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1.  Inclusion of multiple fragment types in the Site Identification by Ligand Competitive Saturation (SILCS) approach 
The Site Identification by Ligand Competitive Saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing Molecular Dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors, and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple, diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced Ligand Grid Free Energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show some, but not all experimental trends are predicted, and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed.
doi:10.1021/ci4005628
PMCID: PMC3947602  PMID: 24245913
2.  A Comparative Study of Transferable Aspherical Pseudoatom Databank and Classical Force Fields for Predicting Electrostatic Interactions in Molecular Dimers 
Accurate and fast evaluation of electrostatic interactions in molecular systems is one of the most challenging tasks in the rapidly advancing field of macromolecular chemistry and drug design. Electrostatic interactions are of crucial importance in biological systems. They are well represented by quantum mechanical methods; however, such calculations are computationally expensive. In this study, we have evaluated the University of Buffalo Pseudoatom Databank (UBDB)1,2 approach for approximation of electrostatic properties of macromolecules and their complexes. We selected the S663 and JSCH-20054 data sets (208 molecular complexes in total) for this study. These complexes represent a wide range of chemical and biological systems for which hydrogen bonding, electrostatic, and van der Waals interactions play important roles. Reference electrostatic energies were obtained directly from wave functions at the B3LYP/aug-cc-pVTZ level of theory using the SAPT (Symmetry-Adapted Perturbation Theory) scheme for calculation of electrostatic contributions to total intermolecular interaction energies. Electrostatic energies calculated on the basis of the UBDB were compared with corresponding reference results. Results were also compared with energies computed using a point charge model from popular force fields (AM1-BCC and RESP used in AMBER and CGenFF from CHARMM family). The energy trends are quite consistent (R2 ≈ 0.98) for the UBDB method as compared to the AMBER5 and CHARMM force field methods6(R2 ≈ 0.93 on average). The RSMEs do not exceed 3.2 kcal mol–1 for the UBDB and are in the range of 3.7–7.6 kcal mol–1 for the point charge models. We also investigated the discrepancies in electrostatic potentials and magnitudes of dipole moments among the tested methods. This study shows that estimation of electrostatic interaction energies using the UBDB databank is accurate and reasonably fast when compared to other known methods, which opens potential new applications to macromolecules.
doi:10.1021/ct4011129
PMCID: PMC3985931  PMID: 24803869
3.  Conformational Properties of α- or β-(1→6)-Linked Oligosaccharides: Hamiltonian Replica Exchange MD Simulations and NMR Experiments 
The Journal of Physical Chemistry. B  2014;118(11):2851-2871.
Conformational sampling for a set of 10 α- or β-(1→6)-linked oligosaccharides has been studied using explicit solvent Hamiltonian replica exchange (HREX) simulations and NMR spectroscopy techniques. Validation of the force field and simulation methodology is done by comparing calculated transglycosidic J coupling constants and proton–proton distances with the corresponding NMR data. Initial calculations showed poor agreement, for example, with >3 Hz deviation of the calculated 3J(H5,H6R) values from the experimental data, prompting optimization of the ω torsion angle parameters associated with (1→6)-linkages. The resulting force field is in overall good agreement (i.e., within ∼0.5 Hz deviation) from experimental 3J(H5,H6R) values, although some small limitations are evident. Detailed hydrogen bonding analysis indicates that most of the compounds lack direct intramolecular H-bonds between the two monosaccharides; however, minor sampling of the O6···HO2′ hydrogen bond is present in three compounds. The results verify the role of the gauche effect between O5 and O6 atoms in gluco- and manno-configured pyranosides causing the ω torsion angle to sample an equilibrium between the gt and gg rotamers. Conversely, galacto-configured pyranosides sample a population distribution in equilibrium between gt and tg rotamers, while the gg rotamer populations are minor. Water radial distribution functions suggest decreased accessibility to the O6 atom in the (1→6)-linkage as compared to the O6′ atom in the nonreducing sugar. The role of bridging water molecules between two sugar moieties on the distributions of ω torsion angles in oligosaccharides is also explored.
doi:10.1021/jp412051v
PMCID: PMC3979472  PMID: 24552401
4.  Polarizable Empirical Force Field for Hexopyranose Monosaccharides Based on the Classical Drude Oscillator 
A polarizable empirical force field based on the classical Drude oscillator is presented for the hexopyranose form of selected monosaccharides. Parameter optimization targeted quantum mechanical (QM) dipole moments, solute–water interaction energies, vibrational frequencies, and conformational energies. Validation of the model was based on experimental data on crystals, densities of aqueous-sugar solutions, diffusion constants of glucose, and rotational preferences of the exocylic hydroxymethyl of d-glucose and d-galactose in aqueous solution as well as additional QM data. Notably, the final model involves a single electrostatic model for all sixteen diastereomers of the monosaccharides, indicating the transferability of the polarizable model. The presented parameters are anticipated to lay the foundation for a comprehensive polarizable force field for saccharides that will be compatible with the polarizable Drude parameters for lipids and proteins, allowing for simulations of glycolipids and glycoproteins.
doi:10.1021/jp412696m
PMCID: PMC4143499  PMID: 24564643
5.  Boc-protected 1-(3-oxocycloalkyl)ureas via a one-step Curtius rearrangement: mechanism and scope 
Tetrahedron letters  2013;55(4):842-844.
1-(3-Oxocyclobutyl) carboxylic acid (4a) was converted into N-Boc-protected 1-(3-oxocyclobutyl) urea (5a), a key intermediates for the preparation of agonists of metabotropic glutamate receptor 5, in one-step when treated with diphenyl phosphoryl azide and triethylamine in tert-butanol. The mechanism of the reaction involves a nucleophilic addition of the in situ generated tert-butyl carbamate to the isocyanate intermediate. This reaction is applicable to other 1-(3-oxocycloalkyl) carboxylic acids but not to linear γ-keto carboxylic acids.
doi:10.1016/j.tetlet.2013.12.021
PMCID: PMC3928826  PMID: 24563555
1-(3-Oxo)ureas; Curtius rearrangement; Carbamoylcarbamate; γ-Keto carboxylic acid; 1-(3-Oxocyclobutyl) carboxylic acid
6.  Molecular Mechanics 
Current pharmaceutical design  2014;20(20):3281-3292.
Molecular Mechanics (MM) force fields are the methods of choice for protein simulations, which are essential in the study of conformational flexibility. Given the importance of protein flexibility in drug binding, MM is involved in most if not all Computational Structure-Based Drug Discovery (CSBDD) projects. This section introduces the reader to the fundamentals of MM, with a special emphasis on how the target data used in the parametrization of force fields determine their strengths and weaknesses. Variations and recent developments such as polarizable force fields are discussed. The section ends with a brief overview of common force fields in CSBDD.
PMCID: PMC4026342  PMID: 23947650
Molecular Mechanics; Force Fields; Structure-Based Drug Design
7.  Protonation of Trimethylamine N-Oxide (TMAO) is required for stabilization of RNA tertiary structure 
Biophysical chemistry  2013;184:10.1016/j.bpc.2013.08.002.
The osmolyte trimethylamine N-oxide (TMAO) stabilizes the tertiary but not the secondary structures of RNA. However, molecular dynamics simulations performed on the PreQl riboswitch showed that TMAO destabilizes the tertiary riboswitch structure, leading us to hypothesize that the presence of RNA could result in enhanced population of the protonated form, TMAOP. Constant pH replica exchange simulations showed that a percentage of TMAO is indeed protonated, thus contributing to the stability of the tertiary but not the secondary structure of PreQl. TMAOP results in an unfavorable dehydration of phosphodiester backbone, which is compensated by electrostatic attraction between TMAOP and the phosphate groups. In addition, TMAOP interacts with specific sites in the tertiary RNA structure, mimicking the behavior of positively charged ions and of the PreQl ligand in stabilizing RNA. Finally, we predict that TMAO-induced stabilization of RNA tertiary structures should be strongly pH dependent.
doi:10.1016/j.bpc.2013.08.002
PMCID: PMC3842377  PMID: 24012912
PreQ1 riboswitch; RNA folding; pKa; RNA tertiary structure; constant pH simulations; molecular dynamics simulations; CHARMM
8.  Force Field for Peptides and Proteins based on the Classical Drude Oscillator 
Presented is a polarizable force field based on a classical Drude oscillator framework, currently implemented in the programs CHARMM and NAMD, for modeling and molecular dynamics (MD) simulation studies of peptides and proteins. Building upon parameters for model compounds representative of the functional groups in proteins, the development of the force field focused on the optimization of the parameters for the polypeptide backbone and the connectivity between the backbone and side chains. Optimization of the backbone electrostatic parameters targeted quantum mechanical conformational energies, interactions with water, molecular dipole moments and polarizabilities and experimental condensed phase data for short polypeptides such as (Ala)5. Additional optimization of the backbone φ, ψ conformational preferences included adjustments of the tabulated two-dimensional spline function through the CMAP term. Validation of the model included simulations of a collection of peptides and proteins. This 1st generation polarizable model is shown to maintain the folded state of the studied systems on the 100 ns timescale in explicit solvent MD simulations. The Drude model typically yields larger RMS differences as compared to the additive CHARMM36 force field (C36) and shows additional flexibility as compared to the additive model. Comparison with NMR chemical shift data shows a small degradation of the polarizable model with respect to the additive, though the level of agreement may be considered satisfactory, while for residues shown to have significantly underestimated S2 order parameters in the additive model, improvements are calculated with the polarizable model. Analysis of dipole moments associated with the peptide backbone and tryptophan side chains show the Drude model to have significantly larger values than those present in C36, with the dipole moments of the peptide backbone enhanced to a greater extent in sheets versus helices and the dipoles of individual moieties observed to undergo significant variations during the MD simulations. Although there are still some limitations, the presented model, termed Drude-2013, is anticipated to yield a molecular picture of peptide and protein structure and function that will be of increased physical validity and internal consistency in a computationally accessible fashion.
doi:10.1021/ct400781b
PMCID: PMC3896220  PMID: 24459460
9.  Desmethyl Macrolides: Synthesis and Evaluation of 4,8,10-Tridesmethyl Cethromycin 
ACS medicinal chemistry letters  2013;4(11):1114-1118.
Antibiotic-resistant bacteria are emerging at an alarming rate in both hospital and community settings. Motivated by this issue, we have prepared desmethyl (i.e., replacing methyl groups with hydrogens) analogues of third-generation macrolide drugs telithromycin (TEL, 2) and cethromycin (CET, 6), both of which are semi-synthetic derivatives of flagship macrolide antibiotic erythromycin (1). Herein, we report the total synthesis, molecular modeling, and biological evaluation of 4,8,10-tridesmethyl cethromycin (7). In MIC assays, CET analogue 7 was found to be equipotent with TEL (2) against a wild-type E. coli strain, more potent than previously disclosed desmethyl TEL congeners 3, 4, and 5, but fourfold less potent than TEL (2) against a mutant E. coli A2058G strain.
doi:10.1021/ml400337t
PMCID: PMC3901159  PMID: 24470840
total synthesis; ketolide antibiotics; antibiotic resistance; cethromycin; telithromycin; molecular modeling; desmethyl analogues
10.  Polarizable Empirical Force Field for Acyclic Poly-Alcohols Based on the Classical Drude Oscillator 
Biopolymers  2013;99(10):724-738.
A polarizable empirical force field for acyclic polyalcohols based on the classical Drude oscillator is presented. The model is optimized with an emphasis on the transferability of the developed parameters among molecules of different sizes in this series and on the condensed-phase properties validated against experimental data. The importance of the explicit treatment of electronic polarizability in empirical force fields is demonstrated in the cases of this series of molecules with vicinal hydroxyl groups that can form cooperative intra- and intermolecular hydrogen bonds. Compared to the CHARMM additive force field, improved treatment of the electrostatic interactions avoids overestimation of the gas-phase dipole moments, results in significant improvement in the treatment of the conformational energies, and leads to the correct balance of intra- and intermolecular hydrogen bonding of glycerol as evidenced by calculated heat of vaporization being in excellent agreement with experiment. Computed condensed phase data, including crystal lattice parameters and volumes and densities of aqueous solutions are in better agreement with experimental data as compared to the corresponding additive model. Such improvements are anticipated to significantly improve the treatment of polymers in general, including biological macromolecules.
doi:10.1002/bip.22286
PMCID: PMC3902549  PMID: 23703219
CHARMM; ethylene glycol; glycerol; carbohydrate; monosaccharide
11.  CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data 
Journal of computational chemistry  2013;34(25):2135-2145.
Protein structure and dynamics can be characterized on the atomistic level with both nuclear magnetic resonance (NMR) experiments and molecular dynamics (MD) simulations. Here we quantify the ability of the recently presented CHARMM36 (C36) force field (FF) to reproduce various NMR observables using MD simulations. The studied NMR properties include backbone scalar couplings across hydrogen bonds, residual dipolar couplings (RDCs) and relaxation order parameter, as well as scalar couplings, RDCs and order parameters for side chain amino- and methyl- containing groups. It is shown that the C36 force field leads to better correlation with experimental data compared to the CHARMM22/CMAP force field, and suggest using C36 in protein simulations. While both CHARMM FFs contains the same nonbond parameters, our results show how the changes in the internal parameters associated with the peptide backbone via CMAP and the χ1 and χ2 dihedral parameters leads to improved treatment of the analyzed nonbond interactions. This highlights the importance of proper treatment of the internal covalent components in modeling nonbond interactions with molecular mechanics FFs.
doi:10.1002/jcc.23354
PMCID: PMC3800559  PMID: 23832629
12.  Synthesis, Modeling, and Pharmacological Evaluation of UMB 425, a Mixed μ Agonist/δ Antagonist Opioid Analgesic with Reduced Tolerance Liabilities 
ACS Chemical Neuroscience  2013;4(9):1256-1266.
Opioid narcotics are used for the treatment of moderate-to-severe pain and primarily exert their analgesic effects through μ receptors. Although traditional μ agonists can cause undesired side effects, including tolerance, addition of δ antagonists can attenuate said side effects. Herein, we report 4a,9-dihydroxy-7a-(hydroxymethyl)-3-methyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one (UMB 425) a 5,14-bridged morphinan-based orvinol precursor synthesized from thebaine. Although UMB 425 lacks δ-specific motifs, conformationally sampled pharmacophore models for μ and δ receptors predict it to have efficacy similar to morphine at μ receptors and similar to naltrexone at δ receptors, due to the compound sampling conformations in which the hydroxyl moiety interacts with the receptors similar to orvinols. As predicted, UMB 425 exhibits a mixed μ agonist/δ antagonist profile as determined in receptor binding and [35S]GTPγS functional assays in CHO cells. In vivo studies in mice show that UMB 425 displays potent antinociception in the hot plate and tail-flick assays. The antinociceptive effects of UMB 425 are blocked by naloxone, but not by the κ-selective antagonist norbinaltorphimine. During a 6-day tolerance paradigm, UMB 425 maintains significantly greater antinociception compared to morphine. These studies thus indicate that, even in the absence of δ-specific motifs fused to the C-ring, UMB 425 has mixed μ agonist/δ antagonist properties in vitro that translate to reduced tolerance liabilities in vivo.
doi:10.1021/cn4000428
PMCID: PMC3778426  PMID: 23713721
Antinociception; conformationally sampled pharmacophore; delta antagonist; mu agonist; opioid receptor; tolerance
13.  A Comparative Kirkwood-Buff Study of Aqueous Methanol Solutions Modeled by the CHARMM Additive and Drude Polarizable Force Fields 
The journal of physical chemistry. B  2013;117(36):10572-10580.
A comparative study on aqueous methanol solutions modeled by the CHARMM additive and Drude polarizable force fields was carried out by employing Kirkwood-Buff analysis. It was shown that both models reproduced the experimental Kirkwood-Buff integrals and excess coordination numbers adequately well over the entire concentration range. The Drude model showed significant improvement over the additive model in solution densities, partial molar volumes, excess molar volumes, concentration-dependent diffusion constants, and dielectric constants. However, the additive model performed somewhat better than the Drude model in reproducing the activity derivative, excess molar Gibbs energy and excess molar enthalpy of mixing. This is due to the additive achieving a better balance among solute-solute, solute-solvent, and solvent-solvent interactions, indicating the potential for improvements in the Drude polarizable alcohol model.
doi:10.1021/jp4061889
PMCID: PMC3787518  PMID: 23947568
molecular dynamics; electronic polarization; mixed solutions; partial molar volume; radial distribution function
14.  Targeting Protein Tyrosine Phosphatase SHP2 for the Treatment of PTPN11- Associated Malignancies 
Molecular cancer therapeutics  2013;12(9):1738-1748.
Activating mutations in PTPN11 (encoding SHP2), a protein tyrosine phosphatase (PTP) that plays an overall positive role in growth factor and cytokine signaling, are directly associated with the pathogenesis of Noonan syndrome and childhood leukemias. Identification of SHP2 selective inhibitors could lead to the development of new drugs that ultimately serve as treatments for PTPN11-associated diseases. As the catalytic core of SHP2 shares extremely high homology to those of SHP1 and other PTPs that play negative roles in cell signaling, to identify selective inhibitors of SHP2 using computer-aided drug design, we targeted a protein surface pocket that is adjacent to the catalytic site, is predicted to be important for binding to phosphopeptide substrates, and has structural features unique to SHP2. From computationally selected candidate compounds, #220–324 effectively inhibited SHP2 activity with an IC50 of 14 μM. Fluorescence titration experiments confirmed its direct binding to SHP2. This active compound was further verified for its ability to inhibit SHP2-mediated cell signaling and cellular function with minimal off-target effects. Furthermore, mouse myeloid progenitors with the activating mutation (E76K) in PTPN11 and patient leukemic cells with the same mutation were more sensitive to this inhibitor than wild-type cells. This study provides evidence that SHP2 is a “druggable” target for the treatment of PTPN11-associated diseases. Since the small molecule SHP2 inhibitor identified has a simple chemical structure, it represents an ideal lead compound for the development of novel anti-SHP2 drugs.
doi:10.1158/1535-7163.MCT-13-0049-T
PMCID: PMC3769526  PMID: 23825065
PTPN11 (SHP2); Phosphatase; Inhibitor; Drug development; Leukemia
15.  Site-Specific Fragment Identification Guided by Single-Step Free Energy Perturbation Calculations 
The in-silico Site Identification by Ligand Competitive Saturation (SILCS) approach identifies the binding sites of representative chemical entities on the entire protein surface, information that can be applied for computational fragment-based drug design. In this study, we report an efficient computational protocol that uses sampling of the protein-fragment conformational space obtained from the SILCS simulations and performs single step free energy perturbation (SSFEP) calculations to identify site-specific favorable chemical modifications of benzene involving substitutions of ring hydrogens with individual non-hydrogen atoms. The SSFEP method is able to capture the experimental trends in relative hydration free energies of benzene analogues and for two datasets of experimental relative binding free energies of congeneric series of ligands of the proteins α-thrombin and P38 MAP kinase. The approach includes a protocol in which data obtained from SILCS simulations of the proteins is first analyzed to identify favorable benzene binding sites following which an ensemble of benzene-protein conformations for that site is obtained. The SSFEP protocol applied to that ensemble results in good reproduction of experimental free energies of the α-thrombin ligands, but not for P38 MAP kinase ligands. Comparison with results from a P38 full-ligand simulation and analysis of conformations reveals the reason for the poor agreement being the connectivity with the remainder of the ligand, a limitation inherent in fragment-based methods. Since the SSFEP approach can identify favorable benzene modifications as well as identify the most favorable fragment conformations, the obtained information can be of value for fragment linking or structure-based optimization.
doi:10.1021/ct300088r
PMCID: PMC3490637  PMID: 23144598
16.  A hybrid mechanism of action for BCL6 in B-cells defined by formation of functionally distinct complexes at enhancers and promoters 
Cell reports  2013;4(3):10.1016/j.celrep.2013.06.016.
SUMMARY
The BCL6 transcriptional repressor is required for development of germinal center (GC) B-cells and diffuse large B-cell lymphomas (DLBCL). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B-cells is unknown. We find that in B-cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are: i) formation of a unique ternary BCOR-SMRT complex at promoters with each corepressor binding to symmetrical sites on BCL6 homodimers, linked to specific epigenetic chromatin features, and ii) the “toggling” of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 de-acetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B-cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.
doi:10.1016/j.celrep.2013.06.016
PMCID: PMC3854650  PMID: 23911289
17.  A Polarizable Force Field of Dipalmitoylphosphatidylcholine based on the Classical Drude Model for Molecular Dynamics Simulations of Lipids 
The journal of physical chemistry. B  2013;117(31):9142-9160.
A polarizable force field of saturated phosphatidylcholine-containing lipids based on the classical Drude oscillator model is optimized and used in molecular dynamics simulations of bilayer and monolayer membranes. The hierarchical parameterization strategy involves the optimization of parameters for small molecules representative of lipid functional groups, followed by their application in larger model compounds and full lipids. The polar head group is based on molecular ions tetramethyl ammonium and dimethyl phosphate, the esterified glycerol backbone is based on methyl acetate, and the aliphatic lipid hydrocarbon tails are based on linear alkanes. Parameters, optimized to best represent a collection of gas and liquid properties for these compounds, are assembled into a complete model of dipalmitoylphosphatidylcholine (DPPC) lipids that is tested against the experimental properties of bilayer and monolayer membranes. The polarizable model yields average structural properties that are in broad accord with experimental data. The area per lipid of the model is 60 Å2, slightly smaller than the experimental value of 63 Å2. The order parameters from nuclear magnetic resonance deuterium quadrupolar splitting measures, the electron density profile, and the monolayer dipole potential are in reasonable agreement with experimental data, and with the non-polarizable CHARMM C36 lipid force field.
doi:10.1021/jp402860e
PMCID: PMC3799809  PMID: 23841725
DPPC; Bilayer; Monolayer; Drude Force Field
18.  Intrinsic energy landscapes of amino acid side chains 
Amino acid side chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side chain χ1 and χ2 torsions in dipeptides in the alphaR, beta and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ1 and χ2 free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima in the QM surfaces as expected for sp3 (or sp2 for χ2 of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side chain energetics can influence backbone preference identified a strong correlation for residues in the AlphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the AlphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.
doi:10.1021/ci300079j
PMCID: PMC3398815  PMID: 22582825
19.  Molecular Details of the Activation of the μ Opioid Receptor 
The journal of physical chemistry. B  2013;117(26):7907-7917.
Molecular details of μ opioid receptor activations were obtained using molecular dynamics simulations of the receptor in the presence of 3 agonists, 3 antagonists, a partial agonist and on the constitutively active T279K mutant. Agonists have a higher probability of direct interactions of their basic nitrogen (N) with Asp147 as compared to antagonists, indicating that direct ligand-Asp147 interactions modulate activation. Medium size substituents on the basic N of antagonists lead to steric interactions that perturb N-Asp147 interactions, while additional favorable interactions occur with larger basic N substituents, such as in N-phenethylnormorphine, restoring N-Asp147 interactions, leading to agonism. With the orvinols, the increased size of the C19 substituent in buprenorphine over diprenorphine leads increased interactions with residues adjacent to Asp147, partially overcoming the presence of the cyclopropyl N substituent, such that buprenorphine is a partial agonist. Results also indicate different conformational properties of the intracellular regions of the transmembrane helices in agonists versus antagonists.
doi:10.1021/jp404238n
PMCID: PMC3735350  PMID: 23758404
Structure-activity relationship; molecular dynamics; binding orientations; agonists and antagonists
20.  Bifurcated Hydrogen Bonding and Asymmetric Fluctuations in a Carbohydrate Crystal Studied via X-ray Crystallography and Computational analysis 
The journal of physical chemistry. B  2013;117(25):7546-7553.
The structure of the O-methyl glycoside of the naturally occurring 6-O-[(R)-1-carboxyethyl]-α-d-galactopyranose, C10H18O8, has been determined by X-ray crystallography at 100 K, supplementing the previously determined structure obtained at 293 K (Acta Cryst., 1996, C52, 2285-2287). Molecular dynamics simulations of this glycoside were performed in the crystal environment with different numbers of units cells included in the primary simulation system at both 100 K and 293 K. The calculated unit cell parameters and the intra-molecular geometries (bonds, angles, and dihedrals) agree well with experimental results. Atomic fluctuations, including B-factors and anisotropies, are in good agreement with respect to the relative values on an atom-by-atom basis. In addition, the fluctuations increase with increasing simulation system size, with the simulated values converging to values lower than those observed experimentally indicating that the simulation model is not accounting for all possible contributions to the experimentally observed B-factors which may be related to either the simulation time scale or size. In the simulations the hydroxyl group of O7 is found to form bifurcated hydrogen bonds with O6 and O8 of an adjacent molecule, with the interactions dominated by the HO7-O6 interaction. Quantum mechanical calculations support this observation.
doi:10.1021/jp403719g
PMCID: PMC3771504  PMID: 23738792
CHARMM force field; carbohydrates; molecular dynamics simulation; molecular modeling; monosaccharides
21.  Estimation of ligand efficacies of metabotropic glutamate receptors from conformational forces obtained from molecular dynamics simulations 
Group 1 metabotropic glutamate receptors (mGluR) are G-protein coupled receptors with a large bilobate extracellular ligand binding region (LBR) that resembles a Venus fly trap. Closing of this LBR in the presence of a ligand is associated with the activation of the receptor. From conformational sampling of the LBR-ligand complexes using all-atom molecular dynamics (MD) simulations, we characterized the conformational minima related to the hinge like motion associated with the LBR closing/opening in the presence of known agonists and antagonists. By applying a harmonic restraint on the LBR, we also determined the conformational forces generated by the different ligands. The change in the location of the minima and the conformational forces were used to quantify the efficacies of the ligands. This analysis shows that efficacies can be estimated from the forces of a single conformation of the receptor, indicating the potential of MD simulations as an efficient and useful technique to quantify efficacies thereby facilitating the rational design of mGluR agonists and antagonists.
doi:10.1021/ci400160x
PMCID: PMC3693859  PMID: 23688150
metabotropic glutamate receptors; conformational sampling; molecular dynamics; conformational force; efficacy
22.  Structure and thermodynamic insights on acetylaminofluorene-modified deletion DNA duplexes as models for frameshift mutagenesis 
Chemical research in toxicology  2013;26(6):937-951.
2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF–induced SMIs formed during a translesion synthesis. We performed spectroscopic, thermodynamic, and molecular dynamics studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and −1, −2, and −3 deletion duplexes of the 5′-CTCTCGATG[FAAF]CCATCAC-3′ sequence and an additional −1 deletion duplex of the 5′-CTCTCGGCG[FAAF]CCATCAC-3′ NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being ‘GC’ −1 (73%) > ‘AT’ −1 (72%) > full (60%) > −2 (55%) > −3 (37%). Thermodynamic stability was in the order of −1 deletion > −2 deletion > fully paired > −3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs are thermodynamically more stable than the conformationally flexible external B conformer. Results from the molecular dynamics simulations indicate perturbation of base stacking dominate the relative stability along with contributions from bending, duplex dynamics, solvation effects that are important in specific cases. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.
doi:10.1021/tx400116n
PMCID: PMC3733349  PMID: 23688347
23.  Structural Modifications of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione that Improve Selectivity for the Inhibition of Melanoma Cells Containing Active ERK Signaling 
Organic & biomolecular chemistry  2013;11(22):3706-3732.
Towards the development of potent and selective inhibitors of melanoma cells containing active ERK signaling, we herein report on the pharmacophore determination and optimization of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione.
doi:10.1039/c3ob40199e
PMCID: PMC3663054  PMID: 23624850
24.  Conformational determinants of the activity of Antiproliferative factor glycopeptide 
The antiproliferative factor (APF) involved in interstitial cystitis is a glycosylated nonapeptide (TVPAAVVVA) containing a sialylated core α-O-disaccharide linked to the N-terminal threonine. The chemical structure of APF was deduced using spectroscopic techniques and confirmed using total synthesis. The synthetic APF provided a platform to study amino acid modifications and their effect on APF activity, based on which a structure-activity relationship (SAR) for APF activity was previously proposed. However, this SAR model could not explain the change in activity associated with minor alterations in the peptide sequence. Presented is computational analysis of 14 APF derivatives to identify structural trends from which a more detailed SAR is obtained. The APF activity is found to be dictated by the close interplay between carbohydrate-peptide and peptide-peptide interactions. The former involves hydrogen bond and hydrophobic interactions and the latter is dominated by hydrophobic interactions. The highly flexible hydrophobic peptide adopts collapsed conformations separated by low energy barriers. APF activity correlates with hydrophobic clustering associated with amino acids 4A, 6V and 8V. Peptide conformations are highly sensitive to single point mutations, which explain the experimental trends. The presented SAR will act as a guide for lead optimization of more potent APF analogues of potential therapeutic utility.
doi:10.1021/ci400147s
PMCID: PMC3690088  PMID: 23627670
25.  (Ala)4-X-(Ala)4 as a model system for the optimization of the χ1 and χ2 amino acid side-chain dihedral empirical force field parameters 
Amino acid side-chain fluctuations play an essential role in the structure and function of proteins. Accordingly, in theoretical studies of proteins it is important to have an accurate description of their conformational properties. Recently, new side-chain torsion parameters were introduced into the CHARMM and Amber additive force fields and evaluated based on the conformational properties of the individual side-chains using protein simulations in explicit solvent. While effective for validation, MD simulations of proteins must be extended into the microsecond regime to obtain full convergence of the side-chain conformations, limiting their use for force field optimization. To address this, we systematically test the utility of explicit solvent simulations of (Ala)4-X-(Ala)4 peptides, where X represents the amino acids, as model systems for the optimization of χ1 and χ2 side-chain parameters. The effect of (Ala)4-X-(Ala)4 backbone conformation was tested by constraining the backbone in the α-helical, C5, C7eq and PPII conformations and performing exhaustive sampling using Hamiltonian replica exchange simulations. Rotamer distributions from protein and the (Ala)4-X-(Ala)4 simulations showed the highest correlation for the C7eq and PPII conformations, though agreement was best for the α-helical conformation for Asn. Hydrogen bond analysis indicate the utility of the C7eq and PPII conformations to be due to specific side-chain-backbone hydrogen bonds not being oversampled, thereby allowing sampling of a range of side-chain conformations consistent with the distributions occurring in full proteins. It is anticipated that the (Ala)4-X-(Ala)4 model system will allow for iterative force field optimization targeting condensed phase conformational distributions of side-chains.
doi:10.1002/jcc.23178
PMCID: PMC3570682  PMID: 23197420

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