Foundations of biomolecular modeling.

The 2013 Nobel Prize in Chemistry has been awarded to Martin Karplus, Michael Levitt, and Arieh Warshel for "development of multiscale models for complex chemical systems." The honored work from the 1970s has provided a foundation for the widespread activities today in modeling organic and biomolecular systems.

Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid.

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

Enthalpies and entropies of hydration from Monte Carlo simulations.

The changes in free energy, enthalpy, and entropy for transfer of a solute from the gas phase into solution are the fundamental thermodynamic quantities that characterize the solvation process. Owing to the development of methods based on free-energy perturbation theory, computation of free energies of solvation has become routine in conjunction with Monte Carlo (MC) statistical mechanics and molecular dynamics (MD) simulations. Computation of the enthalpy change and by inference the entropy change is more challenging. Two methods are considered in this work corresponding to direct averaging for the solvent and solution and to computing the temperature derivative of the free energy in the van't Hoff approach. The application is for neutral organic solutes in TIP4P water using long MC simulations to improve precision. Definitive results are also provided for pure TIP4P water. While the uncertainty in computed free energies of hydration is ca. 0.05 kcal mol-1, it is ca. 0.4 kcal mol-1 for the enthalpy changes from either van't Hoff plots or the direct method with sampling for 5 billion MC configurations. Partial molar volumes of hydration are also computed by the direct method; they agree well with experimental data with an average deviation of 3 cm3 mol-1. In addition, the results permit breakdown of the errors in the free energy changes from the OPLS-AA force field into their enthalpic and entropic components. The excess hydrophobicity of organic solutes is enthalpic in origin.

Design, synthesis, and biological testing of biphenylmethyloxazole inhibitors targeting HIV-1 reverse transcriptase.

We report non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) using a biphenylmethyloxazole pharmacophore. A crystal structure of benzyloxazole 1 was obtained and suggested the potential viability of biphenyl analogues. In particular, 6a, 6b, and 7 turned out to be potent NNRTIs with low-nanomolar activity in enzyme inhibition and infected T-cell assays, and with low cytotoxicity. Though modeling further suggested that analogues with fluorosulfate and epoxide warheads might provide covalent modification of Tyr188, synthesis and testing did not find evidence for this outcome.

Computation of Absolute Binding Free Energies for Noncovalent Inhibitors with SARS-CoV-2 Main Protease.

Accurate, routine calculation of absolute binding free energies (ABFEs) for protein-ligand complexes remains a key goal of computer-aided drug design since it can enable screening and optimization of drug candidates. For development and testing of related methods, it is important to have high-quality datasets. To this end, from our own experimental studies, we have selected a set of 16 inhibitors of the SARS-CoV-2 main protease (Mpro) with structural diversity and well-distributed BFEs covering a 5 kcal/mol range. There is also minimal structural uncertainty since X-ray crystal structures have been deposited for 12 of the compounds. For methods testing, we report ABFE results from 2 µs molecular dynamics (MD) simulations using free energy perturbation (FEP) theory. The correlation of experimental and computed results is encouraging, with a Pearson's r2 of 0.58 and a Kendall τ of 0.24. The results indicate that current FEP-based ABFE calculations can be used for identification of active compounds (hits). While their accuracy for lead optimization is not yet sufficient, this activity remains addressable in separate lead series by relative BFE calculations.

Assessing Metadynamics and Docking for Absolute Binding Free Energy Calculations Using Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors.

Absolute binding free energy (ABFE) calculations can be an important part of the drug discovery process by identifying molecules that have the potential to be strong binders for a biomolecular target. Recent work has used free energy perturbation (FEP) theory for these calculations, focusing on a set of 16 inhibitors of the severe acute respiratory syndrome coronavirus 2 main protease (Mpro). Herein, the same data set is evaluated by metadynamics (MetaD), four different docking programs, and molecular mechanics with generalized Born and surface area solvation. MetaD yields a Kendall τ distance of 0.28 and Pearson r2 of 0.49, which reflect somewhat less accuracy than that from the ABFE FEP results. Notably, it is demonstrated that an ensemble docking protocol by which each ligand is docked into the 13 crystal structures in this data set provides improved performance, particularly when docking is carried out with Glide XP (Kendall τ distance = 0.20, Pearson r2 = 0.71), Glide SP (Kendall τ distance = 0.19, Pearson r2 = 0.66), or AutoDock 4 (Kendall τ distance = 0.21, Pearson r2 = 0.55). The best results are obtained with "superconsensus" docking by averaging the 52 results for each compound using the 4 docking protocols and all 13 crystal structures (Kendall τ distance = 0.18, Pearson r2 = 0.73).

Ensemble Geometric Deep Learning of Aqueous Solubility.

Geometric deep learning is one of the main workhorses for harnessing the power of big data to predict molecular properties such as aqueous solubility, which is key to the pharmacokinetic improvement of drug candidates. Two ensembles of graph neural network architectures were built, one based on spectral convolution and the other on spatial convolution. The pretrained models, denoted respectively as SolNet-GCN and SolNet-GAT, significantly outperformed the existing neural networks benchmarked on a validation set of 207 molecules. The SolNet-GCN model demonstrated the best performance on both the training and validation sets, with RMSE values of 0.53 and 0.72 log molar unit and Pearson r2 values of 0.95 and 0.75, respectively. Further, the ranking power of the SolNet models agreed well with a QM-based thermodynamic cycle approach at the PBE-vdW level of theory on a series of benzophenylurea derivatives and a series of benzodiazepine derivatives. Nevertheless, testing the resultant models on a set of inhibitors of the macrophage migration inhibitory factor (MIF) illustrated that the inclusion of atomic attributes to discriminate atoms with a higher tendency to form intermolecular hydrogen bonds in the crystalline state and to identify planar or nonplanar substructures can be beneficial for the prediction of aqueous solubility.

Indoloxytriazines as binding molecules for the JAK2 JH2 pseudokinase domain and its V617F variant.

Small molecules that selectively bind to the pseudokinase JH2 domain over the JH1 kinase domain of JAK2 kinase are sought. Virtual screening led to the purchase of 17 compounds among which 9 were found to bind to V617F JAK2 JH2 with affinities of 40 - 300 µM in a fluorogenic assay. Ten analogues were then purchased yielding 9 additional active compounds. Aminoanilinyltriazine 22 was particularly notable as it shows no detectable binding to JAK2 JH1, and it has a 65-µM dissociation constant K d with V617F JAK2 JH2. A crystal structure for 22 in complex with wild-type JAK2 JH2 was obtained to elucidate the binding mode. Additional de novo design led to the synthesis of 19 analogues of 22 with the most potent being 33n with K d values of 2-3 µM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

From in silico hit to long-acting late-stage preclinical candidate to combat HIV-1 infection.

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.

Targeting the TS dimer interface in bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa: Virtual screening identifies novel TS allosteric inhibitors.

Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.

Metadynamics as a Postprocessing Method for Virtual Screening with Application to the Pseudokinase Domain of JAK2.

With standard scoring methods, top-ranked compounds from virtual screening by docking often turn out to be inactive. For this reason, metadynamics, a method used to sample rare events, was studied to further evaluate docking poses with the aim of reducing false positives. Specifically, virtual screening was performed with Glide SP to seek potential molecules to bind to the ATP site in the pseudokinase domain of JAK2 kinase, and promising compounds were selected from the top-ranked 1000 based on visualization. Rescoring with Glide XP, GOLD, and MM/GBSA was unable to differentiate well between active and inactive compounds. Metadynamics was then used to gauge the relative binding affinity from the required time or the potential of mean force needed to dissociate the ligand from the bound complex. With consideration of previously known binders of varying affinities, metadynamics was able to differentiate between the most active compounds and inactive or weakly active ones, and it could identify correctly most of the selected virtual screening compounds as false positives. Thus, metadynamics has the potential to be a viable postprocessing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds.

Covalent inhibitors for eradication of drug-resistant HIV-1 reverse transcriptase: From design to protein crystallography.

Development of resistance remains a major challenge for drugs to treat HIV-1 infections, including those targeting the essential viral polymerase, HIV-1 reverse transcriptase (RT). Resistance associated with the Tyr181Cys mutation in HIV-1 RT has been a key roadblock in the discovery of nonnucleoside RT inhibitors (NNRTIs). It is the principal point mutation that arises from treatment of HIV-infected patients with nevirapine, the first-in-class drug still widely used, especially in developing countries. We report covalent inhibitors of Tyr181Cys RT (CRTIs) that can completely knock out activity of the resistant mutant and of the particularly challenging Lys103Asn/Tyr181Cys variant. Conclusive evidence for the covalent modification of Cys181 is provided from enzyme inhibition kinetics, mass spectrometry, protein crystallography, and antiviral activity in infected human T-cell assays. The CRTIs are also shown to be selective for Cys181 and have lower cytotoxicity than the approved NNRTI drugs efavirenz and rilpivirine.

Novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa identified by virtual screening.

Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.

Molecular and cellular studies evaluating a potent 2-cyanoindolizine catechol diether NNRTI targeting wildtype and Y181C mutant HIV-1 reverse transcriptase.

The development of efficacious NNRTIs for HIV/AIDS therapy is commonly met with the emergence of drug resistant strains, including the Y181C variant. Using a computationally-guided approach, we synthesized the catechol diether series of NNRTIs, which display sub-nanomolar potency in cellular assays. Among the most potent were a series of 2-cyanoindolizine substituted catechol diethers, including Compound 1. We present here a thorough evaluation of this compound, including biochemical, cellular, and structural studies. The compound demonstrates low nanomolar potency against both WT and Y181C HIV-1 RT in in vitro and cellular assays. Our crystal structures of both the wildtype and mutant forms of RT in complex with Compound 1 allow the interrogation of this compound's features that allow it to maintain strong efficacy against the drug resistant mutant. Among these are compensatory shifts in the NNRTI binding pocket, persistence of multiple hydrogen bonds, and van der Waals contacts throughout the binding site. Further, the fluorine at the C6 position of the indolizine moiety makes multiple favorable interactions with both RT forms. The present study highlights the indolizine-substituted catechol diether class of NNRTIs as promising therapeutic candidates possessing optimal pharmacological properties and significant potency against multiple RT variants.

QM/MM Calculations for the Cl- + CH3Cl SN2 Reaction in Water Using CM5 Charges and Density Functional Theory.

The prototypical SN2 reaction of chloride ion with methyl chloride has been reinvestigated in aqueous solution using QM/MM methodology featuring MO6-2X/6-31+G(d) calculations with the TIP4P water model, and partial charges were computed with the CM5 method. Though the DFT method yields excellent gas-phase energetics for the reaction, the QM/MM approach is found to yield overestimation of the activation barrier by ca. 12 kcal/mol. The discrepancy is traced to underestimate of the magnitude of the partial charges on the chlorine atoms in the transition structure. When CM1 or CM3 charges based on semiempirical wave functions are used instead, the agreement with experiment is much improved. The findings emphasize the sensitivity of the results of QM/MM calculations to the choice of QM method, the MM force field, and implementation of the QM/MM interface.

LigParGen web server: an automatic OPLS-AA parameter generator for organic ligands.

The accurate calculation of protein/nucleic acid-ligand interactions or condensed phase properties by force field-based methods require a precise description of the energetics of intermolecular interactions. Despite the progress made in force fields, small molecule parameterization remains an open problem due to the magnitude of the chemical space; the most critical issue is the estimation of a balanced set of atomic charges with the ability to reproduce experimental properties. The LigParGen web server provides an intuitive interface for generating OPLS-AA/1.14*CM1A(-LBCC) force field parameters for organic ligands, in the formats of commonly used molecular dynamics and Monte Carlo simulation packages. This server has high value for researchers interested in studying any phenomena based on intermolecular interactions with ligands via molecular mechanics simulations. It is free and open to all at jorgensenresearch.com/ligpargen, and has no login requirements.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.

Improved Treatment of Nucleosides and Nucleotides in the OPLS-AA Force Field.

DFT calculations have been used to develop improved descriptions of the torsional energetics for nucleosides and nucleotides in the OPLS-AA force field. Scans of nucleotide dihedral angles (γ, χ, and ß) and methyl phosphates provided the bases for the new torsional parameters. In addition, the angle-bending parameters of phosphodiesters and ribose were updated, and adjustments were made to existing carbohydrate torsions to better capture the sugar puckering landscape of ribose. MD simulations of nucleosides with the new parameters demonstrate a significant improvement in the ribose sugar puckering and χ angle distributions. Additionally, energy-minimization of protein-nucleotide crystal structures with the new parameters produced accurate poses.

Molecular dynamics and Monte Carlo simulations for protein-ligand binding and inhibitor design.

BACKGROUND: Non-nucleoside inhibitors of HIV reverse transcriptase are an important component of treatment against HIV infection. Novel inhibitors are sought that increase potency against variants that contain the Tyr181Cys mutation. METHODS: Molecular dynamics based free energy perturbation simulations have been run to study factors that contribute to protein-ligand binding, and the results are compared with those from previous Monte Carlo based simulations and activity data. RESULTS: Predictions of protein-ligand binding modes are very consistent for the two simulation methods; the accord is attributed to the use of an enhanced sampling protocol. The Tyr181Cys binding pocket supports large, hydrophobic substituents, which is in good agreement with experiment. CONCLUSIONS: Although some discrepancies exist between the results of the two simulation methods and experiment, free energy perturbation simulations can be used to rapidly test small molecules for gains in binding affinity. GENERAL SIGNIFICANCE: Free energy perturbation methods show promise in providing fast, reliable and accurate data that can be used to complement experiment in lead optimization projects. This article is part of a Special Issue entitled "Recent developments of molecular dynamics".

A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors.

Human macrophage migration inhibitory factor (MIF) is both a keto-enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein-ligand hydrogen bonding, aryl-aryl, and cation-π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders.