The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. In this study, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogs of GC-376. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

Among the drug targets being investigated for SARS-CoV-2, the viral main protease (M pro ) is one of the most extensively studied. M pro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites. It is highly conserved and has a unique substrate preference for glutamine in the P1 position. Therefore, M pro inhibitors are expected to have broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as M pro inhibitors. In this study, we investigated the mechanism of action of six previously reported M pro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12, using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of M pro by these six compounds is nonspecific and that the inhibition is abolished or greatly reduced with the addition of reducing reagent 1,4-dithiothreitol (DTT). Without DTT, these six compounds inhibit not only M pro but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease and 2A pro and 3C pro from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC 50 values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that these six compounds are nonspecific SARS-CoV-2 M pro inhibitors and urge the scientific community to be stringent with hit validation.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1’ pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1’ pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1’, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A and AARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A/A, ARs, or AAR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A/A, A/A, A/A/A, and A). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A receptor.

Author Contributions:KB, AK and GL conceived and designed the research; KB performed the mammalian assays; KK conducted radioligand binding experiments, PL, DS, EV and AK performed the molecular dynamic simulations; SH derived the equations for the 'Rapid competitor dissociation kinetics' model; KB, GL and AK analysed data; KB and GL wrote manuscript, AK revised and edited the manuscript. Summary Background and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task.Experimental approach 39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding 2 free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Word count: 241

Adenosine A3 receptor (A3R) is a promising drug target cancer and for a number of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no experimentally determined structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]­carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calculations of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helices (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calculations distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32 in the low region and the remote L2647.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.

The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no experimental structure of A3R. Here, we have generated a molecular model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)­methyl]­amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA). Molecular dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, molecular mechanics-generalized Born surface area calculations were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.