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Nirmatrelvir is a specific antiviral drug that targets the main protease (M pro ) of SARS-CoV-2 and has been approved to treat COVID-19 1 , 2 . As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations 3 . The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of M pro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 M pro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4′ subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously 3 . Such a profile was also observed for ensitrelvir, another clinically relevant M pro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation M pro inhibitors. A biochemical and structural analysis demonstrates that alterations at the substrate-binding pocket of the main protease of SARS-CoV-2 can allow the virus to develop resistance to nirmatrelvir in two distinct ways.

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC 50 of 2.1 μM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLpro C111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro. The SARS-CoV-2 papain-like protease (PLpro) is of interest as a drug target. Here, the authors identify GRL0617 as a PPI (protein–protein interaction) inhibitor of SARS-CoV-2 PLpro that inhibits its deISGylating activity and present the mechanism of action of the compound through the GRL0617-bound PLpro crystal structure and NMR studies.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process. Analysis of over 6000 single-site variants of SARS-CoV-2 Papain-Like protease uncovers critical mutations involved in enzymatic function and highlight the potential for drug-escape.

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease M pro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the M pro monomer-dimer equilibrium dissociation constant. Since the functional unit of M pro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 M pro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the M pro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The M pro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

The SARS-CoV-2 papain-like protease (PL pro ) is a cysteine protease that cleaves viral polyproteins and antagonizes the host immune response during viral replication. Jun12682 and PF-07957472 are the first-in-class PL pro inhibitors showing potent in vivo antiviral efficacy in mouse models. In this study, we characterize naturally occurring mutations at residues located at the drug-binding site of Jun12682 . The results reveal several PL pro mutants showing significant drug resistance while maintaining comparable enzymatic activity as the wild-type PL pro . The physiological relevance of the identified drug-resistant mutants, including E167G and Q269H, is validated through independent serial viral passage experiments. Molecular dynamics simulations and perturbative free energy calculations show that drug-resistant PL pro mutants weaken hydrogen bonding and π-π stacking interactions. Collectively, this study identifies E167, Y268, and Q269 as drug-resistant hotspots for PL pro inhibitors that bind to the BL2 loop and groove region, which are valuable in informing the design of the next-generation PL pro inhibitors. This study identifies E167, Y268, and Q269 as drug-resistant hotspots for SARS-CoV papain-like protease inhibitors that bind to the BL2 loop and groove region, which are valuable in informing the design of the next-generation PLpro inhibitors.

SARS-CoV-2 main protease, M pro , is responsible for processing the viral polyproteins into individual proteins, including the protease itself. M pro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g., E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g., P252L, T21I, L50F), which restore the fitness of viral replication. To probe the role of the non-active site mutations in fitness rescue, here we use an M pro triple mutant (L50F/E166A/L167F) that confers nirmatrelvir drug resistance with a viral fitness level similar to the wild-type. By comparing peptide and full-length M pro protein as substrates, we demonstrate that the binding of M pro substrate involves more than residues in the active site. Particularly, L50F and other non-active site mutations can enhance the M pro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of M pro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying M pro and substrate interactions, and offers important insights into M pro function, resistance development, and inhibitor design. Non-active site mutations such as L50F are crucial for restoring the viral fitness of SARS-CoV-2 main protease. Here, the authors use full-length Mpro protein as substrate and demonstrate that L50F can promote the formation of enzyme-substrate complex and thus contribute to nirmatrelvir resistance.

The main protease (Mpro) is a major protease having an important role in viral replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that caused the pandemic of 2020. Here, active Mpro was obtained as a 34.5 kDa protein by overexpression in E. coli BL21 (DE3). The optimal pH and temperature of Mpro were 7.5 and 37 °C, respectively. Mpro displayed a Km value of 16 μM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified Mpro. The IC50 values were 137 μg/mL for black garlic extract and 9–197 μM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on Mpro. The structure–activity relationship of these polyphenols revealed that the hydroxyl group in C3′, C4′, C5′ in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on Mpro.

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I–MAVS complex to attenuate the RIG-I–mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

COVID-19 still poses a worldwide health threat due to continuous viral mutations and potential resistance to vaccination. SARS-CoV-2 viral multiplication hindrance by inhibiting the viral main protease (Mpro) deemed propitious. Structure-based virtual screening (SBVS) is a conventional strategy for discovering new inhibitors. Nonetheless, the SBVS efforts against Mpro variants needed to be benchmarked. Herein, in the first stage of the study, we evaluated four docking tools (FRED, PLANTS, AutoDock Vina and CDOCKER) via an in-depth benchmarking approach against SARS-CoV2 Mpro of both the wild type (WTMpro) and the deadly Omicron P132H variant (OMpro). We started by compiling an active dataset of non-covalent small molecule inhibitors of the WTMpro from literature and the COVID-Moonshot database along with generating a high-quality benchmark set via DEKOIS 2.0. pROC-Chemotype plots revealed superior performance for AutoDock Vina against WTMpro, while both FRED and AutoDock Vina demonstrated excellent performance for OMPro. In the second stage, VS was performed on a focused library of 636 compounds transformed from the early-enriched cluster related to perampanel via a scaffold hopping approach. Subsequently, molecular dynamics (MD) simulation and MM GBSA calculations validated the binding potential of the recommended hits against both explored targets. This study provides an example of how to conduct an in-depth benchmarking approach for both WTMPro and OMPro variants and offering an evaluated SBVS protocol for them both.