3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Critical assessment of popular biomolecular force fields for molecular dynamics simulations of folding and enzymatic activity of main protease of coronavirus SARS-CoV-2.

The main cysteine protease (Mpro) of coronavirus SARS-CoV-2 has become a promising target for computational development in anti-COVID-19 treatments. Here, we benchmarked the performance of six biomolecular molecular dynamics (MD) force fields (OPLS-AA, CHARMM27, CHARMM36, AMBER03, AMBER14SB and GROMOS G54A7) and three water models (TIP3P, TIP4P and SPC) for reproducing the native fold and the enzymatic activity of Mpro as monomeric and dimeric units. The MD sampling up to 1 µs suggested that the proper choice of the force fields and water models plays an essential role in reproducing the tertiary structure and the inter-residue distance between the catalytic dyad His41-Cys145. We found that while most benchmarked all-atom force fields reproduce well the native fold of Mpro, the CHARMM27/TIP3P and OPLS-AA/TIP4P setups revealed a good performance in reproducing the structure of the catalytic domain. In addition, these FF setups were also well-adopted for MD sampling of Mpro at the physiologic conditions by mimicking the presence of 100 mM NaCl and the elevated temperature of 310 K. Finally, both FFs were also performed well in reproducing the native fold of Mpro in a dimeric form. Therefore, comparing the preservation of the native fold of Mpro and the stability of its catalytic site architecture, our MD benchmarking suggests that the OPLS-AA/TIP4P and CHARMM27/TIP3P MD setups at the physiologic conditions may be well-suited for rapid in silico screening and developing broad-spectrum anti-coronaviral therapeutic agents.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease.

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 310-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Deciphering inhibitory activity of marine algae Ecklonia cava phlorotannins against SARS CoV-2 main protease: A coupled in-silico docking and molecular dynamics simulation study.

The onset of COVID-19 due to the SARS CoV-2 virus has spurred an urgent need for potent therapeutics and vaccines to combat this global pandemic. The main protease (Mpro) of the virus, crucial in its replication, has become a focal point in developing anti-COVID-19 drugs. The cysteine protease Mpro in SARS CoV-2 bears a significant resemblance to the same protease found in SARS CoV-1. Previous research highlighted phlorotannins derived from Ecklonia cava, an edible marine algae, as inhibitors of SARS CoV-1 Mpro activity. However, it remains unclear whether these marine-derived phlorotannins also exert a similar inhibitory effect on SARS CoV-2 Mpro. To unravel this, our study utilized diverse in-silico methodologies. We explored the pharmacological potential of various phlorotannins (phloroglucinol, triphloretol-A, eckol, 2-phloroeckol, 7-phloroeckol, fucodiphloroethol G, dieckol, and phlorofucofuroeckol-A) and assessed their binding efficacies alongside established Mpro inhibitors (N3 and lopinavir) through molecular docking studies. Among these compounds, five phlorotannins (eckol, 2-phloroeckol, 7-phloroeckol, dieckol, and phlorofucofuroeckol-A) exhibited potent binding affinities comparable to or surpassing N3 and lopinavir, interacting especially with the catalytic residues His41 and Cys145 of Mpro. Moreover, molecular dynamics simulations revealed that these five Mpro-phlorotannin complexes displayed enhanced stability and maintained comparable or slightly reduced compactness. They exhibited reduced conformational changes and increased expansion relative to the Mpro-N3 and/or Mpro-lopinavir complex. Our MM-GBSA analysis further supported these findings. Overall, our investigation highlights the potential of these five phlorotannins in inhibiting the proteolytic function of SARS CoV-2 Mpro, offering promise for anti-COVID-19 drug development.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

Repurposing of antimycobacterium drugs for COVID-19 treatment by targeting SARS CoV-2 main protease: An in-silico perspective.

The global mortality rate has been significantly impacted by the COVID-19 pandemic, caused by the SARS CoV-2 virus. Although the pursuit for a potent antiviral is still in progress, experimental therapies based on repurposing of existing drugs is being attempted. One important therapeutic target for COVID-19 is the main protease (Mpro) that cleaves the viral polyprotein in its replication process. Recently minocycline, an antimycobacterium drug, has been successfully implemented for the treatment of COVID-19 patients. But it's mode of action is still far from clear. Furthermore, it remains unresolved whether alternative antimycobacterium drugs can effectively regulate SARS CoV-2 by inhibiting the enzymatic activity of Mpro. To comprehend these facets, eight well-established antimycobacterium drugs were put through molecular docking experiments. Four of the antimycobacterium drugs (minocycline, rifampicin, clofazimine and ofloxacin) were selected by comparing their binding affinities towards Mpro. All of the four drugs interacted with both the catalytic residues of Mpro (His41 and Cys145). Additionally, molecular dynamics experiments demonstrated that the Mpro-minocyline complex has enhanced stability, experiences reduced conformational fluctuations and greater compactness than other three Mpro-antimycobacterium and Mpro-N3/lopinavir complexes. This research furnishes evidences for implementation of minocycline against SARS CoV-2. In addition, our findings also indicate other three antimycobacterium/antituberculosis drugs (rifampicin, clofazimine and ofloxacin) could potentially be evaluated for COVID-19 therapy.

De novo design of potential peptide analogs against the main protease of Omicron variant using in silico studies.

SARS-CoV-2 and its variants are crossing the immunity barrier induced through vaccination. Recent Omicron sub-variants are highly transmissible and have a low mortality rate. Despite the low severity of Omicron variants, these new variants are known to cause acute post-infectious syndromes. Nowadays, novel strategies to develop new potential inhibitors for SARS-CoV-2 and other Omicron variants have gained prominence. For viral replication and survival the main protease of SARS-CoV-2 plays a vital role. Peptide-like inhibitors that mimic the substrate peptide have already proved to be effective in inhibiting the Mpro of SARS-CoV-2 variants. Our systematic canonical amino acid point mutation analysis on the native peptide has revealed various ways to improve the native peptide of the main protease. Multi mutation analysis has led us to identify and design potent peptide-analog inhibitors that act against the Mpro of the Omicron sub-variants. Our in-depth analysis of all-atom molecular dynamics studies has paved the way to characterize the atomistic behavior of Mpro in Omicron variants. Our goal is to develop potent peptide-analogs that could be therapeutically effective against Omicron and its sub-variants.

In vitro biological evaluation and in silico insights into the antiviral activity of standardized olive leaves extract against SARS-CoV-2.

There is still a great global need for efficient treatments for the management of SARS-CoV-2 illness notwithstanding the availability and efficacy of COVID-19 vaccinations. Olive leaf is an herbal remedy with a potential antiviral activity that could improve the recovery of COVID-19 patients. In this work, the olive leaves major metabolites were screened in silico for their activity against SARS-CoV-2 by molecular docking on several viral targets such as methyl transferase, helicase, Plpro, Mpro, and RdRp. The results of in silico docking study showed that olive leaves phytoconstituents exhibited strong potential antiviral activity against SARS-CoV-2 selected targets. Verbacoside demonstrated a strong inhibition against methyl transferase, helicase, Plpro, Mpro, and RdRp (docking scores = -17.2, -20, -18.2, -19.8, and -21.7 kcal/mol.) respectively. Oleuropein inhibited 5rmm, Mpro, and RdRp (docking scores = -15, -16.6 and -18.6 kcal/mol., respectively) respectively. Apigenin-7-O-glucoside exhibited activity against methyl transferase and RdRp (docking score = -16.1 and -19.4 kcal/mol., respectively) while Luteolin-7-O-glucoside inhibited Plpro and RdRp (docking score = -15.2 and -20 kcal/mol., respectively). The in vitro antiviral assay was carried out on standardized olive leaf extract (SOLE) containing 20% oleuropein and IC50 was calculated. The results revealed that 20% SOLE demonstrated a moderate antiviral activity against SARS-CoV-2 with IC50 of 118.3 µg /mL. Accordingly, olive leaf could be a potential herbal therapy against SARS-CoV-2 but more in vivo and clinical investigations are recommended.

SARS-CoV-2 Mpro oligomerization as a potential target for therapy.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

Discovery of Covalent Lead Compounds Targeting 3CL Protease with a Lateral Interactions Spiking Neural Network.

Covalent drugs exhibit advantages in that noncovalent drugs cannot match, and covalent docking is an important method for screening covalent lead compounds. However, it is difficult for covalent docking to screen covalent compounds on a large scale because covalent docking requires determination of the covalent reaction type of the compound. Here, we propose to use deep learning of a lateral interactions spiking neural network to construct a covalent lead compound screening model to quickly screen covalent lead compounds. We used the 3CL protease (3CL Pro) of SARS-CoV-2 as the screen target and constructed two classification models based on LISNN to predict the covalent binding and inhibitory activity of compounds. The two classification models were trained on the covalent complex data set targeting cysteine (Cys) and the compound inhibitory activity data set targeting 3CL Pro, respected, with good prediction accuracy (ACC > 0.9). We then screened the screening compound library with 6 covalent binding screening models and 12 inhibitory activity screening models. We tested the inhibitory activity of the 32 compounds, and the best compound inhibited SARS-CoV-2 3CL Pro with an IC50 value of 369.5 nM. Further assay implied that dithiothreitol can affect the inhibitory activity of the compound to 3CL Pro, indicating that the compound may covalently bind 3CL Pro. The selectivity test showed that the compound had good target selectivity to 3CL Pro over cathepsin L. These correlation assays can prove the rationality of the covalent lead compound screening model. Finally, covalent docking was performed to demonstrate the binding conformation of the compound with 3CL Pro. The source code can be obtained from the GitHub repository (https://github.com/guzh970630/Screen_Covalent_Compound_by_LISNN).

An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.

The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Chemo- and Site-Selective Lysine Modification of Peptides and Proteins under Native Conditions Using the Water-Soluble Zolinium.

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.

Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.