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Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.

Rate and equilibrium constants for the conversion of (CO)5CrC(OCH2CH2OH)Ph (8-CrOOH), (CO)5WC(OCH2CH2OH)Ph (8-WOOH), (CO)5CrC(SCH2CH2OH)Ph (8-CrSOH), and (CO)5WC(SCH2CH2OH)Ph (8-WSOH) into the corresponding cyclic adducts (9-CrOO- for M = Cr, X = O; 9-WOO- for M = W, X = O; 9-CrSO- for M = Cr, X = S; 9-WSO- for M = W, X = S) are reported. Under basic conditions the reaction occurs via the respective oxyanions, (CO)5CrC(OCH2CH2O-)Ph (8-CrOO-), etc.; under acidic conditions the reaction may either be concerted with water acting as a general base catalyst or stepwise via a zwitterionic intermediate. The absence of buffer catalysis suggests but does not prove the operation of the stepwise mechanism. The equilibrium constants for cyclization of 8-CrSO- and 8-WSO- are about 1 order of magnitude higher than for 8-CrOO- and 8-WOO-, respectively. For the rate constants the situation is reversed, with 8-CrOO- and 8-WOO- being substantially more reactive than 8-CrSO- and 8-WSO-, respectively. These results reflect t...

Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.