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Probing the Dynamic Structure-Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory.

ABSTRACT: The SARS-CoV-2 main protease (M^pro) is of major interest as an antiviral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 M^pro crystal structures, are underway in many laboratories. However, little is known about the dynamic enzyme mechanism, which is needed to inform both assay development and structure-based inhibitor design. Here, we apply biodynamics theory to characterize the structural dynamics of substrate-induced M^pro activation under nonequilibrium conditions. The catalytic cycle is governed by concerted dynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132-147) on which Cys145 (comprising the thiolate nucleophile and half of the oxyanion hole) and Gly143 (comprising the second half of the oxyanion hole) reside. In particular, we observed the following: (1) Domain 3 undergoes dynamic rigid-body rotation about the domain 2-3 linker, alternately visiting two primary conformational states (denoted as M₁ ^pro ? M₂ ^pro); (2) The Gly143-containing crest of the m-shaped loop undergoes up and down translations caused by conformational changes within the rising stem of the loop (Lys137-Asn142) in response to domain 3 rotation and dimerization (denoted as M_1/down ^pro ? 2·M_2/up ^pro) (noting that the Cys145-containing crest is fixed in the up position). We propose that substrates associate to the M_1/down ^pro state, which promotes the M_2/down ^pro state, dimerization (denoted as 2·M_2/up ^pro-substrate), and catalysis. Here, we explore the state transitions of M^pro under nonequilibrium conditions, the mechanisms by which they are powered, and the implications thereof for efficacious inhibition under in vivo conditions.

SUBMITTER: Wan H

PROVIDER: S-EPMC7671103 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Probing the Dynamic Structure-Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory.

Wan Hongbin H Aravamuthan Vibhas V Pearlstein Robert A RA

ACS pharmacology & translational science 20201106 6

The SARS-CoV-2 main protease (M<sup>pro</sup>) is of major interest as an antiviral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 M<sup>pro</sup> crystal structures, are underway in many laboratories. However, little is known about the dynamic enzyme mechanism, which is needed to inform both assay development and structure-based inhibitor design. Here, we apply biodynamics theory to characterize the structural dynamics ...[more]

PMID: 33330838

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Project description:The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Dataset Information

Probing the Dynamic Structure-Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory.

Publications

Probing the Dynamic Structure-Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory.

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