Method for the identification of active site protease inactivators

A method for identifying an active site protease inhibitor comprising:

• determining the optimal pH range for substrate cleavage conditions for a target protease;
• contacting each of a plurality of substrates with the target protease within the optimal pH range to identify at least one high kcat substrate (high kcat substrate) and at least one noncleavable low kcat substrate;
• performing a series of first competitive binding assays within the optimal pH range using the target protease, a first population of high kcat substrates, and each of a selected number of noncleavable substrates to identify a plurality of noncleavable inhibitors each comprising a different peptide core;
• performing a series of second competitive binding assays using the target protease, a second population of high kcat substrates and each of the noncleavable inhibitors, wherein the second population of high kcat substrates includes a greater number of substrates that the first population of high kcat substrates;
• quantifying the inhibitory effect of the noncleavable inhibitors to yield a ranked list of noncleavable inhibitors;
• assessing the selectivity of the noncleavable inhibitors with respect to proteases of the same class as the target protease in order to determine whether the inhibitor is selective for the target protease;
• covalently linking the peptide core to a plurality of inactivating reactants to yield a plurality of protease inactivators, each protease inactivator comprising a homologous peptide core but a different inactivating reactant;
• performing a competitive binding assay for each protease inactivator using the target protease, at least one high kcat substrate, and the protease inactivator;
• quantifying the inhibitory effect of the protease inactivators to yield a ranked list of protease inactivators, each protease inactivator comprising a different inactivating reactant;
• providing at least one set of protease inactivators, the set comprising a protease inactivator selected from the ranked list and a plurality of other protease inactivators comprising a different peptide core and the same inactivating reactant as the selected protease inactivator;
• determining kinetic constants Ki, kcat and km for each member of the set of protease inactivators;
• performing a first linear regression on first points (x,y) representing (log(Ki),log(Km/Kcat)) for selected members of the set of protease inactivators to yield a first line represented by y=MT*x+BT and having a first regression coefficient RT, wherein MT is the slope of the line and BT is the y-intercept value;
• performing a second linear regression on second points (x,y) representing (log (Ki),log(Km)) for selected members of the set of protease inactivators to yield a second line represented by y=MG*X+BG and having a second regression coefficient RG, wherein MG is the slope of the line and BG is the y-intercept value;
• and
• comparing the RT and RG to determine whether the inactivating reactant functions as a transition state protease inactivator or a ground state protease inactivator wherein, if the inactivating reactant functions as a transition state protease inactivator, the method further comprises calculating a transition state score TSS for the inactivating reactant, wherein TSS=RT/(ABS (1-MT)*1/P) where P is the number of points on the line and calculating a transition state inhibitor score ITS for each member of the set of protease inactivators, wherein ITS=log(TSS/(Ki)), whereas if the inactivating reactant functions as a ground state protease inactivator, the method further comprises calculating a ground state score GSS for the inactivating reactant, wherein GSS=RG/(ABS (1-MG) *1/P) where P is the number of points on the line calculating a ground state nhibitor score IGS for each member of the set of protease inactivators, wherein IGS=log(GSS/(Ki));
• and
• assessing the selectivity of the protease inactivator with respect to proteases of the same class as the target protease in order to determine whether the inhibitor is selective for the target protease.