| US 7,351,690 B2 | ||
| Knockout identification of target-specific sites in peptides | ||
| Shubh D. Sharma, Cranbury, N.J. (US); Yi-Qun Shi, East Brunswick, N.J. (US); Margarita Bastos, Plainsboro, N.J. (US); Ramesh Rajpurohit, Hillsboro, N.J. (US); and Hui-Zhi Cai, East Brunswick, N.J. (US) | ||
| Assigned to Palatin Technologies, Inc., Cranbury, N.J. (US) | ||
| Filed on Jan. 30, 2004, as Appl. No. 10/769,695. | ||
| Application 10/769695 is a continuation in part of application No. 10/464117, filed on Jun. 17, 2003. | ||
| Application 10/464117 is a continuation of application No. PCT/US01/50075, filed on Dec. 19, 2001. | ||
| Claims priority of provisional application 60/256842, filed on Dec. 19, 2000. | ||
| Claims priority of provisional application 60/304835, filed on Jul. 11, 2001. | ||
| Claims priority of provisional application 60/327835, filed on Oct. 04, 2001. | ||
| Claims priority of provisional application 60/444129, filed on Jan. 31, 2003. | ||
| Prior Publication US 2004/0248212 A1, Dec. 09, 2004 | ||
| This patent is subject to a terminal disclaimer. | ||
| Int. Cl. A61K 38/16 (2006.01) | ||
| U.S. Cl. 514—6 [530/300; 530/326; 530/327; 530/328; 530/329; 530/330; 530/331; 435/7.1] | 26 Claims |
| 1. A method of determining the specific residues binding to a target of interest, such residues being within a known parent
polypeptide that binds to the target of interest, comprising the steps of:
(a) providing a known parent polypeptide with a known primary structure, such primary structure consisting of n residues where
n is 3 to about 20 amino acid residues, which parent polypeptide binds to a target of interest;
(b) constructing a first peptide of the formula R1—Z—R2,
wherein
R1 comprises from 2 to n residues, such residues being the same as residues in the parent polypeptide and in the same order as
residues in the parent polypeptide primary structure, provided that any proline residue in the two residue positions immediately
adjacent the amino-terminus side of Z is substituted with glycine, alanine, serine, 2-aminoisobutyric acid (Aib), 1-amino,
1-cyclopentane carboxylic acid, or dehydroalanine, and any cysteine residue in R1 is S-protected or substituted with glycine, alanine, serine, 2-aminoisobutyric acid, 1-amino, 1-cyclopentane carboxylic acid,
or dehydroalanine;
Z is an amino acid residue providing both a nitrogen atom (N) and a sulfur atom (S) for metal ion complexation;
R2 comprises from 0 to n−2 residues, such residues being the same as residues in the parent polypeptide and in the same order
as residues in the parent polypeptide primary structure, provided that any cysteine residue is S-protected or substituted
with glycine, alanine, serine, 2-aminoisobutyric acid, 1-amino, 1-cyclopentane carboxylic acid, or dehydroalanine, and forming
with R1 a sequence in the same order as in the parent polypeptide primary structure with Z either inserted between two adjacent residues
corresponding to two adjacent residues in such primary structure or substituting for a single residue corresponding to a single
residue in such primary structure, and wherein the residues comprising R1—Z—R2 are equal to either n or n+1;
(c) complexing the first peptide of the formula R1—Z—R2 to a rhenium (Re) or technetium (Tc) metal ion, thereby forming a first R1—Z—R2 metallopeptide;
(d) screening the first R1—Z—R2 metallopeptide for binding to the target of interest;
(e) repeating steps (b) through (d), wherein the resulting R1—Z—R2 metallopeptide differs in at least either R1 or R2; and
(f) selecting the R1—Z—R2 metallopeptide exhibiting decreased binding to the target of interest as compared to the binding of the parent polypeptide
to the target of interest, whereby at least one residue of the sequence binding to the metal ion of such R1—Z—R2 metallopeptide is identified as the specific residues of the parent polypeptide binding to the target of interest.
|