Stabilized low affinity conformation of integrins for drug discovery

Abstract

The methods and compositions described herein are based, in part, on the discovery that the introduction of a disulfide bond into an integrin polypeptide by the substitution of at least one cysteine residue in the polypeptide permits stabilization of the integrin in a “closed/inactive” state. This stabilizing disulfide bond permits integrins to be screened for a candidate molecule that can bind to the closed state. In particular, this approach can be used to screen for agents that bind to the closed state of an integrin polypeptide, and are useful as therapeutic treatments to prevent integrin activation.

1 . A method for identifying a candidate modulator of integrin activity, the method comprising (a) contacting an integrin polypeptide with a candidate agent, wherein the integrin polypeptide is locked into a desired conformation; and (b) detecting binding of the candidate agent to the integrin polypeptide, wherein binding of the candidate agent to the integrin polypeptide is indicative that the candidate agent is a candidate modulator of integrin activity. 
     
     
         2 . The method of  claim 1 , wherein the candidate agent is selected from the group consisting of an antibody, a small molecule, a chemical, a peptide, and a peptidomimetic. 
     
     
         3 . The method of  claim 1 , wherein the candidate modulator stabilizes the integrin polypeptide into a closed conformation. 
     
     
         4 . The method of  claim 1 , wherein the candidate modulator inhibits binding of an integrin ligand to the integrin polypeptide. 
     
     
         5 . The method of  claim 1 , wherein the integrin polypeptide is selected from the group consisting of α V β 3 , α II bβ 3 , α V β 5 , α V β 1 , α V β 5 , α M β 2 , α X β 2 , α L β 2 , and α V β 8 . 
     
     
         6 . The method of  claim 1 , wherein locking the integrin polypeptide into the desired conformation comprises introducing a stabilizing disulfide bond into the integrin polypeptide. 
     
     
         7 . The method of  claim 6 , wherein the disulfide bond is formed by a cysteine residue substitution of at least one amino acid residue of the integrin polypeptide. 
     
     
         8 . The method of  claim 6 , wherein the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), I955C (human α V ), Q956C (human α V ), V664C (human (β 3 ), P688C (human (β 3 ), L662C (human (β 6 ), P686C (human (β 6 ), A619C (human (β 8 ), and F636C (human (β 3 ). 
     
     
         9 . The method of  claim 8 , wherein an optimal sequence alignment is used to identify homologous residues for a cysteine substitution in an integrin polypeptide selected from the group consisting of α V β 3 , α II bβ 3 , α V β 6 , α V β 5 , α M β 2 , α X β 2 , α L β 2 , and α V β 8 . 
     
     
         10 . The method of  claim 1 , wherein the candidate agent is assayed for activation or inhibition of integrin activity. 
     
     
         11 . The method of  claim 9 , wherein a cell-based assay is used to determine integrin activity. 
     
     
         12 . An integrin polypeptide composition comprising: a modified integrin polypeptide, wherein the integrin polypeptide is locked in a closed conformation. 
     
     
         13 . The composition of  claim 12 , wherein the integrin polypeptide is modified by substitution of at least one amino acid residue for a cysteine residue, whereby a disulfide bond is formed. 
     
     
         14 . The composition of  claim 13 , wherein the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), 1955C (human α V ), Q956C (human α V ), V664C (human (β 3 ), P688C (human (β 3 ), L662C (human (β 6 ), P686C (human (β 6 ), A619C (human (β 8 ), and F636C (human (β 3 ).