Biochemical and Structural Studies of Viral Proteases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Characterization of the heterodimer intermediate in the N-terminal autoprocessing pathway: A mechanism was proposed based on our analysis of various precursor constructs of MPro with cleavage sites at both termini. In that mechanism, the MPro precursor undergoes N-terminal intramolecular cleavage from either the monomer or the dimer. The product derived from N-terminal cleavage with a free N-terminus enables forming a heterodimeric intermediate and thereby increasing the catalytic activity and faster processing of the second N-terminal nsp4/nsp5 site of the heterodimer. To validate the occurrence of such association, we now show that a mixture of monomeric, MPro10-306 and MPro1-199, constructs assemble to form a heterodimer with increased catalytic activity. The heterodimer is formed through cooperative assembly mediated by the interaction of a single N-finger region of MPro1-199 with domain III of MPro10-306 (Reference 1). Subsequent cleavages at the C-terminus of MPro occur via an intermolecular mechanism. Current investigations involve studying the influence of the interface formed by the C-terminal residues, and the monomer-dimer equilibrium, on C-terminal intermolecular processing by the mature MPro to understand how this step relates to the biology of virus assembly and maturation. Insights into the molecular basis of drug resistance: Rapid selection of mutations rendering resistance to clinical inhibitors, nirmatrelvir (NMV) and ensitrelvir (ESV), occurs under (A) drug pressure and (B) natural propagation. We investigated five drug resistant mutants (DRM), each carrying 2-3 substitution mutations, belonging to (A), and one mutant belonging to (B), which in addition to a substitution mutation carried an unusual deletion mutation. The effects of drug-resistant mutations on N-terminal autoprocessing, dimer dissociation constant (Kdimer), catalytic activity and thermodynamic parameters of inhibitor binding were systematically examined in comparison with the wild type MPro. The three-dimensional (3D) structures of DRMs yielding crystals in the absence and presence of the above inhibitors were described (in collaboration with ORNL), in a context to provide insights into the molecular basis of drug resistance and development of second-generation antivirals with improved binding affinities to DRMs. Results of these studies are reported in references 2 and 3 and can be summarized as follows. Overall, the decrease in binding affinity appears to parallel the selection pressure corresponding to the chosen drug. Mutations appear not to impair N-terminal autoprocessing that is critical for dimerization and ensuing catalytic activity consistent with this process being intramolecular, and insensitive to inhibition and selection of current clinical drugs. In the mature MPro, co-selected secondary mutations enhance dimer stability, and thereby increase catalytic activity to offset the effects of critical active site subsite primary mutations which drastically decrease catalytic activity and drug binding affinity. Drug resistance mutations also lead to active site opening and changes in the conformational dynamics, to modulate N-terminal autoprocessing and parameters that govern dimerization, catalytic function and inhibition of MPro. Structural differences in the interaction of inhibitors with the mutant’s active site residues and local changes in hydration attributing to weaker binding were also observed. Steric and electronic properties of P2 groups on covalent inhibitor binding (in collaboration with ORNL): The influence of steric and electronic properties of P2 substituents, designed to engage the S2 substrate binding subsite within the MPro active site, on inhibitor binding affinity was explored. Taken together with previously published studies, our results indicate that introduction of aromatic or sterically bulky substituents as P2 groups, which lead to unfavorable interplay of enthalpy and entropy of binding as well as of the active site conformational dynamics, should be avoided in the future MPro inhibitor design (Reference 4). This underscores the feature that the MPro active site malleability may be accompanied by a conformational strain, and it remains to be explored how selection of natural and drug-resistant variants may accommodate mutations in subsites to alleviate the conformational strain for improved catalytic function. Inactive E-state as a target for drug design: Our studies led to the identification of the inactive E-state of monomeric MPro in equilibrium with the active E*-state. The 3D structures of the two states being distinct, provides a new (viable) approach for the development of future drugs to arrest polyprotein maturation. Methods and reagents have been developed to identify and characterize compounds, selected by virtual screening of FDA-approved drug library and Mcule drug discovery platform, that restrict N-terminal autoprocessing. Neutron Crystallography (in collaboration with ORNL): To gain insights into the atomic-level understanding of the E-state and the active site-inhibitor complexes of selected DRMs including H-bond positions and protonation states of residues, studies are underway for optimizing conditions to enable attaining neutron-quality crystals of both hydrogenous (protiated) and deuterated monomeric MPro constructs and selected DRMs. The above understanding will provide unique knowledge for future drug design strategies. Ongoing studies of related proteases: The mechanism of activation and efficacy of MPro inhibitors on chymotrypsin-like cysteine proteases encoded by related viruses are being explored to enable defining broad-spectrum inhibitors of these proteases. Initial characterizations of these enzymes are being carried out using established methods described for MPro.