 PL-1946 ReO[V] Heptanoyl-[(2S,3R),5-phenyl
pyrrolidinyl-2-carbonyl]- 1 81 24 D-Phe(2-Cl)-L-Arg-L-Nal
2-L-Cys-NH.sub.2 PL-1947 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(4-CF.sub.3)-L-Arg-L-Trp-L- 1 98 23
Cys-NH.sub.2 PL-1948 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(4-CH.sub.3)-L-Arg-L-Trp-L- 1 98
50 Cys-NH.sub.2 PL-1949 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(4-Cl)-L-Arg-L-Trp-L- 1 99 77
Cys-NH.sub.2 PL-1950 ReO[V]
Heptanoyl-L-Ser(Bzl)-D-Phe(3-Cl)-L-Arg-L-Trp-L- 1 92 37
Cys-NH.sub.2 PL-1951 ReO[V]
Ac-D-Val-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 60 32
PL-1952 ReO[V]
Ac-D-Ala-L-Chg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 88 46 PL--
ReO[V] Ac-D-Ala-L-Val-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 69
19 1953 PL-1955 ReO[V]
Ac-D-Chg-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 76 68
PL-1956 ReO[V]
Ac-D-Cha-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 58 64
PL-1957 ReO[V]
Ac-D-Leu-L-His-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 66 53
PL-1958 ReO[V]
Ac-D-Val-L-Phg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 60 31
PL-1959 ReO[V] Ac-D-Chg-L-Phg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 55
27 NH.sub.2 PL-1960 ReO[V]
Ac-D-Cha-L-Phg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 38 16 NH.sub.2
PL-1961 ReO[V]
Ac-D-Leu-L-Phg-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 77 38
PL-1962 ReO[V]
Ac-D-Val-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 47 23
PL-1963 ReO[V] Ac-D-Chg-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 31
27 NH.sub.2 PL-1964 ReO[V]
Ac-D-Cha-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp- 1 28 38 NH.sub.2
PL-1965 ReO[V]
Ac-D-Leu-L-Phe-L-Cys-D-Phe(2-Cl)-L-Arg-L-Trp-NH.sub.2 1 62 46
PL-1970 ReO[V] Phenylacetyl-L-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-D-Trp- 1
92 51 NH.sub.2 PL-1971 ReO[V]
3'-bromophenylacetyl-L-Arg-D-Ala-Dphe(4-Cl)-L- 1 33 67
Cys-L-Trp-NH.sub.2 PL-1972 ReO[V]
Phenylacetyl-D-Arg-L-Ala-D-Phe(4-Cl)-L-Cys-L-Trp- 1 17 27
NH.sub.2
[0167] For selected peptides of this invention, a cAMP assay was
also performed, with results as shown in Table 2. Human MC4-R or
B-16 cells were grown to confluence in 96 well plates (plating
approximately 250,000 cells per well). Identical sets of cells in
triplicate were treated with 0.2 mM isobutylmethylxanthine (IBMX)
and the chosen concentration of the rhenium metal ion-complexed
peptide and the rhenium metal ion-complexed peptide in the presence
of 20 nM .alpha.-MSH. Cells similarly treated but with only 20 nM
.alpha.-MSH served as positive control. A buffer blank, as a
negative control, was also included. Incubation was for one hour at
37.degree. C. after which the medium was aspirated and the cells
extarted with 150 microliters of HCl. Total cAMP accumulated in 100
microliters of this solution was quantitated using a commercially
available low pH cAMP assay kit (R&D Systems) by the procedure
specified by the kit supplier. The table shows the amount of cAMP
accumulated in the cells upon exposure to the test compound alone
and the presence of 20 nM .alpha.-MSH. The rhenium metal
ion-complexed peptide showing cAMP accumulation in the same range
as or higher than the positive control (buffer blank in the
presence of .alpha.-MSH) are considered to be agonist ligands. The
rhenium metal ion-complexed peptide showing accumulation in the
same range as the negative control (buffer blank in the absence of
.alpha.-MSH) are ineffective at the test concentration if the
result is similar to the positive control where alpha-MSH is also
present in the assay. The rhenium metal ion-complexed peptide
showing accumulation in the same range as the negative control are
considered to be antagonists if there is inhibition in cAMP when
.alpha.-MSH is present in the assay. Values above the positive
control are due to potent agonism, experimental variance or
synergistic agonistic effects of rhenium metal ion-complexed
peptide and .alpha.-MSH. TABLE-US-00003 TABLE 2 Evaluation of
MC-specific metallopeptides of the primary structure of Table 1 in
cAMP stimulation assay in presence and absence of natural
stimulator .alpha.-MSH cAMP Accumulation (pMol/ml) METAL .mu.M
Absence of Presence of ID ION Concentration .alpha.-MSH .alpha.-MSH
Buffer blank 1 99 PL-815 ReO[V] 10 55 178 PL-836 ReO[V] 10 0.31 68
PL-837 ReO[V] 10 9 63 PL-838 ReO[V] 10 2 53 PL-1102 ReO[V] 10 2 113
PL-1113 ReO[V] 10 123 67 PL-1145 ReO[V] 10 221 277 PL-1160 ReO[V]
10 18 34 PL-1165 ReO[V] 10 15 71 PL-1179 ReO[V] 10 7 50 PL-1183
ReO[V] 1 42 39 PL-1185 ReO[V] 1 30 22 PL-1189 ReO[V] 1 52 34
PL-1190 ReO[V] 1 63 148 PL-1191 ReO[V] 1 8 94 PL-1207 ReO[V] 1 4 3
PL-1208 ReO[V] 10 2 2 PL-1209 ReO[V] 1 5 13 PL-1210 ReO[V] 1 8 5
PL-1211 ReO[V] 1 10 8 PL-1222 ReO[V] 50 177 346 PL-1223 ReO[V] 10
63 85 PL-1233 ReO[V] 10 19 21 PL-1235 ReO[V] 10 29 28 PL-1236
ReO[V] 10 11 30 PL-1237 ReO[V] 10 96 116 PL-1238 ReO[V] 10 26 140
PL-1239 ReO[V] 10 108 131 PL-1249 ReO[V] 10 4 139 PL-1250 ReO[V] 10
2 15 PL-1251 ReO[V] 10 7 21 PL-1255 ReO[V] 10 2 2 PL-1256 ReO[V] 1
17 71 PL-1258 ReO[V] 10 9 63 PL-1259 ReO[V] 1 131 131 PL-1260
ReO[V] 1 5 6 PL-1261 ReO[V] 1 189 214 PL-1264 ReO[V] 1 1 11 PL-1265
ReO[V] 1 3 127 PL-1266 ReO[V] 1 2 131 PL-1268 ReO[V] 1 18 16
PL-1269 ReO[V] 1 4 2 PL-1270 None 1 139 123 PL-1272 None 1 80 75
PL-1276 ReO[V] 1 3 12 PL-1281 ReO[V] 1 158 25 PL-1285 ReO[V] 1 148
158 PL-1292 ReO[V] 1 3 13 PL-1293 ReO[V] 1 3 6 PL-1294 ReO[V] 1 43
91 PL-1295 ReO[V] 1 136 122 PL-1298 ReO[V] 1 1 32 PL-1297 ReO[V] 1
3 7 PL-1301 ReO[V] 1 1 23 PL-1302 ReO[V] 1 12 43 PL-1303 ReO[V] 1 2
59 PL-1305 ReO[V] 1 2 3 PL-1306 ReO[V] 1 189 158 PL-1329 ReO[V] 1 1
63 PL-1332 ReO[V] 1 0 103 PL-1335 ReO[V] 1 0 3 PL-1340 ReO[V] 1 3 7
PL-1342 ReO[V] 10 43 59 PL-1346 ReO[V] 10 3 76 PL-1347 ReO[V] 10 2
81 PL-1351 ReO[V] 10 68 30 PL-1370 ReO[V] 1 2 16 PL-1372 ReO[V] 1
13 149 PL-1373 ReO[V] 1 1 8 PL-1374 ReO[V] 1 37 73 PL-1387 None 1
52 94 PL-1396 ReO[V] 1 36 120 PL-1438 ReO[V] 1 113 126 PL-1440
ReO[V] 1 59 55 PL-1443 ReO[V] 1 4 34 PL-1451 ReO[V] 1 9 90 PL-1493
ReO[V] 1 4 15 PL-1581 ReO[V] 1 111 132 PL-1658 ReO[V] 1 10 67
PL-1659 ReO[V] 1 7 78 PL-1662 ReO[V] 1 6 96 PL-1663 ReO[V] 1 55 122
PL-1664 ReO[V] 1 7 44 PL-1665 ReO[V] 1 10 70 PL-1666 ReO[V] 1 15 28
PL-1667 ReO[V] 1 23 63 PL-1684 ReO[V] 1 5 110 PL-1685 ReO[V] 1 7
102 PL-1686 ReO[V] 1 4 106 PL-1690 ReO[V] 1 13 76 PL-1691 None 1 30
102 PL-1692 None 1 18 106 PL-1694 ReO[V] 1 25 92 PL-1695 ReO[V] 1
30 101 PL-1702 ReO[V] 1 4 134 PL-1703 ReO[V] 1 2 73 PL-1704 ReO[V]
1 3 73 PL-1705 ReO[V] 1 2 74 PL-1706 ReO[V] 1 2 71 PL-1707 ReO[V] 1
15 156 PL-1708 ReO[V] 1 13 54 PL-1709 ReO[V] 1 33 46 PL-1710 ReO[V]
1 3 7 PL-1718 ReO[V] 1 23 105 PL-1722 ReO[V] 1 28 180 PL-1723
ReO[V] 1 30 169 PL-1726 ReO[V] 1 21 171 PL-1727 ReO[V] 1 27 151
PL-1728 ReO[V] 1 15 164 PL-1730 ReO[V] 1 22 111 PL-1731 ReO[V] 1 19
117 PL-1732 ReO[V] 1 14 105 PL-1733 ReO[V] 1 16 114 PL-1734 ReO[V]
1 13 122 PL-1735 ReO[V] 1 20 115 PL-1736 ReO[V] 1 17 152 PL-1737
ReO[V] 1 53 162 PL-1738 ReO[V] 1 7 173 PL-1751 ReO[V] 1 71 96
PL-1752 ReO[V] 1 4 101 PL-1753 ReO[V] 1 39 104 PL-1754 ReO[V] 1 3
100 PL-1755 ReO[V] 1 21 110 PL-1756 ReO[V] 1 43 102 PL-1757 ReO[V]
1 11 96 PL-1758 ReO[V] 1 34 61 PL-1759 ReO[V] 1 24 79 PL-1760
ReO[V] 1 66 80 PL-1761 ReO[V] 1 11 87 PL-1762 ReO[V] 1 49 88
PL-1774 ReO[V] 1 48 163 PL-1775 ReO[V] 1 53 170 PL-1776 ReO[V] 1 26
138 PL-1778 ReO[V] 1 50 141 PL-1779 ReO[V] 1 47 155 PL-1780 ReO[V]
1 8 126 PL-1782 ReO[V] 1 7 108 PL-1788 ReO[V] 1 189 109 PL-1789
ReO[V] 1 96 90 PL-1790 ReO[V] 1 164 149 PL-1791 ReO[V] 1 163 137
PL-1792 ReO[V] 1 187 109 PL-1793 ReO[V] 1 119 98 PL-1794 ReO[V] 1 4
133 PL-1795 ReO[V] 1 71 124 PL-1796 ReO[V] 1 4 133 PL-1799 ReO[V] 1
9 117 PL-1801 ReO[V] 1 42 107 PL-1805 ReO[V] 1 15 78 PL-1806 ReO[V]
1 10 110 PL-1807 ReO[V] 1 103 115 PL-1808 ReO[V] 1 109 137 PL-1809
ReO[V] 1 113 130 PL-1810 ReO[V] 1 101 125 PL-1811 ReO[V] 1 117 66
PL-1812 ReO[V] 1 132 93 PL-1813 ReO[V] 1 36 91 PL-1814 ReO[V] 1 8
80 PL-1815 ReO[V] 1 34 119 PL-1816 ReO[V] 1 69 89 PL-1817 ReO[V] 1
24 110 PL-1818 ReO[V] 1 6 96 PL-1819 ReO[V] 1 93 96 PL-1820 ReO[V]
1 8 102 PL-1821 ReO[V] 1 37 88 PL-1822 ReO[V] 1 13 101 PL-1823
ReO[V] 1 14 114 PL-1824 ReO[V] 1 71 124 PL-1825 ReO[V] 1 4 133
PL-1825 ReO[V] 1 7 106 PL-1838 ReO[V] 1 16 65 PL-1839 ReO[V] 1 63
77 PL-1840 ReO[V] 1 76 77 PL-1841 ReO[V] 1 91 81 PL-1842 ReO[V] 1
25 76 PL-1843 ReO[V] 1 18 81 PL-1844 ReO[V] 1 110 81 PL-1845 ReO[V]
1 82 38 PL-1877 ReO[V] 1 72 125 PL-1879 ReO[V] 1 75 108 PL-1880
ReO[V] 1 16 92 PL-1883 ReO[V] 1 125 115 PL-1884 ReO[V] 1 108 158
PL-1885 ReO[V] 1 135 148 PL-1886 ReO[V] 1 151 161 PL-1900 ReO[V] 1
19 87 PL-1902 ReO[V] 1 56 143 PL-1903 ReO[V] 1 62 108 PL-1905
ReO[V] 1 59 96 PL-1906 ReO[V] 1 68 87 PL-1907 ReO[V] 1 62 98
PL-1909 ReO[V] 1 70 81 PL-1915 ReO[V] 1 123 128 PL-1917 ReO[V] 1
109 116 PL-1931 ReO[V] 10 15 79 PL-1144 ReO[V] 10 215 280 PL-1489
ReO[V] 1 9 16 PL-1605 ReO[V] 1 16 15
[0168] Based on the foregoing studies, potential lead molecules
have been identified, including the following: TABLE-US-00004
PL-1145 MC1-R agonist (Template 2) PL-1144 MC1-R agonist (Template
2) PL-1493 MC4-R antagonist (Template 1) PL-1883 MC4-R agonist
(Template 1) PL-1489 MC4-R antagonist (Template 5) PL-1950 MC4-R
specific (Template 5) PL-1947 MC4-R specific (Template 5) PL-1581
Non-specific agonist (Template 5) PL-1790 Non-specific agonist
(Template 5) PL-1791 Non-specific agonist (Template 5) PL-1792
Non-specific agonist (Template 5) PL-1605 Non-specific antagonist
(Template 6) PL-1758 Non-specific (Template 4) PL-1877 MC4-R
specific (Template 4) PL-1902 MC4-R specific (Template 4) PL-1909
MC4-R specific agonist (Template 4) PL-1888 MC4-R specific
(Template 7) PL-1269 MC4-R specific antagonist (Template 3) PL-1297
MC4-R specific antagonist (Template 3)
[0169] Radiopharmaceutical Applications. In one embodiment,
metallopeptides of this invention that are MC1-R specific can be
used, when complexed to .sup.99mTc as a radiodiagnostic agent, for
imaging melanoma tumor metastases, and when complexed to
rhenium-188 (.sup.188Re), rhenium-186 (.sup.186Re) or other
therapeutic radionuclides as a radiotherapeutic agent for treatment
of melanoma tumors and metastatic tumors.
[0170] Human melanoma has a complex antigenic profile. It is
generally believed that malignant melanoma is derived by UV
activity from DOPA positive melanocytes, the melanin (skin pigment)
producing units. Primary diagnosis involves electron microscopic
examination to reveal the presence or absence of pre-melanosomes.
Melanotic melanoma is classified as dendritic, spindle, bizarre,
large epitheloid, small nevus and so on. Amelanotic melanoma, on
the other hand, is frequently misdiagnosed because the histology of
these cells resembles that of malignant lymphoma, carcinoma or
sarcoma. Therefore, morphological evaluation may not prove reliable
for clinical diagnosis. Thus, there is a need for a
receptor-specific diagnostic test, particularly to identify and
locate metastatic melanoma tumors.
[0171] Several studies have documented the presence of melanotropin
receptors on primary human melanoma cells. Melanotropin receptors
have been reported as markers for melanotic and amelanotic human
melanoma tumors. In particular, the presence of MC1-R has been
demonstrated in human melanoma cells by an antibody to MC1-R.
[0172] The melanotropin bioactive sequence, or message segment, is
a tetrapeptide, His-Phe-Arg-Trp (SEQ ID NO:1), that exist as a
reverse turn. Within the reverse turn, the His, Phe, and Trp
residues have been postulated to form a hydrophobic receptor
binding surface. The His residue has recently been identified as a
signal residue that helps discriminate between MC1-R and MC4-R.
Thus, it is possible to design metallopeptides of this invention
which are specific for MC1-R, and bind MC1-R with high affinity,
but which are not specific for MC4-R, or which bind MC4-R with low
affinity. One result is a .sup.99mTc-labeled radioimaging agent
with high specific affinity and selectivity for the MC1-R. In
addition, both agonists and antagonists metallopeptides of this
invention are contemplated for comparative evaluation in imaging
melanoma tumors.
[0173] The product can be formulated as a single-vial, lyophilized
radiolabeling kit containing the peptide in an uncomplexed state,
buffer, and a reducing agent for pertechnetate. To induce
radiolabeling, resulting in a metallopeptide, the vial is incubated
after the addition of sodium pertechnetate.
[0174] In one method of labeling with .sup.99mTc, a 5-10 .mu.g
sample of the peptide taken in 0.001 N aq. HCl is mixed with 1-30
mCi of generator-eluted Na.sup.99mTcO.sub.4 in a 5 ml serum vial.
The volume of the resulting mixture is adjusted to 600 .mu.l using
injectable saline. A 400 .mu.l volume of freshly prepared and
nitrogen-purged phthalate-tartrate-Sn(II) buffer (40:10:1 mM) is
added to the vial under a nitrogen head space. The vial is
immediately sealed and placed in a shielded boiling water bath.
After 15 minutes the vial is removed from the water bath and
allowed to come to room temperature. The radiochemical purity, as
calculated from HPLC profiles, ranges from 90-99%.
[0175] The peptides of this invention may alternatively be labeled
with .sup.99mTc by other means, including use of
stannous-tartrate-succinate buffer, stannous-EDTA-succinate buffer,
stannous stabilized in glucoheptonate, or a
stannous-borate-tartrate buffer, as well as other means of labeling
with .sup.99mTc known in the art.
[0176] For radiopharmaceutical and other medical applications, the
metallopeptides of this invention may be delivered to a subject by
any means known in the art. This includes intravenous injection,
subcutaneous injection, administration through mucous membranes,
oral administration, dermal administration, regional administration
to an organ, cavity or region, and the like.
[0177] Imaging may be any means known in the art, including gamma
camera and SPECT imaging. Imaging may commence immediately after
administration, and may include time course radiographic studies,
and imaging may continue so long as images may be obtained.
[0178] The MC1-R specific metallopeptides of this invention may be
used as melanoma specific tumor imaging and staging agent. These
uses include early detection and localization of primary and
disseminated lesions, identification of lymph nodes containing
lesions, radioimmunoguided surgery applications and the like. Tumor
imaging using a .sup.99mTc-labeled metallopeptide of this invention
selective for the MC1-R will further help in formulating the
optimal clinical treatment modality, whether surgical, radiation or
chemotherapeutic.
[0179] Chemoprevention Applications. In another embodiment,
metallopeptides of this invention that are MC1-R specific can be
used as chemoprevention agents against sun-induced, such as by UV
radiation, neoplastic activity in human skin. MC1-R agonist
metallopeptides of this invention may be employed to stimulate
epidermal melanocytes to produce melanin as well as to convert
pheomelanin to eumelanin. Eumelanin, which is dark brown or black
pigmentation, is considered more photo-protective than pheomelanin,
which is yellow or red pigmentation.
[0180] In general, darker skinned individuals have lower incidences
of skin cancer than lighter skinned people. The dark pigment
eumelanin, a brown/black pigment incorporating dopa-based
structural units, is the main photoprotective agent in skin.
Lighter colored people have higher levels of pheomelanin, a
red/yellow pigment having predominantly cysteine and related
sulfur-based structural units, which is an inefficient UV absorber.
The process of melanogenesis is believed to involve stimulation of
MC1-R in epidermal melanocytes, thereby mediating the stimulation
of tyrosinase enzymes within these pigment cells, inducing the
conversion of tyrosine to dopa and then through dopaquinone to
eumelanin. Sun tanning due to direct sun exposure is also proposed
to result from the same pathway by local production of melanotropic
peptide from a POMC gene in the epidermis. Thus stimulation of
eumelanin production and conversion of pheomelanin to eumelanin may
be a desirable chemoprevention modality in blocking sun, or UV,
induced neoplastic activity in skin.
[0181] A potent, high-affinity and highly selective MC1-R agonist
metallopeptide of this invention can accordingly be used as a
therapeutic chemoprevention agent for combating harmful sun, or UV,
exposure that induces neoplastic activity in skin melanocytes.
[0182] Particularly for individuals previously diagnosed with
melanoma or diagnosed as highly susceptible to melanoma, avoidance
of any significant UV exposure is medically necessary. Currently,
both physical screens, such as hats, long sleeves and so on, and
various sun- or UV-protecting creams and formulations are utilized.
However, the efficacy, both absolute and as a function of time, of
even the best chemical sun block is limited, and physical screens
are inappropriate for many activities. This results in individuals
either receiving unacceptably high UV doses or foregoing normal
activities, such as swimming, hiking, skiing, attending sporting
events, employment in outdoor settings and so on.
[0183] Eumelanin production through activation of the MC1-R on
epidermal melanocytes provides a better natural shield against sun
(UV) induced mutations and DNA damage than does any chemical sun
block. Eumelanin is a better UV absorber over a wide range than any
commercially available sun-protecting cream or formulation.
[0184] The metallopeptides for this application may be made with
non-radioactive isotopes of rhenium, or other metals as specified
herein, with metal ion complexation by any means specified herein
or known in the art. The metallopeptides may be formulated by any
means known in the art, including but not limited to tablets,
capsules, caplets, suspensions, powders, lyophilized forms and
aerosols and may be mixed and formulated with buffers, binders,
stabilizers, anti-oxidants and other agents known in the art. The
metallopeptides may be administered by any systemic or partially
systemic means known in the art, including but not limited to
intravenous injection, subcutaneous injection, administration
through mucous membranes, oral administration, dermal
administration, skin patches, aerosols and the like.
[0185] Therapeutic Applications. In another embodiment,
metallopeptides of this invention that are MC4-R agonists can be
used as a therapeutic agent to modify energy metabolism and feeding
behavior, including treatment of pathological obesity and related
conditions. Metallopeptides of this invention that are MC4-R
antagonists can also be used as a therapeutic agent in eating
disorders, such as treatment of anorexia.
[0186] Control centers for eating and satiety reside in the
hypothalamus. These responses are determined by diverse hormones
and soluble factors that signal through specific receptors in the
hypothalamus. MC4-R is known to be expressed in the brain, and
inactivation of this receptor by gene targeting has resulted in
mice with the maturity-onset obesity syndrome that is associated
with hyperphagia, hyperinsulinemia and hyperglycemia.
[0187] In yet another embodiment, metallopeptides of this invention
may used as therapeutic agents for treatment of sexual dysfunction,
including treatment of both male erectile dysfunction and female
sexual dysfunction. In yet another embodiment, metallopeptides of
this invention may be used as therapeutic agents for treatment of
inflammation, including specifically MC1-R and MC3-R agonist
metallopeptides.
[0188] The metallopeptides for this application may be made with
non-radioactive isotopes of rhenium, or other metals as specified
herein, with metal ion complexation by any means specified herein
or known in the art. The metallopeptides may be formulated by any
means known in the art, including but not limited to tablets,
capsules, caplets, suspensions, powders, lyophilized forms and
aerosols and may be mixed and formulated with buffers, binders,
stabilizers, anti-oxidants and other agents known in the art. The
metallopeptides may be administered by any systemic or partially
systemic means known in the art, including but not limited to
intravenous injection, subcutaneous injection, administration
through mucous membranes, oral administration, dermal
administration, skin patches, aerosols and the like.
[0189] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Development of a Prototype Metallopeptide Library for the
Melanocortin Receptor
[0190] The library design was based on the tetrapeptide message
sequence, His-Phe-Arg-Trp (6-9 sequence) (SEQ ID NO:1), of
.alpha.-MSH. This sequence exists as a reverse turn, making it
suitable for conversion into a metallopeptide format of this
invention. In this approach metallopeptides were designed around a
tripeptide N.sub.3S.sub.1 MBD designed for a rhenium metal ion. The
MBD was derivatized to yield the pentapeptide
Ac-His-Phe-Arg-Cys-Trp-NH.sub.2 (SEQ ID NO:8) as a putative
candidate for melanocortin ("MC") receptors. Further refinements in
the structure were made in response to other considerations,
including the chirality of amino acid side chains, yielding a
template structure Ac-His-D-Phe-Arg-Cys-Trp-NH.sub.2. The structure
of this peptide after binding to rhenium is: ##STR3##
[0191] The template structure was used to define a small
combinatorial library utilizing split synthesis methodologies. The
final template selected for the combinatorial library was
Ac-D-His-Xaa-D-Cys-Trp-NH.sub.2, where Xaa was D-(2')
Naphthylalanine, D-Trp, D-HomoPhe, or D-Phenylglycine. For this
library, the peptide resin, Cys(S.sup.tBu)-Trp(Boc)-Resin was split
in four equal parts. Each part was reacted with one of the four Xaa
types. After coupling, the resin pools were mixed and synthesis
continued in a single pool to couple the His residue. The final
result was four separate peptides in a single pool, each peptide
varying by one amino acid, in the Xaa position.
[0192] An S.sup.tBu OSPG group was used to protect the SH group
during synthesis. After solid-phase assembly of the peptide chain
using Fmoc chemistry with acid labile side chain protecting groups,
the S.sup.tBu group was split using tributylphosphine. The
resulting free SH-containing peptide-resin was treated with the
rhenium transfer agent Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the
presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene as base. The
resulting metallopeptide resin was then treated with TFA to cleave
it from the resin and de-protect all the side chain protecting
groups. The products were analyzed by mass spectrometry. HPLC
analysis was performed and individual peaks collected and subjected
to mass analysis. The resulting peptides were analyzed by electron
spray mass spectrometry, yielding the predicted mass, including the
rhenium complexed to the peptide.
EXAMPLE 2
Design and Synthesis of Melanocortin Receptor-Specific
Metallopeptide Library
[0193] The library was rationally designed based upon data relating
to melanocortin receptors and peptide sequences specific to the
melanocortin receptors, including melanotropin side-chain
pharmacophores, D-Phe.sup.7 and Trp.sup.9, that interact with a
hydrophobic network of receptor aromatic residues in transmembrane
regions 4, 5, 6, and 7. Based on this design criterion, a
pharmacophore for the melanocortin receptor was preliminarily
defined, and a combinatorial library designed for identification of
potent and receptor-selective agonists.
[0194] Based on the design criteria, the putative structure
R-Aaa-Baa-L-Cys-Caa-NH.sub.2 was selected, in which each of Aaa,
Baa and Caa are selected from L- or D-isomers of 2-Nal (1), Phe
(2), Trp (3), Tyr (4) and Ala (5), so that any one of the foregoing
can be substituted for any one of Aaa, Baa or Caa. In the
nomenclature adopted for the library design, the five amino acids
were designated 1 through 5, with the isomerism conventionally
notated, so that, for example, Baa.sub.2L refers to L-Phe in the
Baa position.
[0195] The terminal R group was selected from Ac,
C.sub.6H.sub.5OOH, CH.sub.3(CH.sub.2).sub.5--COOH,
C.sub.6H.sub.5CH.dbd.CH--COOH (trans) and Pyridine-3-carboxylate.
The terminal R group represents a truncated amino acid, and offers
additional structural diversity.
[0196] A pool and split library synthesis scheme was employed such
that 5,000 separate compounds were synthesized, resulting in 200
final pools each containing 25 different compounds, with the
compounds differing solely by the amino acids in the Aaa and Baa
position. Using this methodology, binding characteristics relating
to the Caa amino acid or R terminal group can be identified through
inter-group comparison, thereby simplifying the deconvolution
strategy.
[0197] The library synthesis steps are set forth in FIG. 8. The
resin of step 1 was divided into 10 groups. At step 2 each of
Caa.sub.1L through Caa.sub.5D were coupled to an individual resin
group, and L-Cys was coupled to each resin group, resulting in 10
groups and 20 couplings. Each of the resin groups of step 2 was
then divided into 10 sub-groups as shown at step 3 (with only one
subgroup illustrated at step 3, and for each subgroup of step 3,
each of Baa.sub.1L through Baa.sub.5D were coupled to one group
within the subgroup, resulting in 100 groups in 10 subgroups and
100 couplings. For each subgroup of step 3, the five Baa.sub.xL
members and the five Baa.sub.xD members were separately pooled in
step 4, resulting in 20 subgroups, with each subgroup containing
five different sequences differing by the Baa.sub.x member. Each of
the 20 subgroups of step 4 were then in step 5 divided into 10
groups (with only one shown for illustration purposes in FIG. 8),
and for each subgroup, each of Aaa.sub.1L through Aaa.sub.5D were
coupled to one group within the subgroup, resulting in 200 groups
in 20 subgroups and 200 couplings. For each subgroup of step 5, the
five Aaa.sub.xL members and the five Aaa.sub.xD members were
separately pooled in step 6, resulting in 40 subgroups, with each
subgroup containing twenty-five different sequences differing by
the Baa.sub.x and Aaa.sub.x member. In step 7, each of the 40
subgroups of step 6 was divided into five groups, and each of
R.sub.1 through R.sub.5 were coupled to one group within the
subgroup, resulting in 200 groups in 40 subgroups, with each group
containing 25 different sequences differing by the Baa.sub.x and
Aaa.sub.x member.
[0198] Peptides were synthesized using Fmoc chemistry, with side
chain functionalities protected using acid labile groups. The SH
group of the Cys residue was protected by a S.sup.tBu OSPG
cleavable in presence of both base and acid labile groups using
tributylphosphine as the reducing agent. The peptide chain was
assembled on the solid phase using
1-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium
tetrafluoroborate (TBTU) as a coupling agent. The SH group was then
selectively unprotected and rhenium metal ion complexed using the
rhenium transfer agent Re(O)Cl.sub.3(PPh.sub.3).sub.2 in the
presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) as base. In
this manner, the metal-peptide complex was formed with the peptide
chain still tethered to the solid support. The metallopeptide was
then liberated from the solid support by treatment with TFA. This
solid phase approach to metal ion complexation is fully compatible
with split synthesis methodologies employed in combinatorial
libraries.
[0199] The synthesis process was performed using commercial
automated synthesizers. Multiple manual synthesizers (such as those
commercially available from SynPep Corporation, Dublin, Calif.)
allow parallel synthesis of ten peptides simultaneously.
[0200] Quality control protocols were employed as required, and
include HPLC, mass spectral analysis, and amino acid analysis on
each individual pool of 25 compounds. The presence of each of pool
constituent is established by molecular ion mass spectral analysis.
Negative ion mode electron spray (ES) and matrix-assisted laser
desorption (MALDI) techniques were employed. Using mass spectral
analysis, three different measures were made: (a) the presence of
up to 25 individual compounds by molecular ion peak measurement
(assuming different masses for each compound), (b) confirmation
that the molecular ion peaks show complexation to a rhenium metal
ion, and (c) absence of peaks with molecular masses corresponding
to peptides uncomplexed with metal ion. Rhenium is a mixture of two
isotopes that differ in mass by 2 units (186 and 188) with a
relative abundance of these isotopes of 1:2. The molecular ion
profile of a metallopeptide appears as two peaks that differ by 2
mass units with integrated area ratios of 1:2. Rhenium thus acts as
an internal mass spectral reference for these metallopeptides. A
spectral analysis of one such pool of 25 compounds synthesized by
the methods of this claim is shown at FIG. 9. Five sets of two
metallopeptides in this pool have similar masses due to the
presence of the same amino acids assembled in different sequences.
The relative intensities of the peaks is due to differential
ionization of individual compounds in the pool and does not reflect
the relative amounts in the mixture. Each pair of peaks with mass
unit differences of 2 and relative ratios of 1:2 are due to the
relative abundance of two stable isotopes of rhenium (Re-185 and
Re-187). The spectral analysis did not reveal any free uncomplexed
linear peptides, which would be approximately 197 to 199 mass units
less than the corresponding metallopeptide, due to the absence of
the rhenium-oxo core.
[0201] Amino acid analysis of each pool of 25 metallopeptides was
also employed, and was used to determine the relative equimolar
ratio of each of 25 compounds in a pool. The synthetic protocols of
split synthesis were designed to assure equimolar amounts of pool
constituents.
EXAMPLE 3
Screening of Melanocortin Receptor-Specific Library
[0202] Metallopeptide library pools are screened for MC4-R receptor
and MC1-R receptor binding activity in high throughput screening
assays. The MC receptor-binding assay uses membrane preparations
from B16-F1 or B16-F10 melanoma cells as the source of MC receptor.
Cell membranes prepared from MC4-R-expressing 293 cells and
negative control, untransfected 293 cells, are substituted for
B16-F1 or B16-F10 melanoma cell membranes in MC4-R specific binding
assays. The MC receptor-binding assays use the Millipore
Multi-Screen System and are performed in 96-well Millipore filter
plates (Durapore, 0.45 mm porosity) pre-blocked with 0.5% bovine
serum albumin in phosphate buffered saline. Cell membrane
preparations (12.5 .mu.g/well) are incubated with 0.4 nM
.sup.125I-NDP-MSH in HEPES Buffer containing 0.2% bovine serum
albumin. Non-specific binding is determined by addition of
10.sup.-6 M .alpha.-MSH or 10.sup.-7 M NDP-MSH. Metallopeptides to
be tested are added to reaction wells at a final concentration of 1
mM. After incubation for 90 minutes at room temperature, the
binding reaction is rapidly terminated by filtration to capture the
membranes. Filters are washed 3 times with ice-cold PBS and
air-dried. Individual filters are then punched from the plates and
distributed into gamma counter tubes. Radioactivity associated with
the membranes is determined in a Packard Cobra gamma counter.
Specific binding is determined as the radioactivity in wells
containing .sup.125I-NDP-MSH alone minus the radioactivity in wells
containing 10.sup.-6M .alpha.-MSH. Test compounds are screened in
duplicate wells and are considered to be active where 1 .mu.M
concentrations inhibit >50% of the specific binding. Standard
curves of unlabeled NDP-MSH will be included on each plate as an
internal assay control.
[0203] A commercially available cAMP kit (R&D Systems, DE0350,
low pH) is employed to evaluate agonist potential of
metallopeptides that bind to MC4-R. 293 cells stably transfected
with hMC-4 receptor, or B16-F1 melanoma cells, are grown to
confluence in 96-well dishes. Cells are washed and fresh RPMI
containing 0.2 mM isobutylmethylxanthine (cAMP phosphodiesterase)
and varying concentrations of metallopeptides, or .alpha.-MSH as a
positive control, are added, and the cells are incubated for 1 hour
at 37.degree. C. Medium is aspirated, and cell layers extracted
with 150 .mu.l of 0.1 M HCl. Total cAMP accumulation in 100 .mu.l
of cell extract is quantitated in 96-well plates by competitive
immunoassay with the cAMP kit, using an acetylation modification.
EC.sub.50 values for test compounds are calculated based on cAMP
accumulation in cells treated with 10.sup.-6 M .alpha.-MSH. The
capabilities of both of these cell types to accumulate cAMP in the
presence of .alpha.-MSH and MSH analog peptides are documented in
the scientific literature; see, for example, Ollman M M et al:
Science 278:135-138, 1997.
EXAMPLE 4
Deconvolution of Melanocortin Receptor-Specific Library
[0204] Deconvolution of a positive pool is done by iterative
re-synthesis and screening deconvolution approaches. The individual
25 constituents are synthesized separately, or alternatively in 5
smaller pools of 5 compounds each, with each pool screened in
receptor binding assays. The latter approach is preferred where
there is a high hit frequency in the preliminary screen. The
compounds in pools with the best results (closest to receptor
affinity in the nanomolar range and MC4-R to MC1-R selectivity of
at least 100) are individually synthesized and screened.
EXAMPLE 5
Alternative Method of Deconvolution of Melanocortin
Receptor-Specific Library
[0205] In this example, an alternative method of mass spectral
deconvolution of metallopeptide libraries is employed. The method
is based on the internal signature of rhenium-complexed peptides
(two isotopic peaks in 1:2 ratios differing by 2 mass units), which
generally permits metallopeptide identification even in mixed
solutions. A positive pool is incubated with receptor-bearing
cells, the excess unbound compounds washed away under controlled
conditions, and the cells treated with a solvent to disrupt
metallopeptide binding and extract the metallopeptide in the
solvent. Mass spectral analysis of the solvent reveals the
metallopeptide or metallopeptides which are bound to the
receptor-bearing cells, and through comparison to the quality
control data it is possible to ascertain the specific
metallopeptide or metallopeptides which are bound. This process
provides high throughput of metallopeptide library screening.
EXAMPLE 6
Single Pot Synthesis of a Library of Four Metallopeptides of the
General Structure Ac-His-Xaa-Cys-Trp-NH.sub.2
[0206] A synthesis procedure similar to that described in Example 1
was used in making this library. A NovaSyn TGR resin for making
peptide amides (substitution 0.2 mM/gm) was used. Fmoc synthetic
strategy was employed using the following protected amino acids:
Fmoc-Trp(Boc), Fmoc-Cys(S.sup.tBu), Fmoc-Xaa, and Fmoc-His(Trityl).
The Xaa amino acids were Trp, HomoPhe, 2'-Naphthylalanine, and
Phenylglycine. The peptide resin Cys(S.sup.tBu)-Trp-NH.sub.2 was
split into four equal pools and one of the Xaa amino acids was
coupled to one individual pool. After completion of the coupling
reaction, the four resin pools were mixed again. The synthesis
proceeded with the coupling of His followed by acetylation of the
N-terminus. After the complete assembly of the peptide chain
Ac-His(Trt)-Xaa-Cys(S.sup.tBu)-Trp(Boc)-NH.sub.2, the S.sup.tBu
group was removed by treatment with DMF/tributylphosphine and
rhenium-oxo metal ion was complexed as generally described above.
The fully protected metallopeptide was deblocked and liberated from
the solid support by treatment with a cleavage cocktail (95:5
mixture of trifluoroacetic acid-triisopropylsilane) for three
hours. The metallopeptide library was recovered by precipitation
using cold ether. The resulting pellet was washed twice and 0.5 ml
of 95% acetic acid was added. After one-half hour 5 ml of water was
added and the solution was freeze-dried yielding the desired
library in solid form. Mass spectrometric analysis of the library
pool confirmed the correct masses for all four members of the
library: TABLE-US-00005 TABLE 3 Compound Structure Calculated Mass
Mass (M + 1) found 1 Ac-His-Phg-Cys-Trp-NH.sub.2 815.7 and 817.6
815.2 and 816.7 (SEQ ID NO: 6) 2 Ac-His-Trp-Cys-Trp-NH.sub.2 868.8
and 870.7 868.0 and 870.1 (SEQ ID NO: 3) 3
Ac-His-HPhe-Cys-Trp-NH.sub.2 843.8 and 845.7 842.8 and 845.2 (SEQ
ID NO: 4) 4 Ac-His-2'-Nal-Cys-Trp-NH.sub.2 880.0 and 881.9 879.1
and 880.9 (SEQ ID NO: 5)
[0207] As noted in Table 3, two molecular ion peaks differing in
mass units of 2 were calculated and observed for each structure;
this difference is presumptively due to the presence of two natural
isotopes of rhenium, Re-185 and Re-187, in the complexation step.
In addition, the area under the observed peaks in the spectrometric
analysis showed that for each structure the area was in a 1:2
ratio, which is identical to and presumptively related to the
relative abundance of Re-185 and Re-187 isotopes. These results
confirmed the complexation of rhenium to the peptides. The spectral
analysis is shown at FIG. 10.
[0208] These results were also confirmed by HPLC analysis of this
library of four compounds, which results were compared to HPLC
analysis of each of the four individual members performed under
identical HPLC conditions. As is evident from FIG. 11A, each of the
four member components is present in the library mixture. In the
HPLC profile each of the metallopeptide has resolved into two
isomers due to alternate orientations (syn and anti) of the rhenium
oxo core. The HPLC analysis also revealed the lack of uncomplexed
linear peptides in the preparation. All four compounds used for
this comparison were individually prepared using methods identical
to that described above for synthesis of the library. The HPLC
profiles are shown as FIGS. 11B to 11E.
EXAMPLE 7
Synthesis of MC1-R Specific Metallopeptides for Use as
Chemoprevention Agent
[0209] High potency metallopeptides are also provided with
N-terminal modifications. Systematic N-terminal modifications are
made based upon the limited data available in the literature
related to receptor-binding affinities of peptide analogs for
various MC receptor types. In general, these studies indicate that
the His.sup.6 residue may be a critical factor in determining
receptor selectivity for MC1-R (peripheral) versus MC4-R (brain).
Three-dimensional molecular models of the human melanocortin
receptor have been developed based upon the electron
cryo-microscopic structure of bacteriorhodopsin and the electron
density footprint of bovine rhodopsin. By modeling known potent
agonists into the proposed binding sites, specific ligand-receptor
interactions have been identified. By this means, researchers have
tentatively identified melanotropin side-chain pharmacophores,
D-Phe.sup.7 and Trp.sup.9, and have proposed that these interact
with a hydrophobic network of receptor aromatic residues in
transmembrane regions 4, 5, 6, and 7. The findings of these results
are utilized in selecting N-terminal modifications for the
metallopeptide core. Groups with hydrophobic and/or hydrogen
bonding potential are investigated in a systematic and
stereospecific manner, with contemporaneous assaying of potency and
MC1-R specificity of the resulting metallopeptides. The influence
of the His residue on bioactivity is also investigated. Two series
of metallopeptides are synthesized: [0210]
Rheniumoxo-[R-His-D-Phe-Arg-Cys-Trp-NH.sub.2] and [0211]
Rheniumoxo-[R-D-Phe-Arg-Cys-Trp-NH.sub.2] where R is a pair
consisting of a hydrophobic side chain and hydrophilc side chain
with hydrogen bonding potential which is selected from the
following groups: ##STR4## and where n is from 2 to 9 and X and/or
Y are selected from H, OH, Cl, Br, I, NH.sub.2, OCH.sub.3, NO.sub.2
and similar groups.
EXAMPLE 8
Skin Darkening in Anolis Carolinensis
[0212] PT-1145 complexed with rhenium (0.65 mg taken in a 50 mL
vehicle) was injected intraperitoneally in a lizard (Anolis
carolinensis) that was pre-conditioned for a skin darkening
experiment. The pre-conditioning involved leaving the lizards in a
well-lit white background for 24 hours. Within 10-15 minutes after
injection, the skin coat color turned from bright green to dark
brown to black. The skin coat color remained dark during the
five-hour observation period. Lizards injected with the vehicle
alone (PBS buffer containing 1% each of DMF and beta-cyclodextran)
did not show any change in their skin color during the 5-hour
observation period.
[0213] Each of the foregoing is merely illustrative, and other
equivalent embodiments are possible and contemplated.
[0214] Although this invention has been described with reference to
these preferred embodiments, other embodiments can achieve the same
results. Variations and modifications of the present invention will
be obvious to those skilled in the art and it is intended to cover
in the appended claims all such modifications and equivalents. The
entire disclosures of all applications, patents, and publications
cited above are hereby incorporated by reference.
[0215] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
Sequence CWU 1
1
69 1 4 PRT Artificial 6-9 sequence of alpha-melanocyte stimulating
hormone 1 His Phe Arg Trp 1 2 4 PRT Artificial Synthesized
metal-binding sequence, not species derived 2 Gly Gly Gly Gly 1 3 4
PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 3 His Trp Cys Trp 1 4 4 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 4 His Xaa Cys
Trp 1 5 4 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 5 His Xaa Cys Trp 1 6 4 PRT
Artificial Synthetic metal-binding melanocortin-receptor specific
sequence 6 His Xaa Cys Trp 1 7 4 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 7 His Phe Cys
Trp 1 8 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 8 His Phe Arg Cys Trp 1 5 9
4 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 9 His Phe Trp Cys 1 10 4 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 10 Phe Gly
Cys Trp 1 11 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 11 Phe His Gly Cys Trp 1 5
12 4 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 12 Ala Xaa Cys Trp 1 13 4 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 13
Ala Xaa Cys Trp 1 14 4 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 14 Ala Xaa Cys Trp 1 15 5
PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 15 Xaa Gly Xaa Cys Trp 1 5 16 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 16
Xaa Gly Xaa Cys Trp 1 5 17 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 17 Xaa Gly Xaa Cys Trp 1 5
18 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 18 Xaa Gly Xaa Cys Trp 1 5 19 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 19
Xaa Gly Xaa Cys Trp 1 5 20 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 20 Xaa Gly Leu Cys Trp 1 5
21 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 21 Lys His Phe Cys Trp 1 5 22 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 22
Xaa His Phe Cys Trp 1 5 23 6 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 23 Glu His Gly Arg Trp Cys
1 5 24 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 24 Xaa Xaa Arg Cys Trp 1 5
25 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 25 Xaa Ala Arg Cys Trp 1 5 26 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 26
Xaa Cys Arg Cys Trp 1 5 27 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 27 Arg Trp Xaa Cys Phe 1 5
28 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 28 Xaa Arg Trp Cys Xaa 1 5 29 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 29
Xaa Xaa Trp Cys Arg 1 5 30 6 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 30 Xaa Arg Phe His Cys Trp
1 5 31 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 31 Xaa His Arg Cys Trp 1 5
32 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 32 Arg His Phe Cys Trp 1 5 33 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 33
Arg Trp Phe Cys His 1 5 34 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 34 Trp Phe His Cys Arg 1 5
35 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 35 His Phe Trp Cys Arg 1 5 36 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 36
His Arg Trp Cys Phe 1 5 37 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 37 Xaa Phe His Cys Trp 1 5
38 4 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 38 Phe His Cys Trp 1 39 7 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 39
Xaa Ala His Phe Arg Cys Trp 1 5 40 7 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 40 Xaa Ala
His Phe Arg Cys Trp 1 5 41 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 41 Arg Phe Phe Cys Ser 1 5
42 6 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 42 Arg Phe Xaa Asn Cys Phe 1 5 43 6 PRT
Artificial Synthetic metal-binding melanocortin-receptor specific
sequence 43 Arg Phe Phe Asn Cys Phe 1 5 44 7 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 44
Arg Phe Cys Phe Asn Ala Phe 1 5 45 7 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 45 Arg Phe
Phe Cys Asn Ala Phe 1 5 46 7 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 46 Arg Phe Phe Asn Cys Ala
Phe 1 5 47 7 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 47 Arg Phe Phe Asn Ala Cys
Phe 1 5 48 6 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 48 Arg Phe Phe Asn Phe Cys
1 5 49 7 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 49 Xaa Ala His Xaa Arg Cys
Trp 1 5 50 6 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 50 Xaa Arg Ala Xaa Cys Trp
1 5 51 7 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 51 Xaa Ala His Xaa Arg Cys
Trp 1 5 52 6 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 52 Xaa Arg Ala Xaa Cys Trp
1 5 53 7 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 53 Xaa Ala His Xaa Arg Cys
Trp 1 5 54 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 54 His Gly Gly Cys Trp 1 5
55 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 55 His Xaa Arg Trp Cys 1 5 56 12 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 56
Tyr Val Xaa Gly His Phe Arg Trp Asp Arg Cys Phe 1 5 10 57 12 PRT
Artificial Synthetic metal-binding melanocortin-receptor specific
sequence 57 Tyr Val Xaa Gly His Phe Arg Trp Asp Cys Arg Phe 1 5 10
58 12 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 58 Tyr Val Xaa Gly His Phe Arg Trp Cys Asp Arg
Phe 1 5 10 59 12 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 59 Tyr Val Xaa Gly His Phe
Arg Cys Trp Asp Arg Phe 1 5 10 60 12 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 60 Tyr Val
Xaa Gly His Phe Cys Arg Trp Asp Arg Phe 1 5 10 61 12 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 61
Tyr Val Xaa Gly His Cys Phe Arg Trp Asp Arg Phe 1 5 10 62 12 PRT
Artificial Synthetic metal-binding melanocortin-receptor specific
sequence 62 Tyr Val Xaa Gly Cys His Phe Arg Trp Asp Arg Phe 1 5 10
63 7 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 63 Xaa Ala His Arg Phe Trp Cys 1 5 64 5 PRT
Artificial Synthetic metal-binding melanocortin-receptor specific
sequence 64 Phe Phe Cys Xaa Lys 1 5 65 5 PRT Artificial Synthetic
metal-binding melanocortin-receptor specific sequence 65 Phe Phe
Cys Xaa Lys 1 5 66 5 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 66 Phe Phe Cys Xaa Lys 1 5
67 5 PRT Artificial Synthetic metal-binding melanocortin-receptor
specific sequence 67 Phe Phe Cys Xaa Lys 1 5 68 5 PRT Artificial
Synthetic metal-binding melanocortin-receptor specific sequence 68
Phe Phe Cys Xaa Lys 1 5 69 4 PRT Artificial Synthetic metal-binding
melanocortin-receptor specific sequence 69 Xaa Arg Trp Cys 1
* * * * *