US 7,351,690 B2Granted

Knockout identification of target-specific sites in peptides

AssigneePalatin Technologies, Inc.

InventorsShubh D. Sharma, Yi-Qun Shi, Margarita Bastos, Ramesh Rajpurohit, Hui-Zhi Cai

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Abstract

The invention provides methods for identification and determination of target-specific sites in peptides and proteins, including a method for determining the primary sequence of a secondary structure within a known parent polypeptide that binds to the target of interest. In one embodiment of the invention, a residue or mimetic containing a nitrogen atom and a sulfur atom available for binding to a metal ion is serially substituted for single residues in or inserted between adjacent residues in a known primary sequence of a peptide or protein. The resulting sequence is complexed with a metal ion thereby forming a metallopeptide. The resulting metallopeptides are then used in binding or functional assays related to the target of interest, and the metallopeptide(s) which result in significant or substantially decreased or changed binding or functionality are determined to identify the primary sequence involved in such binding or functionality.

Claims26

Dependent claims

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.

8 more elements

A method of determining the specific residues binding to a target of interest within a known parent polypeptide that binds to the target of interest, comprising the steps of:

(a) providing a parent polypeptide with a known primary sequence consisting of from three to about twenty amino acid residues;
(b) making a series of peptides, wherein each peptide in the series includes the known primary sequence of the parent polypeptide and a single inserted L- or D-3-mercapto amino acid residue, with the single L- or D-3-mercapto amino acid inserted for each peptide at each position along the primary sequence from the position between the second and third residues from the N-terminus through the C-terminus position;
(c) complexing each peptide in the series with a rhenium or technetium metal ion to form a series of metallopeptides;
(d) determining the binding of each metallopeptide of the series of metallopeptides to the target of interest;
(e) selecting the metallopeptide or metallopeptides of the series exhibiting decreased binding to the target of interest as compared to the binding of the parent polypeptide to the target of interest;
and
(f) identifying the amino acid residues involved in rhenium or technetium metal ion complexation other than the inserted L- or D-3-mercapto amino acid residue;
whereby at least one of the identified amino acid residues involved in rhenium or technetium metal ion complexation is the specific residues binding to a target of interest within the known parent polypeptide that binds to the target of interest.

5 more elements

A method of determining the specific residues binding to a target of interest within a known parent polypeptide that binds to the target of interest, comprising the steps of:

(a) providing a parent polypeptide with a known primary sequence consisting of from three to about twenty amino acid residues;
(b) making a series of peptides, wherein each peptide in the series includes the known primary sequence of the parent polypeptide with a single substitution, the single substituent consisting of an L- or D-3-mercapto amino acid residue substituted at each position along the primary sequence from the third residue from the N-terminus through the C-terminus residue;
(c) complexing each peptide in the series with a rhenium or technetium metal ion to form a series of metallopeptides;
(d) determining the binding of each metallopeptide of the series of metallopeptides to the target of interest;
(e) selecting the metallopeptide or metallopeptides of the series exhibiting decreased binding to the target of interest as compared to the binding of the parent polypeptide to the target of interest;
and
(f) identifying the amino acid residues involved in rhenium or technetium metal ion complexation;
whereby at least one of the identified amino acid residues involved in rhenium or technetium metal ion complexation and/or the amino acid residue substituted with an L- or D-3-mercaptoamino acid residue are the specific residues binding to a target of interest within the known parent polypeptide that binds to the target of interest.

5 more elements

Dependencies are inferred from the claim text.

Facts

Application: 10/769,695Filed 30 Jan 2004 · continuation in part
Priority: 19 Dec 2000
Published: US 2004/0248212 A19 Dec 2004
Granted: 1 Apr 2008
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C07K 5/1008
And aliphatic: the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
C07K 1/047
On carriers: simultaneous synthesis of different peptide species; Peptide libraries
C07K 5/1016
With the first amino acid being neutral: and aromatic or cycloaliphatic
C07K 5/1024
Tetrapeptides: with the first amino acid being heterocyclic

Family

Application60/256,842Us provisional application · filed 19 Dec 2000
Application60/304,835Us provisional application · filed 11 Jul 2001
Application60/327,835Us provisional application · filed 4 Oct 2001
PCT/US01/50075Parent of this continuation · filed 19 Dec 2001
Application60/444,129Us provisional application · filed 31 Jan 2003
Application10/464,117Parent of this continuation-in-part · filed 17 Jun 2003
US 2004/0248212 A1Pre-grant publication · published 9 Dec 2004
Application10/464,117Parent of this continuation
US 2005/0014193 A1Published application
US 7,351,690 B2This patent

From the US filing history. Foreign family members aren’t listed.