Specific binding sites in collagen for integrins and use thereof

Abstract

The present invention identified a high affinity binding sequence in collagen type III for the collagen-binding integrin I domains. Provided herein are the methods used to characterize the sequence, the peptides comprising this novel sequence and the use of the peptides in enabling cell adhesion. Also provided herein are methods to identify specific integrin inhibitors, sequences of these inhibitors and their use in inhibiting pathophysiological conditions that may arise due to integrin-collagen interaction.

1 - 2 . (canceled) 
     
     
         3 . A method of identifying an inhibitor of an integrin-collagen interaction, comprising:
 designing a test compound comprising a sequence that is specifically recognized by the I domain of the integrin, wherein said design is based on computer-aided molecular modeling; and   comparing the level of binding of the integrin to the collagen in the presence or absence of the test compound, wherein a decrease in binding of the integrin to the collagen in presence of the test compound indicates that the test compound is an inhibitor of the integrin-collagen interaction.   
     
     
         4 . The method of  claim 3 , wherein said integrin is an α 1 β 1 , α 2 β 1 , α 10 β 1  or α 11 β 1 . 
     
     
         5 . The method of  claim 3 , wherein said collagen is a type I, type II or type III collagen. 
     
     
         6 . The method of  claim 3 , wherein the inhibitor has a triple helical structure comprising an amino acid sequence GFPGER (SEQ ID NO: 6), GFOGEN (SEQ ID NO: 7), GFOGEK (SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ ID NO: 10), GVOGER (SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12). 
     
     
         7 . An inhibitory compound identified by the method of  claim 3 . 
     
     
         8 . A recombinant collagen or collagen like protein comprising the inhibitory sequence identified by method of  claim 3 . 
     
     
         9 . An expression vector, comprising:
 a DNA sequence encoding the recombinant collagen or collagen-like protein of  claim 8 .   
     
     
         10 . A host cell comprising and expressing the expression vector of  claim 9 . 
     
     
         11 . A synthetic collagen or collagen-like peptide comprising the inhibitory sequence identified by method of  claim 3 . 
     
     
         12 . The synthetic peptide of  claim 11 , wherein the inhibitory sequence in a triple helical structure is GFPGER (SEQ ID NO: 6), GFOGEN (SEQ ID NO: 7), GFOGEK (SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ ID NO: 10), GVOGER (SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12). 
     
     
         13 . The synthetic peptide of  claim 12 , wherein the peptide is specific for α 1 β 1 , α 2 β 1 , α 10 β 1  or α 11 β 1  integrins. 
     
     
         14 . A method of inhibiting integrin-collagen interaction, comprising:
 contacting a sample comprising the integrin and the collagen with the peptide of  claim 11 , wherein said peptide binds the integrin with a greater affinity than the collagen, thereby inhibiting the integrin-collagen interaction.   
     
     
         15 . The method of  claim 14 , wherein the integrin-collagen interaction contributes to cell adhesion, cell migration, cell proliferation, cell differentiation, angiogenesis, platelet aggregation or extracellular matrix assembly. 
     
     
         16 . A pharmaceutical composition, comprising:
 the inhibitory compound of  claim 7  and a pharmaceutically acceptable carrier.   
     
     
         17 . A method of treating an individual having a pathophysiological condition resulting from integrin-collagen interaction, comprising the step of:
 administering to the individual a pharmacologically effective amount of the composition of  claim 16  such that said composition inhibits the integrin-collagen interaction thereby treating the individual with the pathophysiological condition.   
     
     
         18 . The method of  claim 17 , wherein the pathophysiological condition is inflammation, tumor growth, metastasis or angiogenesis. 
     
     
         19 . An expression vector, comprising:
 a DNA sequence encoding a recombinant collagen or collagen-like protein of SEQ ID NO: 15; wherein amino acid Xaa in both sequences is hydroxyproline.   
     
     
         20 . An expression vector, comprising:
 a DNA sequence encoding a recombinant collagen or collagen-like protein of SEQ ID NO: 21; wherein amino acid Xaa in both sequences is hydroxyproline.   
     
     
         21 . An expression vector, comprising:
 a DNA sequence encoding a recombinant collagen or collagen-like protein of SEQ ID NO: 25.