Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Abstract

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M−1 s−1, achieves sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

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

“…In crystal structures of WT PLpro bound to GRL0617, the L162 sidechain adopts the “L162 in” conformation, wherein it forms a contact with the ortho -methyl group of GRL0617, thereby blocking access to the catalytic residue C111. , These static structures suggest that extending the ortho -methyl group of GRL0617 would not be successful due to the steric clashes. In fact, previous studies on structure–activity relationship have demonstrated that extending or substituting the methyl group resulted in a significant loss of inhibition. , On the other hand, a promising inhibitor of PLpro has been synthesized by extending the ortho -methyl group of PLpro with a peptidomimetic linker and electrophile to form a covalent bond with C111 . Designing such a compound solely based on the static structure adopting the ″L162 in″ conformation appears irrational.…”

Main and papain-like proteases as prospective targets for pharmacological treatment of coronavirus SARS-CoV-2

“…Papain-like protease (PLpro) is a 35 kDa domain of Nsp3, part of a 215 kDa multidomain protein. 145 This protease cleaves the peptide bonds between Nsp1 and Nsp2, Nsp2 and Nsp3, and Nsp3 and Nsp4, releasing three proteins: Nsp1, Nsp2, and Nsp3. In addition, PLpro is involved in counteracting the host's immune response during viral infection.…”

“…147,157 Of all PLpro cysteines, Cys111 is the most susceptible to oxidation, indicating its unique reactivity toward electrophiles. 145 PLpro binds to viral proteins through its active site to fulfill its biological role. The catalytically active site of PLpro is a conservative drug target among SARS-CoV-2 variants.…”

“…Nevertheless, the growing number of recent studies on PLpro inhibitors indicate that it remains one of the promising targets for the development of small molecule therapeutics against SARS-CoV-2. 145,155,159…”

Landscape of In Silico Tools for Modeling Covalent Modification of Proteins: A Review on Computational Covalent Drug Discovery

“…Thus, the covalent drug design approach was intensely pursued subsequently in the case of PL pro targeting the catalytic C111 residue. For example, Sanders et al were among the first ones to report rational design of drug-like covalent SARS-CoV-2 PL pro inhibitors using a N , N ′-diacetylhydrazine linker conjugating the GRL0617 methyl group with different reactive warheads. In this report, the authors used an ensemble-based approach through Phenix to generate 50 conformations that better fit the data rather than a single PDB structure.…”