A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals

Eur J Med Chem. 2022 Oct 5:240:114570. doi: 10.1016/j.ejmech.2022.114570. Epub 2022 Jun 27.


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.

Keywords: COVID-19; Covalent inhibitors; Main protease; SARS-CoV-2; Tripeptides.

MeSH terms

  • Aldehydes / pharmacology
  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology
  • COVID-19*
  • Coronavirus 3C Proteases
  • Humans
  • Protease Inhibitors / chemistry
  • SARS-CoV-2*
  • Threonine


  • Aldehydes
  • Antiviral Agents
  • Protease Inhibitors
  • Threonine
  • Coronavirus 3C Proteases