Method for integrin ligand discovery

Abstract

This invention relates generally to integrin ligand discovery and to a method of integrin ligand discovery based upon induction of ligand-induced epitopes. Such ligands have the potential to be active agent as anti-inflammatory, anti-angiogenesis and/or anti-thrombotic agents and for the treatment of integrin mediated diseases and/or conditions.

1 . A method for determining whether a compound of unknown integrin activity is a competitive ligand of an integrin molecule comprising exposing said integrin molecule to a compound of unknown integrin activity detection of the integrin ligand binding affinity of a compound of unknown integrin activity, allowing a sufficient time for said compound to bind to said integrin and expose at least one ligand induced epitope if said compound is a ligand of said integrin; exposing said integrin molecule to an antibody comprising a reporter which is specific for said ligand induced epitope and determining whether said compound is a ligand of said integrin by measuring the concentration of antibody bound to said integrin and comparing it to at least one control measurement. 
     
     
         2 . The method according to  claim 1  wherein said control measurement is a measurement of the specific binding of said antibody to said integrin in the presence of a known integrin ligand. 
     
     
         3 . The method according to  claim 1  wherein said control measurement is a measure of the non-specific binding of said antibody to said integrin in the absence of ligand. 
     
     
         4 . The method according to  claim 1  wherein said integrin is expressed on a wild-type cell. 
     
     
         5 . The method according to  claim 1  wherein said integrin is expressed on an engineered cell. 
     
     
         6 . The method according to  claim 1  wherein said cell expresses more than one integrin. 
     
     
         7 . The method according to  claim 1  wherein said integrin is expressed on a cell selected from the group consisting of human peripheral blood granulocytes, monocytes, lymphocytes and human platelet cells. 
     
     
         8 . The method according to  claim 1  wherein said integrin is expressed on a cell selected from the group consisting of U937 cells, MOLT-4 cells, Jurkat cells, THP-1 cells, HL-60 cells, JY cells and MEG-01 cells. 
     
     
         9 . The method according to any of  claim 1  wherein said integrin is Beta1 integrin (CD29), Beta2 integrin (CD18), AlphaM integrin (CD11b), Beta2 integrin (CD11a), Beta3 integrin (CD61), gpilla (a common subunit for alphaIIb and alphaV integrins), AlphaIIb beta3 integrin, CD41/CD61, glycoprotein.IIb/IIa (gpIIb/IIa) or α 4 β 1  integrin (CD49d/CD29). 
     
     
         10 . The method according to  claim 1  wherein said integrin is α 4 β 1  integrin (CD49d/CD29). 
     
     
         11 . The method according to  claim 1  wherein said antibody is a monoclonal antibody (mAb). 
     
     
         12 . The method according to  claim 11  wherein said mAb is selected from the group consisting of B44, HUTS-4, HUTS-21, AG89, 9EG7, MEM-148, MEM-48, KIM185, 127, CBRM1/5, MEM-83; CRC54 and PAC-1. 
     
     
         13 . The method according to  claim 1  wherein said integrin is said integrin is α 4 β 1  integrin (CD49d/CD29) and said antibody is monoclonal antibody HUTS-21 comprising a fluorophore reporter. 
     
     
         14 . The method according to  claim 1  wherein said reporter is a fluorophore. 
     
     
         15 . The method according to  claim 14  wherein said fluorophore is selected from the group consisting of fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde label, fluorescamine, tetramethylrhodamine, dipyrrometheneboron difluoride dyes (available from Molecular Probes, Inc, among others), near infrared dyes and lanthanide chelates. 
     
     
         16 . The method according to  claim 14  wherein said fluorophore is fluorescein or phycoerythrin. 
     
     
         17 . The method according to  claim 16  wherein said fluorophore is phycoerythrin. 
     
     
         18 . The method according to  claim 2  wherein said method is adapted for use in flow cytometry. 
     
     
         19 . The method according to  claim 2  wherein said known ligand is selected from any of compounds set forth in  FIG. 1  or an enantiomer thereof. 
     
     
         20 . The method according to  claim 1  wherein said method is adapted for use in high throughput flow cytometry. 
     
     
         21 . The method according to  claim 1  wherein said compound, identified as a competitive ligand, is subjected to a further assay to determine whether the compound is an agonist or antagonist of integrin. 
     
     
         22 . A method for determining whether or not a compound of unknown integrin activity is an allosteric inhibitor of an integrin molecule comprising exposing said integrin molecule to a known competitive ligand of said integrin molecule in the presence of a integrin-epitope binding antibody comprising a reporter moiety, measuring the binding of said antibody to the integrin molecule in the presence of the competitive ligand; thereafter exposing said bound integrin molecule in the presence of known competitive ligand and bound antibody to a compound of unknown activity and measuring the binding of the antibody to the integrin molecule, wherein the decrease in binding of antibody to integrin is evidence that the compound of unknown activity is an allosteric inhibitor of integrin. 
     
     
         23 . The method according to  claim 22  wherein the decrease in binding of antibody to integrin is compared to a control wherein the control is a measurement of the non-specific binding of the antibody to the integrin molecule. 
     
     
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         41 . A pharmaceutical composition comprising an effective amount of a compound according to the chemical structure: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt or enantiomer thereof, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. 
       
     
     
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         47 . A pharmaceutical composition comprising a compound according to the chemical structure I: 
       
         
           
           
               
               
           
         
         Where 
         R 1  is C 1 -C 4  linear or branched alkyl group; and 
         R 2  is an optionally substituted cyclic or bicyclic hydrocarbon or an optionally substituted 5-10 membered heterocyclic ring or fused bicyclic ring system, or 
         a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. 
       
     
     
         48 . The pharmaceutical composition according to  claim 47  wherein said compound has the chemical structure: 
       
         
           
           
               
               
           
         
       
     
     
         49 . The composition according to  claim 47  wherein R 2  is an adamantly group. 
     
     
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