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.

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 1 -Lll-Aaa-Bbb-Ccc-R 2 ,  R 1 -Bbb-Aaa-Ccc-R 2 ,  R 1 -Ddd-Bbb-Aaa-R 3 ,  R 4 -Eee-Bbb-Ccc-R 2 ,  R 1 -Hhh-Aaa-Bbb-Ccc-R 5 , or  R 1 -Iii-Iii-Ccc-Jjj-Kkk-R 2 ,  
       wherein 
 R 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 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 3  is an amino acid with an aromatic side chain that provides an N for metal ion complexation;  
 R 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 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), Tic, Tiq or Tca, or derivative, analog or homolog thereof;  
 Ggg is an L- or D-configuration aromatic amino acid; and  
 R 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), and derivatives, analogs or homologs thereof, including both natural and synthetic amino acids; and  
 R 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 1 -Lll-Aaa-Bbb-Ccc-R 2 ,  R 1 -Bbb-Aaa-Ccc-R 2 ,  R 1 -Ddd-Bbb-Aaa-R 3 ,  R 4 -Eee-Bbb-Ccc-R 2 ,  R 1 -Hhh-Aaa-Bbb-Ccc-R 5 , or  R 1 -Iii-Iii-Ccc-Jjj-Kkk-R 2 ,  
       wherein 
 R 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 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 3  is an amino acid with an aromatic side chain that provides an N for metal ion complexation;  
 R 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 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), Tic, Tiq or Tca, or derivative, analog or homolog thereof;  
 Ggg is an L- or D-configuration aromatic amino acid; and  
 R 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), and derivatives, analogs or homologs thereof, including both natural and synthetic amino acids; and  
 R 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 1 -Lll-Aaa-Bbb-Ccc-R 2 ,  R 1 -Bbb-Aaa-Ccc-R 2 ,  R 1 -Ddd-Bbb-Aaa-R 3 ,  R 4 -Eee-Bbb-Ccc-R 2 ,  R 1 -Hhh-Aaa-Bbb-Ccc-R 5 , or  R 1 -Iii-Iii-Ccc-Jjj-Kkk-R 2 ,  
       wherein 
 R 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 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 3  is an amino acid with an aromatic side chain that provides an N for metal ion complexation;  
 R 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 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), Tic, Tiq or Tca, or derivative, analog or homolog thereof;  
 Ggg is an L- or D-configuration aromatic amino acid; and  
 R 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 1 -Fff-Aaa-Ggg-Ccc-R 5 ,  
       wherein 
 R 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 2 ), and derivatives, analogs or homologs thereof, including both natural and synthetic amino acids; and  
 R 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.