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.

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.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5′-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.