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The novel SARS-CoV-2 uses ACE2 (Angiotensin-Converting Enzyme 2) receptor as an entry point. Insights on S protein receptor-binding domain (RBD) interaction with ACE2 receptor and drug repurposing has accelerated drug discovery for the novel SARS-CoV-2 infection. Finding small molecule binding sites in S protein and ACE2 interface is crucial in search of effective drugs to prevent viral entry. In this study, we employed molecular dynamics simulations in mixed solvents together with virtual screening to identify small molecules that could be potential inhibitors of S protein -ACE2 interaction. Observation of organic probe molecule localization during the simulations revealed multiple sites at the S protein surface related to small molecule, antibody, and ACE2 binding. In addition, a novel conformation of the S protein was discovered that could be stabilized by small molecules to inhibit attachment to ACE2. The most promising binding site on RBD-ACE2 interface was targeted with virtual screening and top-ranked compounds (DB08248, DB02651, DB03714, and DB14826) are suggested for experimental testing. The protocol described here offers an extremely fast method for characterizing key proteins of a novel pathogen and for the identification of compounds that could inhibit or accelerate spreading of the disease.

Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (19) was 7-O-demethylated by similar high efficiency [21–30 ML/(min·mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (14) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (20) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min·mol CYP)], followed by CYP1A1 [16 ML/(min·mol CYP)] and CYP1A2 [0.6 ML/(min·mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14, 19, and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes. peerReviewed

The estimation of the correct binding mode and affinity of a ligand into a target protein using computational methods is challenging. However, docking can introduce poses from which the correct binding mode could be identified using other methods. Here, we analyzed the reliability of binding energy estimation using the molecular mechanics-generalized Born surface area (MMGBSA) method without and with energy minimization to identify the likely ligand binding modes within docking results. MMGBSA workflow (a) outperformed docking in recognizing the correct binding modes of androgen receptor ligands and (b) improved the correlation coefficient of computational and experimental results of rescored docking poses to phosphodiesterase 4B. Combined with stability and atomic distance analysis, MMGBSA helped to (c) identify the binding modes and sites of metabolism of cytochrome P450 2A6 substrates. The standard deviation of estimated binding energy within one simulation was lowered by minimization in all three example cases. Minimization improved the identification of the correct binding modes of androgen receptor ligands. Although only three case studies are shown, the results are analogous and indicate that these behaviors could be generalized. Such identified binding modes could be further used, for example, with free energy perturbation methods to understand binding energetics more accurately.

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs.To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site.CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85-97 µM, Vmax-values of the oxidation reaction varied 0.25-60 min-1, and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). Km of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) µM and Vmax 0.25 (0.23-0.26) min-1, whereas Km of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) µM and Vmax 60 (58-63) min-1. Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it.The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance.

The COVID-19 pandemic, caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a severe global health crisis now. SARS-CoV-2 utilizes its Spike protein receptor-binding domain (S-protein) to invade human cell through binding to Angiotensin-Converting Enzyme 2 receptor (ACE2). S-protein is the key target for many therapeutics and vaccines. Potential S-protein-ACE2 fusion inhibitor is expected to block the virus entry into the host cell. In many countries, traditional practices, based on natural products (NPs) have been in use to slow down COVID-19 infection. In this study, a protocol was applied that combines mixed solvent molecular dynamics simulations (MixMD) with high-throughput virtual screening (HTVS) to search NPs to block SARS-CoV-2 entry into the human cell. MixMD simulations were employed to discover the most promising stable binding conformations of drug-like probes in the S-protein-ACE2 interface. Detected stable sites were used for HTVs of 612093 NPs to identify molecules that could interfere with the S-protein-ACE2 interaction. In total, 19 NPs were selected with rescoring model. These top-ranked NP-S-protein complexes were subjected to classical MD simulations for 300 ns (3 replicates of 100 ns) to estimate the stability and affinity of binding. Three compounds, ZINC000002128789, ZINC000002159944 and SN00059335, showed better stability in all MD runs, of which ZINC000002128789 was predicted to have the highest binding affinity, suggesting that it could be effective modulator in RBD-ACE2 interface to prevent SARS-CoV-2 infection. Our results support that NPs may provide tools to fight COVID-19.

Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1-mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N-(3,5-dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α-naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10-95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100-fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10-680 times higher than the ones of ANF. DCPCC was a mixed-type inhibitor of CYP1A2. ANF was a competitive tight-binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2-mediated metabolism of procarcinogens and anticancer drugs.

A novel virtual screening methodology called fragment- and negative image-based (F-NiB) screening is introduced and tested experimentally using phosphodiesterase 10A (PDE10A) as a case study. Potent PDE10A-specific small-molecule inhibitors are actively sought after for their antipsychotic and neuroprotective effects. The F-NiB combines features from both fragment-based drug discovery and negative image-based (NIB) screening methodologies to facilitate rational drug discovery. The selected structural parts of protein-bound ligand(s) are seamlessly combined with the negative image of the target's ligand-binding cavity. This cavity- and fragment-based hybrid model, namely its shape and electrostatics, is used directly in the rigid docking of ab initio generated ligand 3D conformers. In total, 14 compounds were acquired using the F-NiB methodology, 3D quantitative structure-activity relationship modeling, and pharmacophore modeling. Three of the small molecules inhibited PDE10A at ~27 to ~67 μM range in a radiometric assay. In a larger context, the study shows that the F-NiB provides a flexible way to incorporate small-molecule fragments into the drug discovery.