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Torsion and Lennard-Jones parameters of the OPLS all-atom force field have been refined for describing thermodynamics and dynamics of a wide range of liquid alkanes. Monte Carlo statistical mechanics (MC) and Molecular Dynamics (MD) simulations were carried out. For thermodynamics properties, MC simulations with truncated electrostatic interactions performed very closely to MD simulations with a Verlet neighbor list and the Particle Mesh Ewald algorithm. The average errors in comparison with experimental data for computed properties were improved with the modified force field (OPLS/2020), especially for long-chain alkanes. For liquid densities, heats of vaporization, and free energies of hydration, the average errors are 0.01 g/cm(3), 0.2 kcal/mol, and ca. 0.5 kcal/mol, respectively; significant gains were made for relative heats of vaporization of isomeric series. Results for self-diffusion coefficients also reproduce well experimental data for linear alkane liquids up to hexadecane. The new force field is suitable for use in improved modeling of myriad systems of importance in chemistry, biology, and materials science.

PDF - 89K, Supplementary Materials and Methods.

Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I–, alkylation-, and chain terminator–induced DNA damage, whereas TDP2 repairs topoisomerase II–induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors. Mol Cancer Ther; 13(8); 2116–26. ©2014 AACR.

PDF - 65K, IC50 value table.

PDF - 188K, Suface Plasmon Resonance (SPR) profiles binding for JLT048 and NCGC00183674 to recombinant TDP1 bound to the surface.

PDF - 119K, Schematic representation of the AlphaScreen 3'-phospho-tyrosine DNA substrate used in the TDP1 qHTS assay.

[Image: see text] The Janus kinase 2 (JAK2) pseudokinase domain (JH2) is an ATP-binding domain that regulates the activity of the catalytic tyrosine kinase domain (JH1). Dysregulation of JAK2 JH1 signaling caused by the V617F mutation in JH2 is implicated in various myeloproliferative neoplasms. To explore if JAK2 activity can be modulated by a small molecule binding to the ATP site in JH2, we have developed several ligand series aimed at selectively targeting the JAK2 JH2 domain. We report here the evolution of a false virtual screen hit into a new JAK2 JH2 series. Optimization guided by computational modeling has yielded analogues with nanomolar affinity for the JAK2 JH2 domain and >100-fold selectivity for the JH2 domain over the JH1 domain. A crystal structure for one of the potent compounds bound to JAK2 JH2 clarifies the origins of the strong binding and selectivity. The compounds expand the platform for seeking molecules to regulate JAK2 signaling, including V617F JAK2 hyperactivation.

Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30-fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.

JAK2 is a non-receptor tyrosine kinase that regulates hematopoiesis through the JAK-STAT pathway. The pseudokinase domain (JH2) is an important regulator of the activity of the kinase domain (JH1). V617F mutation in JH2 has been associated with the pathogenesis of various myeloproliferative neoplasms, but JAK2 JH2 has been poorly explored as a pharmacological target. In light of this, we aimed to develop JAK2 JH2 binders that could selectively target JH2 over JH1 and test their capacity to modulate JAK2 activity in cells. Toward this goal, we optimized a diaminotriazole lead compound into potent, selective, and cell-permeable JH2 binders leveraging computational design, synthesis, binding affinity measurements for the JH1, JH2 WT, and JH2 V617F domains, permeability measurements, crystallography, and cell assays. Optimized diaminotriazoles are capable of inhibiting STAT5 phosphorylation in both WT and V617F JAK2 in cells.

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for Mpro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to Mpro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization., Noncovalent inhibitors with low-nanomolar potency are reported for the main protease of SARS-CoV-2. X-ray crystal structures of complexes are provided, and antiviral activity is also demonstrated using infected cells.

[Image: see text] Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether non-nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC(50) values for in vitro inhibition of WT RT and EC(50) values for cytopathic protection of HIV-1-infected human T-cells in the 5–320 nM range.

The binding energies for cation-π complexation are underestimated by traditional fixed-charge force fields owing to their lack of explicit treatment of ion-induced dipole interactions. To address this deficiency, an explicit treatment of cation-π interactions has been introduced into the OPLS-AA force field. Following prior work with atomic cations, it is found that cation-π interactions can be handled efficiently by augmenting the usual 12-6 Lennard-Jones potentials with 1/r4 terms. Results are provided for prototypical complexes as well as protein-ligand systems of relevance for drug design. Alkali cation, ammonium, guanidinium, and tetramethylammonium were chosen for the representative cations, while benzene and six heteroaromatic molecules were used as the π systems. The required nonbonded parameters were fit to reproduce structure and interaction energies for gas-phase complexes from density functional theory (DFT) calculations at the ωB97X-D/6-311++G(d,p) level. The impact of the solvent was then examined by computing potentials of mean force (pmfs) in both aqueous and tetrahydrofuran (THF) solutions using the free-energy perturbation (FEP) theory. Further testing was carried out for two cases of strong and one case of weak cation-π interactions between druglike molecules and their protein hosts, namely, the JH2 domain of JAK2 kinase and macrophage migration inhibitory factor. FEP results reveal greater binding by 1.5-4.4 kcal/mol from the addition of the explicit cation-π contributions. Thus, in the absence of such treatment of cation-π interactions, errors for computed binding or inhibition constants of 101-103 are expected.

Human immunodeficiency virus (HIV)-1 remains as a global health issue that is primarily treated with highly active antiretroviral therapy, a combination of drugs that target the viral life cycle. One class of these drugs are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral reverse transcriptase (RT). First generation NNRTIs were troubled with poor pharmacological properties and drug resistance, incentivizing the development of improved compounds. One class of developed compounds are the 2-naphthyl phenyl ethers, showing promising efficacy against the Y181C RT mutation. Further biochemical and structural work demonstrated differences in potency against the Y181C mutation and binding mode of the compounds. This work aims to understand the relationship between the binding mode and ability to overcome drug resistance using macromolecular x-ray crystallography. Comparison of 2-naphthyl phenyl ethers bound to Y181C RT reveal that compounds that interact with the invariant W229 are more capable of retaining efficacy against the resistance mutation. Additional modifications to these compounds at the 4-position, computationally designed to compensate for the Y181C mutation, do not demonstrate improved potency. Ultimately, we highlight important considerations for the development of future HIV-1 drugs that are able to combat drug resistance.

[Image: see text] Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC(50) values of 13–72 μM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 μM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC(50) of 8.0 μM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC(50) of 4.8 μM. Structure–activity data are rationalized based on computed structures for DNMT3B complexes.

Non-covalent inhibitors of the main protease (Mpro) of SARS-CoV-2 having a pyridinone core were previously reported with IC50 values as low as 0.018 μM for inhibition of enzymatic activity and EC50 values as low as 0.8 μM for inhibition of viral replication in Vero E6 cells. The series has now been further advanced by consideration of placement of substituted five-membered-ring heterocycles in the S4 pocket of Mpro and N-methylation of a uracil ring. Free energy perturbation calculations provided guidance on the choice of the heterocycles, and protein crystallography confirmed the desired S4 placement. Here we report inhibitors with EC50 values as low as 0.080 μM, while remdesivir yields values of 0.5–2 μM in side-by-side testing with infectious SARS-CoV-2. A key factor in the improvement is enhanced cell permeability, as reflected in PAMPA measurements. Compounds 19 and 21 are particularly promising as potential therapies for COVID-19, featuring IC50 values of 0.044–0.061 μM, EC50 values of ca. 0.1 μM, good aqueous solubility, and no cytotoxicity.

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.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

Accurate methods to estimate free energies play an important role for studying diverse condensed-phase problems in chemistry and biochemistry. The most common methods used in conjunction with molecular dynamics (MD) and Monte Carlo statistical mechanics (MC) simulations are free energy perturbation (FEP) and thermodynamic integration (TI). For common applications featuring the conversion of one molecule to another, simulations are run in stages or multiple "λ-windows" to promote convergence of the results. For computation of absolute free energies of solvation or binding, calculations are needed in which the solute is typically annihilated in the solvent and in the complex. The present work addresses identification of optimal protocols for such calculations, specifically, the creation/annihilation of organic molecules in aqueous solution. As is common practice, decoupling of the perturbations for electrostatic and Lennard-Jones interactions was performed. Consistent with earlier reports, FEP calculations for molecular creations are much more efficient, while annihilations require many more windows and may converge to incorrect values. Strikingly, we find that as few as four windows may be adequate for creation calculations for solutes ranging from argon to ethylbenzene. For a larger druglike molecule, MIF180, which contains 22 non-hydrogen atoms and three rotatable bonds, 10 creation windows are found to be adequate to yield the correct free energy of hydration. Convergence is impeded with procedures that use any sampling in the annihilation direction, and there is no need for postprocessing methods such as the Bennett acceptance ratio (BAR).

Significant improvements have been made to the OPLS-AA force field for modeling RNA. New torsional potentials were optimized based on density functional theory (DFT) scans at the ωB97X-D/6-311++G(d,p) level for potential energy surfaces of the backbone α and γ dihedral angles. In combination with previously reported improvements for the sugar puckering and glycosidic torsion terms, the new force field was validated through diverse molecular dynamics simulations for RNAs in aqueous solution. Results for dinucleotides and tetranucleotides revealed both accurate reproduction of 3 J couplings from NMR and the avoidance of several unphysical states observed with other force fields. Simulations of larger systems with noncanonical motifs showed significant structural improvements over the previous OPLS-AA parameters. The new force field, OPLS-AA/M, is expected to perform competitively with other recent RNA force fields and to be compatible with OPLS-AA models for proteins and small molecules.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs), which bind to an allosteric site 10 −15Å from the polymerase active site, play a central role in anti-HIV chemotherapy. Though NNRTIs have been known for 30 years, the pathways by which they bind and unbind from HIV-RT have not been characterized. In crystal structures for complexes, three channels are found to extend from the NNRTI binding site to the exterior of the protein, while added mystery comes from the fact that the binding site is collapsed in the unliganded protein. To address this issue, metadynamics simulations have been performed to elucidate the unbinding of four NNRTIs from HIV-RT. A general and transferable collective variable (CV) defined by the distance between the center-of-mass (COM) of the binding pocket and COM of the ligand is used to follow the dynamics while minimizing the bias. The metadynamics also allows computation of the barriers to unbinding, which are compared with the observed potencies of the compounds in an antiviral assay.

There is a clinical need for direct-acting antivirals targeting SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, to complement current therapeutic strategies. The main protease (Mpro) is an attractive target for antiviral therapy. However, the vast majority of protease inhibitors described thus far are peptidomimetic and bind to the active-site cysteine via a covalent adduct, which is generally pharmacokinetically unfavorable. We have reported the optimization of an existing FDA-approved chemical scaffold, perampanel, to bind to and inhibit Mpro noncovalently with IC50s in the low-nanomolar range and EC50s in the low-micromolar range. Here, we present nine crystal structures of Mpro bound to a series of perampanel analogs, providing detailed structural insights into their mechanism of action and structure-activity relationship. These insights further reveal strategies for pursuing rational inhibitor design efforts in the context of considerable active-site flexibility and potential resistance mechanisms., Graphical abstract, Deshmukh et al. investigate the structure-activity relationship of a novel pharmacophore targeting the SARS-CoV-2 main protease derived from the chemical scaffold of the FDA-approved drug perampanel. X-ray crystal structures of enzyme-inhibitor complexes and in vitro activity assays provide guidance for rational drug design.

The binding energies for cation-π complexation are underestimated by traditional fixed-charge force fields owing to their lack of explicit treatment of ion-induced dipole interactions. To address this deficiency, explicit treatment of cation-π interactions has been introduced into the OPLS-AA force field. Following prior work with atomic cations, it is found that cation-π interactions can be handled efficiently by augmenting the usual 12–6 Lennard-Jones potentials with 1/r(4) terms. Results are provided for prototypical complexes as well as protein-ligand systems of relevance for drug design. Alkali cation, ammonium, guanidinium, and tetramethylammonium were chosen for the representative cations, while benzene and six heteroaromatic molecules were used as the π systems. The required non-bonded parameters were fit to reproduce structures and interactions energies for gas-phase complexes from DFT calculations at the ωB97X-D/6–311++G(d,p) level. The impact of solvent was then examined by computing potentials of mean force (pmfs) in both aqueous and THF solutions using free-energy perturbation (FEP) theory. Further testing was carried out for two cases of strong and one case of weak cation-π interactions between drug-like molecules and their protein hosts, namely, the JH2 domain of JAK2 kinase and macrophage migration inhibitory factor. FEP results reveal greater binding by 1.5 – 4.4 kcal/mol from addition of the explicit cation-π contributions. Thus, in the absence of such treatment of cation- π interactions, errors for computed binding or inhibition constants of 10(1) −10(3) are expected.

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-μM concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 μM: manidipine (4.8 μM), boceprevir (5.4 μM), lercanidipine (16.2 μM), bedaquiline (18.7 μM), and efonidipine (38.5 μM). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1’, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.

The following joint Editorial was originally published in ACS Applied Materials & Interfaces (DOI: 10.1021/acsami.0c10979). We confront the terrible reality that systemic racism and discrimination impacts the daily personal and professional lives of many members of the scientific community and broader society. In the U.S., the brutal killing of George Floyd while in police custody is one of the most recent examples of the centuries of systemic violence suffered by Black Americans. This moment and its aftermath lay bare the legacies of racism and its exclusionary practices. Let us be clear: we, the Editors, Staff, and Governance Members of ACS Publications condemn the tragic deaths of Black people and stand in solidarity with Black members of the science and engineering community. Moreover, ACS condemns racism, discrimination, and harassment in all forms. We will not tolerate practices and viewpoints that exclude or demean any member of our community. Despite these good intentions, we recognize that our community has not done enough to provide an environment for Black chemists to thrive. Rep. Eddie Bernice Johnson, Chairwoman of the U.S. House Committee on Science, Space, and Technology said, “So far, we have gotten by with a STEM workforce that does not come close to representing the diversity of our nation. However, if we continue to leave behind so much of our nation’s brainpower, we cannot succeed.”(1) Indeed, the U.S. National Science Foundation notes that Blacks and other under-represented minority groups continue to be under-represented in science and engineering education and employment.(2) What is abundantly clear in this moment is that this lack of representation is a symptom of systemic racism across all levels of education and professional life. We know that supportive words are not enough. We must develop and implement a concrete plan for changing our trajectory. Publications and citations are academic currency, and while we like to think publishing a manuscript is “just about the science”, we know that is not true for everyone. We have seen the biases (largely through the lens of gender and in Western countries because of the limitations in bibliometric analyses) and applaud our colleagues at the RSC for their massive study that explored these gender barriers in the publishing pipeline(3) and their recent Inclusion and Diversity Framework.(4) At the present time, unfortunately, less is known about the effects of race and ethnicity on publishing success. A study published in PeerJ, however, found that unprofessional reviewer comments had a disproportionate effect on authors from under-represented groups.(5) As the world’s leading society publisher, we have a responsibility to aggressively combat bias in all aspects of the publishing process, including systemic under-representation of Blacks in this endeavor (no ACS journal is currently led by a Black Editor-in-Chief). Within ACS Publications, we actively track gender and geographic diversity of editors, advisors, authors, and reviewers, and we anecdotally report on race of editors. Diversity encompasses many more dimensions than these, and we acknowledge that we can do much more than we have. We affirm that diversity and inclusion strengthen the research community and its impact, and we are committed to developing, implementing, tracking, and reporting on our progress to ensure that our editors, advisors, reviewers, and authors are more diverse and that all authors receive the same fair treatment and opportunity to publish in our journals. We acknowledge that we do not have all the answers now, but we seek to hear from and listen to our community on how we can improve our journals to be more diverse and inclusive. As first steps, we commit to the taking the following actions: Gathering and making public our baseline statistics on diversity within our journals, encompassing our editors, advisors, reviewers, and authors; annually reporting on progress Training new and existing editors to recognize and interrupt bias in peer review Including diversity of journal contributors as an explicit measurement of Editor-in-Chief performance Appointing an ombudsperson to serve as a liaison between Editors and our Community Developing an actionable diversity plan for each ACS journal These are only initial plans and the start of a conversation: other ideas are beginning to germinate, and we commit to sharing them with you regularly. We invite you contribute your ideas on how we can do better via our Axial website. We are listening carefully. We encourage you to take immediate action in your own circles. In a recent editorial, JACS Associate Editor Melanie Sanford(6) offered practical steps to take now. Take a moment to find out more about these actions and how to bring them into your work and your life. We all have a responsibility to eradicate racism and discrimination in the science and engineering community; indeed, to make a real difference, we need to be antiracist. The tragic events we have seen in the Black community provide great urgency to this goal. The work will be difficult and will force us to confront hard realities about our beliefs and actions. We fully expect that you, and everyone in the community, will hold us accountable.

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 for sampling 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 (pmf) 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 post-processing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules which preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

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.

The generation of a complete ensemble of geometrical configurations is required to obtain reliable estimations of absolute binding free energies by alchemical free-energy methods. Molecular dynamics (MD) is the most popular sampling method, but the representation of large biomolecular systems may be incomplete owing to energetic barriers that impede efficient sampling of the configurational space. Monte Carlo (MC) methods can possibly overcome this issue by adapting the attempted movement sizes to facilitate transitions between alternative local-energy minima. In this study, we present an MC statistical mechanics algorithm to explore the protein-ligand conformational space with emphasis on the motions of the protein backbone and side chains. The parameters for each MC move type were optimized to better reproduce conformational distributions of 18 dipeptides and the well-studied T4-lysozyme L99A protein. Next, the performance of the improved MC algorithms was evaluated by computing absolute free energies of binding for L99A lysozyme with benzene and seven analogs. Results for benzene with L99A lysozyme from MD and the optimized MC protocol were found to agree within 0.6 kcal/mol, while a mean unsigned error of 1.2 kcal/mol between MC results and experiment was obtained for the seven benzene analogs. Significant advantages in computation speed are also reported with MC over MD for similar extents of configurational sampling.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is implicated in the regulation of inflammation, cell proliferation, and neurological disorders. MIF is also an enzyme that functions as a keto-enol tautomerase. Most potent MIF tautomerase inhibitors incorporate a phenol, which hydrogen bonds to Asn97 in the active site. Starting from a 113-μm docking hit, we report results of structure-based and computer-aided design that have provided substituted pyrazoles as phenol alternatives with potencies of 60-70 nm. Crystal structures of complexes of MIF with the pyrazoles highlight the contributions of hydrogen bonding with Lys32 and Asn97, and aryl-aryl interactions with Tyr36, Tyr95, and Phe113 to the binding.

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 Kd 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 Kd values of 2–3 μM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

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 β) 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.

The atomic point-charge model used in most molecular mechanics force fields does not represent well the electronic anisotropy that is featured in many directional non-covalent interactions. Sulfur participates in several types of such interactions with its lone pairs and σ-holes. The current study develops a new model, via the addition of off-atom charged sites, for a variety of sulfur compounds in the OPLS-AA and OPLS/CM5 force fields to address the lack of charge anisotropy. Parameter optimization is carried out to reproduce liquid-state properties, torsional and non-covalent energetics from reliable quantum mechanical calculations, and electrostatic potentials. Significant improvements are obtained for computed free energies of hydration, reducing the mean unsigned errors from ca. 1.4 to 0.4–0.7 kcal/mol. Enhanced accuracy in directionality and energetics is also obtained for molecular complexes with sulfur-containing hydrogen and halogen bonds. Moreover, the new model reproduce the unusual conformational preferences of sulfur-containing compounds with 1,4-intramolecular chalcogen bonds. Transferability of the new force field parameters to cysteine and methionine is verified via molecular dynamic simulations of blocked dipeptides. The study demonstrates the effectiveness of using off-atom charge sites to address electronic anisotropy, and provides a parameterization methodology that can be applied to other systems.

Janus kinases (JAKs) regulate hematopoiesis via the cytokine-mediated JAK-STAT signaling pathway. JAKs contain tandem C-terminal pseudokinase (JH2) and tyrosine kinase (JH1) domains. The JAK2 pseudokinase domain adopts a protein kinase fold and, despite its pseudokinase designation, binds ATP with micromolar affinity. Recent evidence shows that displacing ATP from the JAK2 JH2 domain alters the hyperactivation state of the oncogenic JAK2 V617F protein while sparing the wild type JAK2 protein. In this study, small molecule binders of JAK2 JH2 were identified via an in vitro screen. Top hits were characterized using biophysical and structural approaches. Development of pseudokinase-selective compounds may offer novel pharmacological opportunities for treating cancers driven by JAK2 V617F and other oncogenic JAK mutants.

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

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.

Calculation of the absolute free energy of binding (ΔG(bind)) for a complex in solution is challenging owing to the need for adequate configurational sampling and an accurate energetic description, typically with a force field (FF). In this study, Monte Carlo (MC) simulations with improved side-chain and backbone sampling are used to assess ΔG(bind) for the complex of a drug-like inhibitor (MIF180) with the protein macrophage migration inhibitory factor (MIF) using free energy perturbation (FEP) calculations. For comparison, molecular dynamics (MD) simulations were employed as an alternative sampling method for the same system. With the OPLS-AA/M FF and CM5 atomic charges for the inhibitor, the ΔG(bind) results from the MC/FEP and MD/FEP simulations, −8.80 ± 0.74 and −8.46 ± 0.85 kcal/mol, agree well with each other and with the experimental value of −8.98 ± 0.28 kcal/mol. The convergence of the results and analysis of the trajectories indicate that sufficient sampling was achieved for both approaches. Repeating the MD/FEP calculations using current versions of the CHARMM and AMBER FFs led to a 6-kcal/mol range of computed ΔG(bind). These results show that calculation of accurate ΔG(bind) for large ligands is both feasible and numerically equivalent, within error limits, using either methodology.

Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, thymidylate synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl- l -glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.

Both Monte Carlo statistical mechanics and molecular dynamics are being used to compute relative and absolute free energies of binding for protein-ligand complexes. Applications have included computing absolute free energies of binding for L99A lysozyme with benzene and seven analogs, and a drug-like molecule MIF180 with human macrophage migration inhibitory factor (MIF). It is noted that FEP calculations for molecular creations are much more efficient than for annihilations, and strikingly as few as 10 charge and Lennard-Jones windows are needed for creation of MIF180, which has 22 non-hydrogen atoms. Additional computational issues are considered including performance of OPLS-AA/M, CHARMM, and AMBER force fields, atomic charge models, water equilibration, and convergence. Significant advantages in computation speed are reported with MC over MD for similar extents of configurational sampling.

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.

Thymidylate synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R-enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.

It is widely accepted that structural rigidity is required to achieve high levels of asymmetric induction in catalytic, enantioselective reactions. This fundamental design principle often does not apply to highly selective catalytic peptides that often exhibit conformational heterogeneity. As a result, these complex systems are particularly challenging to study both experimentally and computationally. Herein, we utilize molecular dynamics simulations to investigate the role of conformational mobility on the reactivity and selectivity exhibited by a catalytic, β-turn-biased peptide in an atroposelective bromination reaction. By means of cluster analysis, multiple distinct conformers of the peptide and a catalyst-substrate complex were identified in the simulations, all of which were corroborated by experimental NMR measurements. The simulations also revealed that a shift in the conformational equilibrium of the peptidic catalyst occurs upon addition of substrate, and the degree of change varies among different substrates. On the basis of these data, we propose a correlation between the composition of the peptide conformational ensemble and its catalytic properties. Moreover, these findings highlight the importance of conformational dynamics in catalytic, asymmetric reactions mediated by oligopeptides, unveiled through high-level, state-of-the-art computational modeling.

A quantum mechanical bespoke molecular mechanics force field is derived for the L99A mutant of T4 lysozyme and used to compute absolute binding free energies of six benzene analogs to the protein. Promising agreement between theory and experiment highlights the potential for future use of system-specific force fields in computer-aided drug design.

Catechol diethers that incorporate a 7-cyano-2-naphthyl substituent are reported as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Many of the compounds have 1–10 nM potencies toward wild-type HIV-1. An interesting conformational effect allows two unique conformers for the naphthyl group in complexes with HIV-RT. X-ray crystal structures for 4a and 4f illustrate the alternatives.