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Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

ABSTRACT: As an essential enzyme of SARS-CoV-2, main protease (M^Pro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M^Pro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M^Pro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M^Pro inhibition information to assess an M^Pro inhibitor. We used this assay to analyze 30 known M^Pro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular M^Pro inhibition potency implicating their roles in interfering with key steps other than just the M^Pro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M^Pro inhibition IC₅₀ value of 31 nM that matches closely to its strong antiviral effect with an EC₅₀ value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

SUBMITTER: Cao W

PROVIDER: S-EPMC8848508 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

Cao Wenyue W Cho Chia-Chuan Dean CD Geng Zhi Zachary ZZ Shaabani Namir N Ma Xinyu R XR Vatansever Erol C EC Alugubelli Yugendar R YR Ma Yuying Y Chaki Sankar P SP Ellenburg William H WH Yang Kai S KS Qiao Yuchen Y Allen Robert R Neuman Benjamin W BW Ji Henry H Xu Shiqing S Liu Wenshe Ray WR

ACS central science 20220202 2

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. W ...[more]

PMID: 35229034

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Project description:As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

Dataset Information

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

Publications

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

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