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We introduce an enzyme mechanism-based method, EMBM, aimed at rational design of chemical sites, CS, of reaction coordinate analog inhibitors. The energy of valence reorganization of CS, caused by the formation of the enzyme-inhibitor covalent complex, is accounted for by new covalent descriptors W1 and W2. We considered CS fragments with a carbonyl reactivity center, like in native protease substrates. The W1 and W2 descriptors are calculated quantum mechanically on small molecular clusters simulating the reaction core of the formed covalent tetrahedral complex -anionic TC(O−) or neutral TC(OH). The modeling on a reaction core allows generation of various CS and corresponding TC(O−) and TC(OH) as universal building blocks of real inhibitors and their covalent complexes with serine or cysteine hydrolases. Moreover, the approach avoids the need for 3D structure of the target enzyme, so EMBM may be used for ligand-based design. We have built a Chemical Site of Inhibitors (CSI) databank with pairs of W1 and W2 descriptors pre-calculated for both CH 3 O(−) and CH 3 S(−) nucleophiles for every collected CS fragment. We demonstrated that contribution of a CS fragment to the binding affinity of an inhibitor depends on both its covalent reorganization during the chemical transformation and its non-covalent interactions in the enzyme active site. Consequently, prediction of inhibitors binding trend can be done only by accounting for all of these factors, using W1 and W2 in combination with non-covalent QSAR descriptors.

Mechanistic studies of catalysis and the inhibition of serine and cysteine proteases afford new and sometimes surprising insights, challenging conventional dogmas in enzymology. The intrinsic source of the difference in the catalytic mechanisms of serine and cysteine hydrolases, the origin of the stability of the enzymeinhibitor complex in serine proteases, and the structures and mechanisms of catalysis and inhibition in cysteine proteases are not just intellectually interesting; our findings provide a mechanistic basis to understand the trend in the binding affinity of “warheads” of reversible covalent (reaction coordinate analogue, RCA) inhibitors. The theoretically derived covalent descriptors W1 and W2 differentiate serine and cysteine hydrolases and account for the energetic contribution of the new covalent bond in the enzymeinhibitor complex. The W1 and W2 descriptors are at the heart of our enzyme mechanism based method (EMBM); a new computer‐assisted drug design tool for the filtration of inhibitor warheads by activity. EMBM is unique because it accounts for both covalent and noncovalent interactions of RCA inhibitors with their target enzymes.

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