Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Abstract

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek μM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

SARS-CoV-2 Papain-Like Protease Potential Inhibitors—In Silico Quantitative Assessment

“…In this group, the main direction seems to be focused on the peptide scaffolds [30]. There are also other proposals for nonpeptide, covalent inhibitors, such as ebselen [37], its derivatives [31], and disulfiram [38]. In the case of the peptide inhibitors, the covalent bond is formed with one of the amino acids of the catalytic triad-Cys111.…”

3CLpro and PLpro affinity, a docking study to fight COVID19 based on 900 compounds from PubChem and literature. Are there new drugs to be found?

“…In addition, we provide a set of previously published redocked compounds with reported half maximal inhibitory concentration (IC 50 ) values against 3CL pro [ 38 , 73 , 87 , 88 ] and PL pro [ 40 , [89] , [90] , [91] , [92] ] to give the interested reader more detailed view on the docking score vs. dose concentration relation of in vitro tested compounds. Refer to the supplementary materials in respective .xlsx documents, sheets IC 50 _dockings.…”

Discovery of Chalcone-Based Hybrid Structures as High Affinity and Site-Specific Inhibitors Against COVID-19; a Study Based on Various Host-Based and Viral Targets

“…10) [100][101][102][103][104] . In a recent HTS study performed by Zhengnan Shen et al, candesartan as an ARB was identi ed as one of a few compounds that showed inhibitory effects against SARS-CoV-2 PLpro, out of about 15,000 screened compounds 105 .…”

“…Interestingly, in a recent structure-based drug design attempt, extensive SAR evaluations have led to a chalcone-amide isoster bearing a 2-thienyl at position 3 of ring B for producing the strongest derivatives against SARS-CoV-2 PLpro (Fig. 17A, structure H) 105 . The metaand para-uorophenyl substitutions in positions 2 and 3 of the B-ring also existed in the structure of CHA-7 and CHA-177 which were found to have high docking scores in our compound library.…”