Development of antiviral agents against SARS-CoV-2 proteases

Abstract

The rapid spread of severe acute respiratory coronavirus 2 (SARS-CoV-2) led to the global coronavirus disease 2019 (COVID-19) pandemic that caused over 700 million cases and 7 million deaths. In recent decades, the highly pathogenic SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) also emerged, in 2002-2003 and 2012, respectively. These repeated coronavirus outbreaks have posed substantial threats to healthcare systems and global socioeconomic disruptions. Currently, few effective vaccines and antivirals have been approved for COVID-19. However, the continued emergence of SARS-CoV-2 variants and drug resistance issues have blunted vaccine and antiviral effectiveness. Therefore, additional antivirals are needed urgently to combat current and future coronavirus outbreaks. A promising approach is drugging viral proteases Papain-like protease (PLpro) and Main protease (Mpro) that possess essential biological functions. This thesis hence explores the development of antiviral agents against SARS-CoV-2 proteases.

First, a fluorescence-based high-throughput screening of PLpro inhibitors from a drug library containing >50,000 structure-diverse small molecules was utilized, resulting in the identification of a hit compound F0213 with sub-micromolar inhibitory activities against both SARS-CoV-2 and MERS-CoV PLpro. F0213 potently inhibited wildtype SARS-CoV-2 replication in primary cardiomyocyte cells and multiple variants of concern (Alpha, Beta, Delta, and Omicron). Within non-toxic concentrations, F0213 exhibited in vitro pan coronavirus activities including MERS-CoV, hCoV-229E, and hCoV-OC43. Moreover, F0213 antagonized the deubiquitinating and deISGylating activities of PLpro, thus restoring the NF-κB and IFN-β related antiviral immune responses attenuated by PLpro. F0213 was selective against PLpro without interfering with host protease activities. Mechanistic studies revealed F0213 was placed inside a substrate binding cleft of SARS-CoV-2 PLpro where the thiazolo-quinazoline rings occupied the S3-S4 pockets and tightly attached to the BL2 loop via noncovalent interactions. F0213 was inserted into a shallow cleft formed between α7 helix and β8 strand of MERS-CoV PLpro, probably inducing an allosteric change. Site-mutagenesis studies indicated K157 was a key residue to mediate F0213 and SARS-CoV-2 PLpro interaction, while E271 played an important role in the interaction with MERS-CoV PLpro. Importantly, oral administration of F0213 significantly suppressed lung viral load and alleviated pneumonia in SARS-CoV-2-infected hamsters. Upon MERS-CoV infection, F0213 treatment reduced lung viral load >1 log10 and improved the survival rate in hDPP4-knockin mice.

Next, an in-silico structure-based screening of a library containing >8,000 compounds with known functions in DrugBank was conducted, and Trichostatin A was repurposed as a novel SARS-CoV-2 Mpro inhibitor. Trichostatin A displayed potent anti-SARS-CoV-2 activity with an IC50 of 2.7 μM in Caco-2 cells, resulting in a selectivity index of 27.6. The EC50 of Trichostatin A obtained from plaque reduction assay was 1.5 μM, which is below its Cmax (132 μM). Time-of-drug-addition assay indicated Trichostatin A interrupted the post-entry events of the SARS-CoV-2 life cycle. Molecular docking revealed that Trichostatin A acts as a non-peptidomimetic covalent Mpro inhibitor where it is incorporated into the catalytic site of Mpro with good shape complementarity.

Taken together, F0213 and Trichostatin A may serve as important hit compounds for further development as next-generation antiviral agents.