NMR Characterization of the Papain-like Protease from SARS-CoV-2 Identifies the Conformational Heterogeneity in Its Inhibitor-Binding Site

Abstract

Papain-like protease (PLpro) from severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a prime target for the development of antivirals for Coronavirus disease 2019 (COVID-19). However, drugs that target the PLpro protein have not yet been approved. In order to gain insights into the development of a PLpro inhibitor, conformational dynamics of PLpro in complex with GRL0617, the most well-characterized PLpro inhibitor, were investigated using nuclear magnetic resonance (NMR) spectroscopy in solution. Although mutational analyses demonstrated that the L162 sidechain interaction is responsible for the affinity for GRL0617, NMR analyses revealed that L162 in the inhibitor-binding pocket underwent conformational exchange and was not fixed in the conformation in which it formed a contact with ortho-methyl group of GRL0617. The identified conformational dynamics would provide a rationale for the binding mechanism of a covalent inhibitor designed based on GRL0617.

Discovery of orally bioavailable SARS-CoV-2 papain-like protease inhibitor as a potential treatment for COVID-19

“…, R2 group in Fig. 1C ) extending to the recently identified important residue L162 37 leads to activity drop-off. At this stage, we have successfully achieved potent enzymatic activity which is hundreds of times improved comparing to GRL0617.…”

Mutational Profiling of SARS-CoV-2 PLpro in human cells reveals requirements for function, structure, and drug escape

“…We also see a functional role for Arg166 and Asp302, despite their distance from the site of P4 engagement, that cannot be explained by a structural impairment in our datasets. The importance of these residues towards substrate binding have been described elsewhere (Rut et al 2020; Shin et al 2020; Klemm et al 2020; Fu et al 2021; Perlinska et al 2022; Ratia et al 2008a; Shiraishi and Shimada 2023).…”

Fragment-Based Screen of SARS-CoV-2 Papain-like Protease (PLpro)

“…Characteristic peak shifts of each class of fragment were highlighted in green circles. Labeled peak assignments are given in panels A and B sourced from literature . Representative structures of the (C) class A and (D) class B fragment hits with their binding affinities (K d ) measured by NMR titration and calculated ligand efficiency (LE).…”