Assays and peptide substrate for determining aggrecan degrading metallo protease activity

Abstract

This invention is directed to assays to determine the presence or absence of proteins that exhibit aggrecanase or ADMP activity. This invention also relates to peptides that acts as a substrates for ADMPs, their use in various assays to determine the presence or absence of ADMP activity, and their use as inhibitors of ADMP activity.

1 . A method for the determination of the presence of aggrecan-degrading metalloprotease activity comprising: (a) binding an ADMP substrate peptide to a streptavidin-coated microtiter plate; (b) rinsing the microtiter plate with assay buffer; (c) incubating the microtiter plate with an ADMP-containing sample; (d) rinsing the microtiter plate; (e) incubating the microtiter plate with a neoepitope antibody solution; (f) rinsing the microtiter plate; (g) incubating microtiter plates with secondary-detection antibody solution; (h) incubating the microtiter plate with an appropriate substrate solution; (i) quenching the reaction; and (j) reading the optical density.  
     
     
         2 . The method of  claim 1 , wherein said ADMP peptide substrate comprises a covalently-linked linking-moiety.  
     
     
         3 . A method for the determination of ADMP activity by quantifying the appearance of a product peptide comprising: (a) incubating an ADMP substrate peptide with assay buffer and an ADMP-containing sample; (b) quenching the reaction; (c) injecting a portion of the reaction mixture onto a reverse-phase HPLC column; (d) eluting the peptide with an organic solvent; (e) reading the absorbance; and (f) determining the quantity based on a standard curve.  
     
     
         4 . A method for assaying compounds for activity against an ADMP comprising: (a) providing an ADMP and an ADMP substrate; (b) contacting said ADMP with a candidate inhibitory compound in the presence of said ADMP; and (c) measuring the inhibition of the ADMP activity.  
     
     
         5 . An assay for detecting ADMP activity which comprises: (a) incubating a sample containing soluble ADMPs or aggrecanase activity with an aggrecan substrate; and (b) monitoring production of aggrecan fragments produced by specific cleavage at an ADMP-susceptible site using a neoepitope antibody to the new N-terminus or the new C-terminus generated by specific ADMP-mediated cleavage by the Problot assay comprising: (1) incubating a polyvinyl-denedifluoride (PVDF) cationically charged membrane, secured in a welled filtration plate, with a sample containing ADMP-degraded aggrecan; (2) washing any unbound aggrecan from the filtration plate; (3) coupling any unreacted cationic sites on the PVDF membrane with a solution of bovine serum albumin (BSA); (4) washing any unbound BSA from the filtration plate; (5) removing glycosaminoglycan side chains from the bound aggrecan with deglycosylation enzymes, wash membrane; (6) incubating PVDF membrane with a neoepitope antibody to fragments generated by cleavage at an ADMP-sensitive site, wash membrane; (7) incubating PVDF membrane with secondary detection antibody, wash membrane; (8) incubating PVDF membrane with detection substrate; and (9) draining solution into welled plate, obtain absorbance readings on individual samples; compare values to those obtained for standard curve.  
     
     
         6 . An assay according to  claim 5  wherein the sample is derived from cartilage or chondrocytes.  
     
     
         7 . An assay according to  claim 5  wherein the aggrecan substrate is native aggrecan isolated from human or animal tissue.  
     
     
         8 . An assay according to  5  wherein the aggrecan substrate is a recombinant aggrecan molecule or recombinant portion of the aggrecan molecule containing an aggrecanase-sensitive cleavage site.  
     
     
         9 . An assay according to  claim 5  wherein the recombinant portion of the aggrecan molecule contains the E 374 - 374 A bond.  
     
     
         10 . An assay according to  claim 5  wherein the recombinant aggrecan fragment contains the EL 1545 - 546 G bond.  
     
     
         11 . An assay according to  claim 5  wherein the portion of the aggrecan molecule contains the E 1714 - 1715 G bond.  
     
     
         12 . An assay according to  claim 5  wherein the recombinant portion of the aggrecan molecule contains the E 1819 - 1820 A bond.  
     
     
         13 . An assay according to  claim 5  wherein the recombinant portion of the aggrecan molecule contains the E 1919 - 1920 L bond.  
     
     
         14 . A method according to  claim 5  wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E 373  -A 374  bond.  
     
     
         15 . The method of  claim 5  wherein the neoepitope antibody is a BC-3 monoclonal antibody.  
     
     
         16 . The method of  claim 5  wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E 1545 -G 1546  bond.  
     
     
         17 . The method of  claim 5  wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E 1714 -G 1715  bond.  
     
     
         18 . The method of  claim 5  wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E 1819 -A 1820  bond.  
     
     
         19 . The method of  claim 5  wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E 19 9 -L 1920  bond.