A community effort in SARS-CoV-2 drug discovery.

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against COVID-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors.

4-O-Substituted Glucuronic Cyclophellitols are Selective Mechanism-Based Heparanase Inhibitors.

Degradation of the extracellular matrix (ECM) supports tissue integrity and homeostasis, but is also a key factor in cancer metastasis. Heparanase (HPSE) is a mammalian ECM-remodeling enzyme with β-D-endo-glucuronidase activity overexpressed in several malignancies, and is thought to facilitate tumor growth and metastasis. By this virtue, HPSE is considered an attractive target for the development of cancer therapies, yet to date no HPSE inhibitors have progressed to the clinic.

Synergistic inhibition of SARS-CoV-2 cell entry by otamixaban and covalent protease inhibitors: pre-clinical assessment of pharmacological and molecular properties.

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task.

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2.

Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry.

Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Background: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed.

Discovery of a hidden transient state in all bromodomain families.

Bromodomains (BDs) are small protein modules that interact with acetylated marks in histones. These posttranslational modifications are pivotal to regulate gene expression, making BDs promising targets to treat several diseases. While the general structure of BDs is well known, their dynamical features and their interplay with other macromolecules are poorly understood, hampering the rational design of potent and selective inhibitors.

Molecular mechanism of inhibiting the SARS-CoV-2 cell entry facilitator TMPRSS2 with camostat and nafamostat.

The entry of the coronavirus SARS-CoV-2 into human lung cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. assays confirm that both drugs inhibit the human protein TMPRSS2, a SARS-Cov-2 spike protein activator.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment.

An Epoxide Intermediate in Glycosidase Catalysis.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases.

A Single Point Mutation Converts GH84 -GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence.

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases.

Modeling catalytic reaction mechanisms in glycoside hydrolases.

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.

Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis.

Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease, and mouse knockout and inherited human mutations of GYG impair glycogen synthesis. GYG acts as a 'seed core' for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195.

Molecular-Scale Ligand Effects in Small Gold-Thiolate Nanoclusters.

Because of the small size and large surface area of thiolate-protected Au nanoclusters (NCs), the protecting ligands are expected to play a substantial role in modulating the structure and properties, particularly in the solution phase. However, little is known on how thiolate ligands explicitly modulate the structural properties of the NCs at atomic level, even though this information is critical for predicting the performance of Au NCs in application settings including as a catalyst interacting with small molecules and as a sensor interacting with biomolecular systems. Here, we report a combined experimental and theoretical study, using synchrotron X-ray spectroscopy and quantum mechanics/molecular mechanics simulations, that investigates how the protecting ligands impact the structure and properties of small Au(SR) NCs.

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

Mucin-type -glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear.

Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify").

The Molecular Mechanism of Substrate Recognition and Catalysis of the Membrane Acyltransferase PatA from Mycobacteria.

Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements, and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM/PIM.

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors.

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach.

The varied yet family-specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH-family-specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low-nanomolar inhibitor that is selective for GH family 47 inverting α-mannosidases. Here we show, through quantum-mechanical approaches, that kifunensine is restrained to a "ring-flipped" C conformation with another accessible, but higher-energy, region around the B conformation.

Carba-cyclophellitols Are Neutral Retaining-Glucosidase Inhibitors.

The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine-both covalent retaining β-glucosidase inhibitors-we postulated that the corresponding carba "cyclopropyl" analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the H transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the H conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (K = 8.

The molecular mechanism of the ligand exchange reaction of an antibody against a glutathione-coated gold cluster.

The labeling of proteins with heavy atom clusters is of paramount importance in biomedical research, but its detailed molecular mechanism remains unknown. Here we uncover it for the particular case of the anti-influenza N9 neuraminidase NC10 antibody against a glutathione-coated gold cluster by means of ab initio QM/MM calculations. We show that the labeling reaction follows an associative double S2-like reaction mechanism, involving a proton transfer, with low activation barriers only if one of the two distinct peptide/peptidic ligands (the one that occupies the side position) is substituted.

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism.

The enzymatic cleavage of β-1,4-mannans is achieved by -β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure.

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase.

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively.

Selective Derivatization of N-Terminal Cysteines Using Cyclopentenediones.

The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting materials.