Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity

Abstract

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel “BL2 groove” and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Validation and Invalidation of SARS-CoV-2 Papain-like Protease Inhibitors

“… 22 The most promising PL pro inhibitors are the naphthalene-based GRL0617 series of compounds ( Figure 1 A). 6 , 21 , 23 − 25 However, their antiviral potency and pharmacokinetic properties need to be further optimized for the in vivo animal model study. PL pro specifically recognizes the P4-P1 sequence Leu-X-Gly-Gly that is conserved among the nsp1/2, nsp2/3, and nsp3/4 cleavage sites at the viral polyprotein.…”

DrugDevCovid19: An Atlas of Anti-COVID-19 Compounds Derived by Computer-Aided Drug Design

“…We observed that 76%, 75%, 47%, and 59% recommended compounds in Mpro, S3/S4 pocket of PLpro, RdRp, and S respectively achieved lower docking scores (lower than −7.0 kcal/mol), which suggest stronger binding. Particularly, GRL0617, the ranking-first compounds targeting PLpro S3/S4 pocket (docking score = −10.0 kcal/mol) was confirmed to have strong affinity (KD = 1.93 µM) [58], after predicting in July 2020 [37]. Unlike the four targets, only 2% recommended compounds against ACE2 achieved scores were lower than −7.0 kcal/mol.…”

“…For example, the PLpro-inhibitor complex and PLpro apo structure were remarkably different at BL2 loop (Figure S1a), and open and closed conformations were observed in the exterior residues (e.g., Q24, D30, E35, E37, D38, Y41, Q42, N53, E56, Y83, Q325, E329, Q330, K353, R393) of ACE2 (Figure S1b). Those structural variations have been carefully investigated in some of the CADD studies [58,59].…”

“…The different pockets usually play different roles in a target protein. For example, in the papain-like protease domain of PLpro, the S1/S2 pocket is the binding regions of catalytic sites while S3/S4 pocket (the BL2 loop) is substrate-binding related sites [58], and in nsp13, both the ATP binding pocket and RNA binding pocket are used for screening [61]. In this work, we did not distinguish the detailed impact of the binding pockets, but performed the analysis by comparing the recommended compounds using the same binding pockets but not the same target proteins.…”

Validation and invalidation of SARS-CoV-2 papain-like protease inhibitors

“…22 The most promising PL pro inhibitors are the naphthalene-based GRL0617 series of compounds (Figure 1A). 6,21,[23][24][25] However, their antiviral potency and pharmacokinetic properties need to be further optimized for the in vivo animal model study. PL pro specifically recognize the P1-P4 sequence Gly-Gly-X-Leu that is conserved among the nsp1/2, nsp2/3, and nsp3/4 cleavage sites at the viral polyprotein.…”