Coordination of the ammonium group of Lys32 in the active site of human macrophage migration inhibitory factor (MIF) using a 1,7-naphthyridin-8-one instead of a quinoline is investigated. Both gas- and aqueous-phase DFT calculations for model systems indicate potential benefits for the added hydrogen bond with the lactam carbonyl group, while FEP results are neutral. Three crystal structures are reported for complexes of MIF with 3a, 4a, and 4b, which show that the desired hydrogen bond is formed with O–N distances of 2.8–3.0 Å. Compound 4b is the most potent new MIF inhibitor with Ki and Kd values of 90 and 94 nM; it also has excellent aqueous solubility, 288 μg/mL. Consistent with the FEP results, the naphthyridinones are found to have similar potency as related quinolines in spite of the additional protein–ligand hydrogen bond.Keywords: lysine coordination; MIF inhibitors; protein crystallography;

Aqueous solubilities and activities have been measured for 17 members of the quinolinyltriazole series of inhibitors of human macrophage migration inhibitory factor (MIF). Systematic variation of a solvent-exposed substituent provided increases in solubility from 2 μg/mL for the parent compound 3a up to 867 μg/mL. The low solubility of 3a results from its near-planar structure and an intermolecular hydrogen bond, as revealed in a small-molecule X-ray structure. Removal of the hydrogen bond yields a 3-fold increase in solubility, but a 7-fold drop in activity. 5b emerges as the most potent MIF inhibitor with a Ki of 14 nM and good solubility, 47 μg/mL, while 4e has both high potency and solubility.Keywords: Aqueous solubility; crystallography; MIF inhibitors;

•Representation of nucleosides and nucleotides was improved in the OPLS-AA/M force field.•Validation included comparison of MD results and NMR data for nucleosides in water.•Protein-nucleotide complexes were minimized and compared to crystal data.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.Download high-res image (105KB)Download full-size image

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.

Molecular mechanics force fields, which are commonly used in biomolecular modeling and computer-aided drug design, typically treat nonbonded interactions using a limited library of empirical parameters that are developed for small molecules. This approach does not account for polarization in larger molecules or proteins, and the parametrization process is labor-intensive. Using linear-scaling density functional theory and atoms-in-molecule electron density partitioning, environment-specific charges and Lennard-Jones parameters are derived directly from quantum mechanical calculations for use in biomolecular modeling of organic and biomolecular systems. The proposed methods significantly reduce the number of empirical parameters needed to construct molecular mechanics force fields, naturally include polarization effects in charge and Lennard-Jones parameters, and scale well to systems comprised of thousands of atoms, including proteins. The feasibility and benefits of this approach are demonstrated by computing free energies of hydration, properties of pure liquids, and the relative binding free energies of indole and benzofuran to the L99A mutant of T4 lysozyme.

A review is provided on efforts in our laboratory over the last decade to discover anti-HIV agents. The work has focused on computer-aided design and synthesis of non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) with collaborative efforts on biological assaying and protein crystallography. Numerous design issues were successfully addressed including the need for potency against a wide range of viral variants, good aqueous solubility, and avoidance of electrophilic substructures. Computational methods including docking, de novo design, and free-energy perturbation (FEP) calculations made essential contributions. The result is novel NNRTIs with picomolar and low-nanomolar activities against wild-type HIV-1 and key variants that also show much improved solubility and lower cytotoxicity than recently approved drugs in the class.Figure optionsDownload full-size imageDownload as PowerPoint slide

Inhibitors of human macrophage migration inhibitory factor (MIF) previously reported in the literature have been reexamined by synthesis, assaying for tautomerase activity, and protein crystallography. Substantial inconsistencies between prior and current assay results are noted. They appear to arise from difficulties with the tautomerase substrates, solubility issues, and especially covalent inhibition. Incubation time variation shows that 3, 4, 6, and 9 are covalent or slow-binding inhibitors. Two protein crystal structures are provided; one confirms that the twice-discovered 3 is a covalent inhibitor.

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.Keywords: Anti-HIV agents; NNRTIs; protein crystallography;

Due to the importance of water in chemical and biological systems, a coarse-grained representation of the solvent can greatly simplify the description of the system while retaining key thermodynamic properties of the medium. A multiscale solvation model that couples all-atom solutes and polarizable Martini coarse-grained water (AAX/CGS) is developed to reproduce free energies of hydration of organic solutes. Using Monte Carlo/free energy perturbation (MC/FEP) calculations, results from multiscale and all-atom simulations are compared. Improved accuracy is obtained with the AAX/CGS approach for hydrophobic and sulfur- or halogen-containing solutes, but larger deviations are found for polar solute molecules where hydrogen bonding is featured. Furthermore, solvent effects on conformational and tautomeric equilibria of AA solutes were investigated using AA, CG, and GB/SA solvent models. It is found that the CG solvent model can reproduce well the medium effects from experiment and AA simulations; however, the GB/SA solvent model fails in some cases. A 7–30-fold reduction in computational cost is found for the present AAX/CGS multiscale simulations compared to the AA alternative.

The ability to accurately perform molecular dynamics and free energy perturbation calculations for protein–ligand systems is of broad interest to the biophysical and pharmaceutical sciences. In this work, several popular force fields are evaluated for reproducing experimental properties of the flavodoxin/flavin mononucleotide system. Calculated 3J couplings from molecular dynamics simulations probing φ and χ1 dihedral angles are compared to over 1000 experimental measurements. Free energy perturbation calculations were also executed between different protein mutants for comparison with experimental data for relative free energies of binding. Newer versions of popular protein force fields reproduced 3J backbone and side chain couplings with good accuracy, with RMSD values near or below one hertz in most cases. OPLS-AA/M paired with CM5 charges for the ligand performed particularly well, both for the 3J couplings and FEP results, with a mean unsigned error for relative free energies of binding of 0.36 kcal/mol.

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series.

Partial atomic charges for neutral molecules from quantum mechanical calculations are typically scaled for use in molecular modeling of liquid-phase systems. Optimal scale factors of 1.14 for CM1A and 1.27 for CM5 charges were previously determined for minimizing errors in free energies of hydration. The adequacy of the 1.14*CM1A and 1.27*CM5 models are evaluated here in pure liquid simulations in combination with the OPLS-AA force field. For 22 organic liquids, the 1.14*CM1A and 1.27*CM5 models yield mean unsigned errors (MUEs) of ca. 1.40 kcal/mol for heats of vaporization. Not surprisingly, this reflects overpolarization with the scale factors derived for aqueous media. Prediction of pure liquid properties using CM5 charges is optimized using a scale factor of 1.14, which reduces the MUE for heats of vaporization to 0.89 kcal/mol. However, due to the impracticality of using different scale factors in different explicit-solvent condensed-phase simulations, a universal scale factor of 1.20 emerged for CM5 charges. This provides a balance between errors in computed pure liquid properties and free energies of hydration. Computation of free energies of hydration by the GB/SA method further found that 1.20 is equally suited for use in explicit or implicit treatments of aqueous solvation. With 1.20*CM5 charges, a variety of condensed-phase simulations can be pursued while maintaining average errors of 1.0 kcal/mol in key thermodynamic properties.

The development and validation of new peptide dihedral parameters are reported for the OPLS-AA force field. High accuracy quantum chemical methods were used to scan φ, ψ, χ1, and χ2 potential energy surfaces for blocked dipeptides. New Fourier coefficients for the dihedral angle terms of the OPLS-AA force field were fit to these surfaces, utilizing a Boltzmann-weighted error function and systematically examining the effects of weighting temperature. To prevent overfitting to the available data, a minimal number of new residue-specific and peptide-specific torsion terms were developed. Extensive experimental solution-phase and quantum chemical gas-phase benchmarks were used to assess the quality of the new parameters, named OPLS-AA/M, demonstrating significant improvement over previous OPLS-AA force fields. A Boltzmann weighting temperature of 2000 K was determined to be optimal for fitting the new Fourier coefficients for dihedral angle parameters. Conclusions are drawn from the results for best practices for developing new torsion parameters for protein force fields.

Partial molar volume is an important thermodynamic property that gives insights into molecular size and intermolecular interactions in solution. Theoretical frameworks for determining the partial molar volume (V°) of a solvated molecule generally apply Scaled Particle Theory or Kirkwood–Buff theory. With the current abilities to perform long molecular dynamics and Monte Carlo simulations, more direct methods are gaining popularity, such as computing V° directly as the difference in computed volume from two simulations, one with a solute present and another without. Thermodynamically, V° can also be determined as the pressure derivative of the free energy of solvation in the limit of infinite dilution. Both approaches are considered herein with the use of free energy perturbation (FEP) calculations to compute the necessary free energies of solvation at elevated pressures. Absolute and relative partial molar volumes are computed for benzene and benzene derivatives using the OPLS-AA force field. The mean unsigned error for all molecules is 2.8 cm3 mol−1. The present methodology should find use in many contexts such as the development and testing of force fields for use in computer simulations of organic and biomolecular systems, as a complement to related experimental studies, and to develop a deeper understanding of solute–solvent interactions.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure–activity data.

Orbital theory provides a powerful tool for rationalizing and understanding many phenomena in chemistry. In most introductory chemistry courses, students are introduced to atomic and molecular orbitals in the form of two-dimensional drawings. In this work, we describe a general method for producing 3D printing files of orbital models that can be employed with most popular software packages for performing electronic structure calculations and molecular visualization. Methods for producing both solid and mesh orbitals are provided, including pointers for producing a model that is both informative and structurally sound. Finally, numerous examples of various systems of interest in physical organic chemistry are provided in the .stl format for 3D printing, as well as a fully illustrated tutorial for the process.

With a view to improving the consistency of free energy perturbation calculations in Monte Carlo simulations of protein–ligand complexes, we have implemented the replica exchange with solute tempering (REST) method in the MCPRO software. By augmenting the standard REST approach with regular attempted jumps in selected dihedral angles, our combined method facilitates sampling of ligand binding modes that are separated by high free energy barriers and ensures that computed free energy changes are considerably less dependent on the starting conditions and the chosen mutation pathway than those calculated with standard Monte Carlo sampling. We have applied the enhanced sampling method to the calculation of the activities of seven non-nucleoside inhibitors of HIV-1 reverse transcriptase, and its Tyr181Cys variant, and have shown that a range of binding orientations is possible depending on the nature of the ligand and the presence of mutations at the binding site.

The recently developed Charge Model 5 (CM5) is tested for its utility in condensed-phase simulations. The CM5 approach, which derives partial atomic charges from Hirshfeld population analyses, provides excellent results for gas-phase dipole moments and is applicable to all elements of the periodic table. Herein, the adequacy of scaled CM5 charges for use in modeling aqueous solutions has been evaluated by computing free energies of hydration (ΔGhyd) for 42 neutral organic molecules via Monte Carlo statistical mechanics. An optimal scaling factor for the CM5 charges was determined to be 1.27, resulting in a mean unsigned error (MUE) of 1.1 kcal/mol for the free energies of hydration. Testing for an additional 20 molecules gave an MUE of 1.3 kcal/mol. The high precision of the results is confirmed by free energy calculations using both sequential perturbations and complete molecular annihilation. Performance for specific functional groups is discussed; sulfur-containing molecules yield the largest errors. In addition, the scaling factor of 1.27 is shown to be appropriate for CM5 charges derived from a variety of density functional methods and basis sets. Though the average errors from the 1.27*CM5 results are only slightly lower than those using 1.14*CM1A charges, the broader applicability and easier access to CM5 charges via the Gaussian program are additional attractive features. The 1.27*CM5 charge model can be used for an enormous variety of applications in conjunction with many fixed-charge force fields and molecular modeling programs.

Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Promising compounds are reported that show midpicomolar activity against the wild-type virus and sub-20 nM activity against viral variants bearing Tyr181Cys and Lys103Asn mutations in HIV-RT. An X-ray crystal structure at 2.49 Å resolution is also reported for the key compound 6e with HIV-RT.Keywords: Anti-HIV agents; NNRTIs; protein crystallography

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.

Halogen bonding, due to its directionality and tunable strength, is being increasingly utilized in self-assembling materials and crystal engineering. Using density functional theory (DFT) and molecular mechanics (OPLS/CM1Ax) calculations, multiply halogen bonded complexes of brominated imidazole and pyridine are investigated along with their potential in construction of self-assembling architectures. Dimers with 1–10 halogen bonds are considered and reveal maximal binding energies of 3–36 kcal/mol. Cooperative (nonadditive) effects are found in complexes that extend both along and perpendicular to the halogen bonding axes, with interaction energies depending on polarization, secondary interactions, and ring spacers. Four structural motifs were identified to yield optimal halogen bonding. For the largest systems, the excellent agreement found between the DFT and OPLS/CM1Ax results supports the utility of the latter approach for analysis and design of self-assembling supramolecular structures.

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC50 values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ∼40 μg/mL, which is similar to that for the approved drug efavirenz and ∼1000-fold greater than for rilpivirine.

The frequency of biaryl substructures in a database of approved oral drugs has been analyzed. This led to designation of 20 prototypical biaryls plus 10 arylpyridinones for parametrization in the OPLS all-atom force fields. Bond stretching, angle-bending, and torsional parameters were developed to reproduce the MP2 geometries and torsional energy profiles. The transferability of the new parameters was tested through their application to three additional biaryls. The torsional energetics for the 33 biaryl molecules are analyzed and factors leading to preferences for planar and nonplanar geometries are identified. For liquid biphenyl, the computed density and heat of vaporization at the boiling point (255 °C) are also reported.

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved activity towards Tyr181Cys containing variants was pursued with the assistance of free energy perturbation (FEP) calculations. Optimization of the 4-R substituent in 1 led to ethyl and isopropyl analogs 1e and 1f with 1–7 nM potency towards both the wild-type virus and a Tyr181C variant.Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved activity towards Tyr181Cys containing variants was pursued with the assistance of free energy perturbation (FEP) calculations. Optimization of the 4-R substituent in 1 led to ethyl and isopropyl analogs 1e and 1f with 1–7 nM potency towards both the wild-type virus and a Tyr181C variant.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase are reported that have ca. 100-fold greater solubility than the structurally related drugs etravirine and rilpivirine, while retaining high anti-viral activity. The solubility enhancements come from strategic placement of a morpholinylalkoxy substituent in the entrance channel of the NNRTI binding site. Compound 4d shows low-nanomolar activity similar to etravirine towards wild-type HIV-1 and key viral variants.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.

Graphene, carbon nanotubes, and fullerenes are of great interest due to their unique properties and diverse applications in biology, molecular electronics, and materials science. Therefore, there is demand for methods that can accurately model the interface between carbon surfaces and their environment. In this Letter we compare results for complexes of water, potassium ion, and chloride ion with graphene, carbon nanotube, and fullerene surfaces using a standard nonpolarizable force field (OPLS-AA), a polarizable force field (OPLS-AAP), density functional theory (DFT), and ab initio theory. For interactions with water, OPLS-AA with the TIP3P or TIP4P water models describes the interactions with benzene (C6H6) and coronene (C24H12) well; however, for acenes larger than circumcoronene (C54H18) and especially for C60, the interaction energies are somewhat too weak and polarization is needed. For ions interacting with carbon surfaces, inclusion of polarization is essential, and OPLS-AAP is found to perform well in comparison to the highest-level quantum mechanical methods. Overall, OPLS-AAP provides an accurate and computationally efficient force field for modeling condensed-phase systems featuring carbon surfaces.Keywords: acene; arene; buckyball; carbon nanotube; fullerene; graphene; polarization;

X-ray crystal structures at 2.9 Å resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.

A combined computational and experimental kinetic study on the Henry reaction is reported. The effects of solvation on the transition structures and the rates of reaction between nitromethane and formaldehyde, and between nitropropane and benzaldehyde are elucidated with QM/MM calculations.

The effects of addition of a methyl group to a lead compound on biological activity are examined. A literature analysis of >2000 cases reveals that an activity boost of a factor of 10 or more is found with an 8% frequency, and a 100-fold boost is a 1 in 200 event. Four cases in the latter category are analyzed in depth to elucidate any unusual aspects of the protein–ligand binding, distribution of water molecules, and changes in conformational energetics. The analyses include Monte Carlo/free-energy perturbation (MC/FEP) calculations for methyl replacements in inhibitor series for p38α MAP kinase, ACK1, PTP1B, and thrombin. Methyl substitutions ortho to an aryl ring can be particularly effective at improving activity by inducing a propitious conformational change. The greatest improvements in activity arise from coupling the conformational gain with the burial of the methyl group in a hydrophobic region of the protein.

The Plasmodium falciparum orthologue of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host’s immune response. As for other MIF orthologues, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with Ki of ∼40 nM; they are also highly selective with Ki > 100 μM for human MIF.

The representation of chlorine, bromine, and iodine in aryl halides has been modified in the OPLS-AA and OPLS/CM1A force fields in order to incorporate halogen bonding. The enhanced force fields, OPLS-AAx and OPLS/CM1Ax, have been tested in calculations on gas-phase complexes of halobenzenes with Lewis bases, and for free energies of hydration, densities, and heats of vaporization of halobenzenes. Comparisons with results of MP2/aug-cc-pVDZ(-PP) calculations for the complexes are included. Implementation in the MCPRO software also allowed computation of relative free energies of binding for a series of HIV reverse transcriptase inhibitors via Monte Carlo/free-energy perturbation calculations. The results support the notion that the activity of an unusually potent chloro analog likely benefits from halogen bonding with the carbonyl group of a proline residue.

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with computational guidance. Extension of azine-containing inhibitors into the entrance channel between Lys103 and Glu138 has led to the discovery of potent and structurally novel derivatives including dimeric inhibitors in an NNRTI-linker-NNRTI motif.Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with computational guidance. Extension of azine-containing inhibitors into the entrance channel between Lys103 and Glu138 has led to the discovery of potent and structurally novel derivatives including dimeric inhibitors in an NNRTI-linker-NNRTI motif.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) that interfere with the replication of human immunodeficiency virus (HIV) are being pursued with guidance from molecular modeling including free-energy perturbation (FEP) calculations for protein–inhibitor binding affinities. The previously reported pyrimidinylphenylamine 1 and its chloro analogue 2 are potent anti-HIV agents; they inhibit replication of wild-type HIV-1 in infected human T-cells with EC50 values of 2 and 10 nM, respectively. However, they show no activity against viral strains containing the Tyr181Cys (Y181C) mutation in HIV-RT. Modeling indicates that the problem is likely associated with extensive interaction between the dimethylallyloxy substituent and Tyr181. As an alternative, a phenoxy group is computed to be oriented in a manner diminishing the contact with Tyr181. However, this replacement leads to a roughly 1000-fold loss of activity for 3 (2.5 μM). The present report details the efficient, computationally driven evolution of 3 to novel NNRTIs with sub-10 nM potency toward both wild-type HIV-1 and Y181C-containing variants. The critical contributors were FEP substituent scans for the phenoxy and pyrimidine rings and recognition of potential benefits of addition of a cyanovinyl group to the phenoxy ring.

A 5-μM docking hit has been optimized to an extraordinarily potent (55 pM) non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP) calculations to predict relative free energies of binding aided the optimizations by identifying optimal substitution patterns for phenyl rings and a linker. The most potent resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen bond with Pro95 likely contributes to the extreme potency of compound 42. In addition, several examples are provided illustrating failures of attempted grafting of a substructure from a very active compound onto a seemingly related scaffold to improve its activity.

Optimization of thienopyrimidinone derivatives as FGFR1 kinase inhibitors is being pursued. The present results confirm predictions of computational modeling that an aryl substituent can be introduced at the 2-position in structure 3. The substituent is anticipated to project deeper into the binding site and provide opportunities for enhanced activity and selectivity. The most potent analog reported herein, 13, has a 4-hydroxyphenyl substituent and yields an IC50 of 6 μM for inhibition of phosphorylation by FGFR1 kinase. It was also found that the western anisole-containing substituent in 3 can be replaced by a propionic acid group with no loss in potency and with potentially significant gains in pharmacologically relevant properties.

Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.Graphical abstractSubstituted N-phenylbenzisothiazolones are reported as antagonists of the enzymatic activity and signaling of macrophage migration inhibitory factor (MIF). Two analogs are shown to attenuate MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.Highlights► Benzisothiazolones as inhibitors of macrophage migration inhibitory factor (MIF). ► Inhibition of MIF-CD74 binding and ERK phosphorylation. ► Mass spectral analysis of covalent modification of cysteines. ► Potential anti-inflammatory and anti-proliferative agents.

Application of combined quantum and molecular mechanical (QM/MM) methods focuses on predicting activation barriers and the structures of stationary points for organic and enzymatic reactions. Characterization of the factors that stabilize transition structures in solution and in enzyme active sites provides a basis for design and optimization of catalysts. Continued technological advances allowed for expansion from prototypical cases to mechanistic studies featuring detailed enzyme and condensed-phase environments with full integration of the QM calculations and configurational sampling. This required improved algorithms featuring fast QM methods, advances in computing changes in free energies including free-energy perturbation (FEP) calculations, and enhanced configurational sampling. In particular, the present Account highlights development of the PDDG/PM3 semi-empirical QM method, computation of multi-dimensional potentials of mean force (PMF), incorporation of on-the-fly QM in Monte Carlo (MC) simulations, and a polynomial quadrature method for efficient modeling of proton-transfer reactions. The utility of this QM/MM/MC/FEP methodology is illustrated for a variety of organic reactions including substitution, decarboxylation, elimination, and pericyclic reactions. A comparison to experimental kinetic results on medium effects has verified the accuracy of the QM/MM approach in the full range of solvents from hydrocarbons to water to ionic liquids. Corresponding results from ab initio and density functional theory (DFT) methods with continuum-based treatments of solvation reveal deficiencies, particularly for protic solvents. Also summarized in this Account are three specific QM/MM applications to biomolecular systems: (1) a recent study that clarified the mechanism for the reaction of 2-pyrone derivatives catalyzed by macrophomate synthase as a tandem Michael−aldol sequence rather than a Diels−Alder reaction, (2) elucidation of the mechanism of action of fatty acid amide hydrolase (FAAH), an unusual Ser-Ser-Lys proteolytic enzyme, and (3) the construction of enzymes for Kemp elimination of 5-nitrobenzisoxazole that highlights the utility of QM/MM in the design of artificial enzymes.

The Thorpe−Ingold hypothesis for the gem-dimethyl effect in the cyclization reactions of 2-chloroethoxide derivatives has been investigated computationally in the gas phase and in aqueous solution. Ab initio MP2/6-311+G(d,p) and CBS-Q calculations reveal little intrinsic difference in reactivity with increasing α-methylation for the series of reactants 1−3. However, inclusion of continuum hydration or of explicit hydration through mixed quantum and statistical mechanics (MC/FEP) simulations does reproduce the substantial, experimentally observed rate increases with increasing α-methylation. Analysis of the MC/FEP results provides clear evidence that the rate increases stem primarily from increased steric hindrance to hydration of the nucleophilic oxygen atom with increasing α-methylation. Thus, the gem-dimethyl acceleration of oxirane formation for 1−3 is found to be predominantly a solvent effect.

Quantum and molecular mechanics calculations for the Diels−Alder reactions of cyclopentadiene with 1,4-naphthoquinone, methyl vinyl ketone, and acrylonitrile have been carried out at the vacuum−water interface and in the gas phase. In conjunction with previous studies of these cycloadditions in dilute solution, a more complete picture of aqueous environmental effects emerges with implications for the origin of observed rate accelerations using heterogeneous aqueous suspensions, “on water” conditions. The pure TIP4P water slab maintains the bulk density and hydrogen-bonding properties in central water layers. The bulk region merges to vacuum over a ca. 5 Å band with progressive diminution of the density and hydrogen bonding. The relative free energies of activation and transition structures for the reactions at the interface are found to be intermediate between those calculated in the gas phase and in bulk water; i.e., for the reaction with 1,4-naphthoquinone, the ΔΔG⧧ values relative to the gas phase are −3.6 and −7.3 kcal/mol at the interface and in bulk water, respectively. Thus, the results do not support the notion that a water surface is more effective than bulk water for catalysis of such pericyclic reactions. The trend is in qualitative agreement with expectations based on density considerations and estimates of experimental rate constants for the gas phase, a heterogeneous aqueous suspension, and a dilute aqueous solution for the reaction of cyclopentadiene with methyl vinyl ketone. Computed energy pair distributions reveal a uniform loss of 0.5−1.0 hydrogen bond for the reactants and transition states in progressing from bulk water to the vacuum−water interface. Orientational effects are apparent at the surface; e.g., the carbonyl group in the methyl vinyl ketone transition structure is preferentially oriented into the surface. Also, the transition structure for the 1,4-naphthoquinone case is buried more in the surface, and the free energy of activation for this reaction is most similar to the result in bulk water.

Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC50 values of 23 and 50 μM. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 μM. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

Monte Carlo free energy perturbation (MC/FEP) calculations have been applied to compute the relative binding affinities of 17 congeneric pyridazo-pyrimidinone inhibitors of the protein p38α MAP kinase. Overall correlation with experimental data was found to be modest when the complexes were hydrated using a traditional procedure with a stored solvent box. Significant improvements in accuracy were obtained when the MC/FEP calculations were repeated using initial solvent distributions optimized by the water placement algorithm JAWS. The results underscore the importance of accurate placement of water molecules in a ligand binding site for the reliable prediction of relative free energies of binding.

Thermochemical data have been obtained from G3B3 quantum mechanical calculations for 18 prototypical organic molecules, which exhibit E/Z conformational equilibria. The results are fundamentally important for molecular design including evaluation of structures from protein−ligand docking. For the 18 E/Z pairs, relative energies, enthalpies, free energies, and dipole moments are reported; the E − Z free-energy differences at 298 K range from +8.2 kcal/mol for 1,3-dimethyl carbamate to −6.4 kcal/mol for acetone oxime. A combination of steric and electronic effects can rationalize the variations. Free energies of hydration were also estimated using the GB/SA continuum solvent model. These results indicate that differential hydration is unlikely to qualitatively change the preferred direction of the E/Z equilibria, though further study with free-energy methods using explicit solvent is desirable.

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with the assistance of free energy perturbation (FEP) calculations to predict relative free energies of binding. Extension of azole-containing inhibitors into an ‘eastern’ channel between Phe227 and Pro236 has led to the discovery of potent and structurally novel derivatives.Synthesis, assaying, and computational results are reported for new anti-HIV agents that exhibit high potency and low cytotoxicity. Extension into an eastern channel in the HIV-1 reverse transcriptase binding site is investigated.

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto–enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC50 values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF–CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.Substituted benzoxazol-2-ones are reported as antagonists of the signaling by macrophage migration inhibitory factor (MIF). One of the potent analogues is shown to attenuate MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF–CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.Substituted aryl-1,2,3-triazoles are reported as the first agonists of the binding of the cytokine macrophage migration inhibitory factor (MIF) to its receptor CD74. The contrasting behavior of MIF antagonists and agonists is also demonstrated in MIF-dependent ERK1/2 phosphorylation using human fibroblasts.

There is great interest in molecules capable of inhibiting the interactions between p53 and its negative regulators hDM2 and hDMX, as these molecules have validated potential against cancers in which one or both oncoproteins are overexpressed. We reported previously that appropriately substituted β3-peptides inhibit these interactions and, more recently, that minimally cationic β3-peptides are sufficiently cell permeable to upregulate p53-dependent genes in live cells. These observations, coupled with the known stability of β-peptides in a cellular environment, and the recently reported structures of hDM2 and hDMX, motivated us to exploit computational modeling to identify β-peptides with improved potency and/or selectivity. This exercise successfully identified a new β3-peptide, β53-16, that possesses the highly desirable attribute of high affinity for both hDM2 and hDMX and identifies the 3,4-dichlorophenyl moiety as a novel determinant of hDMX affinity.

Macrophage migration inhibitory factor (MIF) is a cytokine that is involved in the regulation of inflammation as well as cell proliferation and differentiation. Deactivation of MIF by antibodies or inhibition of MIF binding to its receptor, CD74, attenuates tumor growth and angiogenesis. To discover small-molecule inhibitors of MIF’s biological activity, virtual screening was performed by docking 2.1 million compounds into the MIF tautomerase active site. After visual inspection of 1200 top-ranked MIF-ligand complexes, 26 possible inhibitors were selected and purchased and 23 of them were assayed. The in vitro binding assay for MIF with CD74 revealed that 11 of the compounds have inhibitory activity in the micromolar regime, including four compounds with IC50 values below 5 μM. Inhibition of MIF tautomerase activity was also established for many of the compounds with IC50 values as low as 0.5 μM; Michaelis−Menten analysis was performed for two cases and confirmed the competitive inhibition.

To discover non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) that are effective against both wild-type (WT) virus and variants that encode the clinically troublesome Tyr181Cys (Y181C) RT mutation, virtual screening by docking was carried out using three RT structures and more than 2 million commercially available compounds. Two of the structures are for WT-virus with different conformations of Tyr181, while the third structure incorporates the Y181C modification. Eventually nine compounds were purchased and assayed. Three of the compounds show low-micromolar antiviral activity toward either or both the wild-type and Y181C HIV-1 strains. The study illustrates a viable protocol to seek anti-HIV agents with enhanced resistance profiles.

In order to seek vancomycin analogs with improved performance against VanA and VanB resistant bacterial strains, extensive computational investigations have been performed to examine the effects of side-chain and backbone modifications. Changes in binding affinities for tripeptide cell-wall precursor mimics, Ac2-l-Lys-d-Ala-d-Ala (3) and Ac2-l-Lys-d-Ala-d-Lac (4), with vancomycin analogs were computed with Monte Carlo/free energy perturbation (MC/FEP) calculations. Replacements of the 3-hydroxyl group in residue 7 with small alkyl or alkoxy groups, which improve contacts with the methyl side chain of the ligands’ d-Ala residue, are predicted to be the most promising to enhance binding for both ligands. The previously reported amine backbone modification as in 5 is shown to complement the hydrophobic modifications for binding monoacetylated tripeptides. In addition, replacement of the hydroxyl groups in residues 5 and 7 by fluorine is computed to have negligible impact on binding the tripeptides, though it may be pharmacologically advantageous.

To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with d-Ala-d-Ala- and d-Ala-d-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH2NH2+ (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the d-Ala-d-Lac sequence and acceptable affinity for the d-Ala-d-Ala sequence.

An efficient molecular simulation methodology has been developed to determine the positioning of water molecules in the binding site of a protein or protein−ligand complex. Occupancies and absolute binding free energies of water molecules are computed using a statistical thermodynamics approach. The methodology, referred to as Just Add Water Molecules (JAWS), features “θ-water” molecules that can appear and disappear on a binding-site grid. Key approximations render the technique far more efficient than conventional free energy simulations. Testing of JAWS on five diverse examples (neuraminidase, scytalone dehydratase, major urinary protein 1, β-lactoglobulin, and COX-2) demonstrates its accuracy in locating hydration sites in comparison to results from high-resolution crystal structures. Possible applications include aid in refinement of protein crystal structures, drug lead optimization, setup of docking calculations, and simulations of protein−ligand complexes.

Following computational analyses, potential non-nucleoside inhibitors of HIV-1 reverse transcriptase have been pursued through synthesis and assaying for anti-viral activity. The general class Het–NH–Ph–U has been considered, where Het is an aromatic heterocycle and U is an unsaturated, hydrophobic group. Results for compounds with Het = 2-thiazoyl and 2-pyrimidinyl are the focus of this report.

Design principles are delineated for non-nucleoside inhibitors for HIV-1 reverse transcriptase (NNRTIs). Simultaneous optimization of binding affinity for wild-type RT, tolerance for viral mutations, and physical properties is pursued. Automated lead generation with the growing program BOMB, Monte Carlo simulations with free-energy perturbation theory for lead optimization, and property analysis with QikProp are featured. An initial 30 μM lead has been optimized rapidly to the 10 nM level.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase are being pursued through synthesis and assaying for anti-viral activity. Following computational analyses, the focus has been on the motif Het–NH–Ph–U, where Het is an aromatic heterocycle and U is an unsaturated, hydrophobic group. Previous investigations with Het = 2-thiazoyl and 2-pyrimidinyl are extended here to triazinyl derivatives. The result is several NNRTIs in the 2–20 nM range with negligible cytotoxicity and auspicious predicted pharmacological properties.Synthesis, assaying, and computational results are reported for new anti-HIV agents that exhibit high potency, low cytotoxicity, and promising pharmacological properties.

An overview is provided on the development and status of potential energy functions that are used in atomic-level statistical mechanics and molecular dynamics simulations of water and of organic and biomolecular systems. Some topics that are considered are the form of force fields, their parameterization and performance, simulations of organic liquids, computation of free energies of hydration, universal extension for organic molecules, and choice of atomic charges. The discussion of water models covers some history, performance issues, and special topics such as nuclear quantum effects.

Partial molar volume is an important thermodynamic property that gives insights into molecular size and intermolecular interactions in solution. Theoretical frameworks for determining the partial molar volume (V°) of a solvated molecule generally apply Scaled Particle Theory or Kirkwood–Buff theory. With the current abilities to perform long molecular dynamics and Monte Carlo simulations, more direct methods are gaining popularity, such as computing V° directly as the difference in computed volume from two simulations, one with a solute present and another without. Thermodynamically, V° can also be determined as the pressure derivative of the free energy of solvation in the limit of infinite dilution. Both approaches are considered herein with the use of free energy perturbation (FEP) calculations to compute the necessary free energies of solvation at elevated pressures. Absolute and relative partial molar volumes are computed for benzene and benzene derivatives using the OPLS-AA force field. The mean unsigned error for all molecules is 2.8 cm3 mol−1. The present methodology should find use in many contexts such as the development and testing of force fields for use in computer simulations of organic and biomolecular systems, as a complement to related experimental studies, and to develop a deeper understanding of solute–solvent interactions.