Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against M^pro and cathepsin L

Abstract

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1’ pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1’ pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1’, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

“…For many coronaviruses, no live virus assay exists, limiting the ability to test compounds within mammalian cell systems to a small subset of all coronaviruses 16 . Furthermore, compounds with activity against live virus may function through a number of mechanisms other than protease inhibition which cannot be readily determined, and may lead to undesired off-target activities which are not realized until much later in the drug development process 17,18 .…”

Potentin vitroanti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

“…Recent studies have shown that peptidic inhibitors of CatL and other related cysteine proteases also possess inhibitory activity against SARS-CoV-2 Mpro ( 40, 41 ). In order to assess the comparative activity of gallinamide A against human CatL and the viral proteases Mpro and PLpro, we incubated these proteases with 10 μM of the natural product, and quantified the remaining activity using fluorogenic substrates.…”

SARS-CoV-2 protein drug targets landscape: a potential pharmacological insight view for the new drug development

“…They have also found that The S2 pocket of cathepsin L to be superficial and different from cathepsin B [109] and it will help to design the specific and effective inhibitors. However, the cathepsin L inhibitors may serve as a therapeutic choice for SARS-CoV-2 and drug design using this enzyme may prevent the progression of pulmonary fibrosis [110]. Cathepsin L inhibitors such as E64d or MDL28170 have been shown to inhibit SARS-CoV-2 replication.…”