Biochemical and Structural Studies of Viral Proteases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Goals 1 and 3 were completed and published as reported in last years progress report. New goals (7-8) were initiated. Progress in areas 2 and 4 to 7 of the goals are summarized below. Goals 2 and 6: The dynamic reorganization of the active site oxyanion loop from an inactive (unwound E-state) to an active conformation (wound E-state) upon dimerization was described. Clinical drugs with high specificity to the E*-state were shown to shift the E-E* equilibrium of the monomeric E-state to the E*-state, independent of dimerization. These observations were possible using the dissected monomeric catalytic domain which allowed its crystallization and structure determination in the absence and presence of inhibitors. Efforts to crystallize the full-length monomeric MPro were unsuccessful. Understanding the dynamic structural features of the active site loop may enable defining high affinity inhibitors, targeting the inactive E-state of the monomeric MPro precursor prior to its maturation, through a combination of virtual screening of compound libraries as well as structure-assisted design. Goal 4: The thermodynamics of binding the clinical covalent (nirmatrelvir, NMV) and noncovalent (ensitrelvir, ESV) inhibitors and the contribution of the catalytic dyad for binding affinity together with the crystal structures were described. When lacking the nucleophilic C145, NMV binding is 400-fold weaker corresponding to 3.5 kcal/mol and 13.3 degree C decreases in free energy (delta G) and thermal stability (Tm), respectively, relative to MProWT. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. The binding of NMV, which carries a reactive warhead targeting the active site C145, occurs via a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Importantly, ESV exhibits a binding affinity to MPro that is similar to NMV but differs in its thermodynamic profile from NMV. The improved binding enthalpy for ESV binding is offset by a positive entropy change, which contrasts NMV, resulting in a net delta G being similar to NMV. Goal 5: As a prelude to understanding the mechanism of catalytic maturation of MPro from its polyprotein precursor, the influence of nsp4 and nsp6 sequences flanking the N- and C-termini, respectively, of MPro (nsp5) on stable dimer formation and appearance of mature-like catalytic activity was investigated. Key findings are 1) MPro catalyzes its own cleavage at its termini from polyproteins, 2) N-terminal cleavage precedes C-terminal cleavage, 3) Nsp4 residues appended to MProH41A (miniprecursor) decrease Tm and increase Kdimer, 4) Miniprecursor dimer reveals asymmetry, semi-open conformation, and disorder of termini, 5) Potent inhibitors of mature MPro dimer bind weakly to the miniprecursor. Studies to describe the early mechanisms of catalytic maturation of MPro from its polyprotein precursor were completed and will be included in the next reporting cycle. Our understanding of the MPro regulation thus far, have led to including two additional goals (7 and 8). One aspect is on understanding the molecular basis of drug resistance by analyzing variants of MPro that are selected upon treatment with inhibitors, NMV and ESV. Precursor processing, enzyme kinetics, inhibitor binding thermodynamic profiles and structures are being compared to provide a comprehensive understanding of MPro evolution under drug pressure and insights into designing second generation inhibitors effective on such drug resistant mutants. The efficacy of MPro inhibitors on related proteases encoded by Norovirus and Dengue virus are also being explored to enable defining the mechanism of activation and broad-spectrum inhibitors of these proteases. Initial studies for their characterization will involve implementing goals 1-3.