David M. Ferguson

AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules

We describe the development, current features, and some directions for future development of the AMBER package of computer programs. This package has evolved from a program that was constructed to do Assisted Model Building and Energy Refinement to a group of programs embodying a number of the powerful tools of modern computational chemistry-molecular dynamics and free energy calculations.

Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication

ABSTRACT Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 μM and a compound concentration of ≥300 μM required to reduce 50% cell viability). Besides WN virus, the compound also inhibited other flaviviruses (dengue, yellow fever, and St. Louis encephalitis viruses), an alphavirus (Western equine encephalitis virus), a coronavirus (mouse hepatitis virus), and a rhabdovirus (vesicular stomatitis virus). However, the compound did not suppress an orthomyxovirus (influenza virus) or a retrovirus (human immunodeficiency virus type 1). Mode-of-action analyses in WN virus showed that the compound did not inhibit viral entry or virion assembly but specifically suppressed viral RNA synthesis. To examine the mechanism of inhibition of dengue virus, we developed two replicon systems for dengue type 1 virus: (i) a stable cell line that harbored replicons containing a luciferase reporter and a neomycin phosphotransferase selection marker and (ii) a luciferase-expressing replicon that could differentiate between viral translation and RNA replication. Analyses of the compound in the dengue type 1 virus replicon systems showed that it weakly suppressed viral translation but significantly inhibited viral RNA synthesis. Overall, the results demonstrate that triaryl pyrazoline exerts a broad spectrum of antiflavivirus activity through potent inhibition of viral RNA replication. This novel inhibitor could be developed for potential treatment of flavivirus infection.

Constant temperature simulations using the Langevin equation with velocity Verlet integration

Abstract An algorithm, which reduces to velocity Verlet in the limit of zero friction, is obtained for the generalized Langevin equation. The formulation presented is unique in that the velocities are based on a direct second order Taylor expansion of the inertial forces. The resulting finite difference equation is compared with a previous formulation of Verlet-based Langevin dynamics. The equations are implemented to study the physical properties of dense neon and liquid water at constant temperatures as a function of the friction rate γ . The results show canonical ensembles can be obtained at moderate friction rates without recurring to switching functions to control energy losses, or overdamping. The LD methodology is further applied to a protein in solution and compared to MD simulations which use a more conventional scaling function for temperature control. Analyses of the MD trajectories show the protein remains at either lower or higher temperature compared to the solvent, depending upon the treatment of the potential function at the systems boundary. On the contrary, LD simulations show a rapid and even equilibration between protein and solvent in all cases.

Parameterization and evaluation of a flexible water model

The thermodynamic, dielectric, and dynamic properties of a newly parameterized flexible water model are studied using molecular dynamics simulations. The potential function developed is based on the popular simple point charge (SPC) rigid model with the addition of appropriate harmonic and anharmonic energy terms for stretching and bending. Care was taken to account for the self‐polarization and gas‐phase monomer energy corrections during the parameterization, which have typically been ignored in past studies. The results indicate that an increased Lennard‐Jones repulsive coefficient and slightly scaled partial charges are required when adding flexibility to the rigid model potential to reliably reproduce the experimental density, energy, and O ⃛ O radial distribution function of water at 298 K and 1 atm. Analysis of the power spectrum derived from the H‐velocity autocorrelation function allowed the water potential to be evaluated further and refined by adjusting the valence forces to fit the vibrational frequencies of the gas and liquid. Once a consistent set of parameters was determined, the static dielectric properties of the water model were calculated at two temperatures using the reaction field method to treat long‐range forces and correlations. The dielectric constant of 75 ± 7 calculated at 300 K is in good agreement with the experimental value of 78.5. The Kirkwood g factor was also examined for temperature dependence and showed the correct increasing behavior with decreasing T. As a final check of the water potential, the free energies of solvation of a flexible water molecule and neon were predicted using thermodynamic perturbation methods. The calculated solvation energies of −7.0 ± 0.8 for water and 2.7 ± 0.7 for neon are both consistent with the experimental values of −6.3 and 2.7 kcal/mol. Comparisons are made throughout the study with the results of previous rigid and flexible model simulations.

High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

ABSTRACT Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.

Molecular Recognition of Opioid Receptor Ligands

The cloning of the opioid receptors and subsequent use of recombinant DNA technology have led to many new insights into ligand binding. Instead of focusing on the structural features that lead to increased affinity and selectivity, researchers are now able to focus on why these features are important. Site-directed mutagenesis and chimeric data have often been at the forefront in answering these questions. Herein, we survey pharmacophores of several opioid ligands in an effort to understand the structural requirements for ligand binding and selectivity. Models are presented and compared to illustrate key sites of recognition for both opiate and nonopiate ligands. The results indicate that different ligand classes may recognize different sites within the receptor, suggesting that multiple epitopes may exist for ligand binding and selectivity.

On the role of extracellular loops of opioid receptors in conferring ligand selectivity

Based on an analysis of results taken from site‐directed mutagenesis studies performed on opioid receptors, a role for the extracellular loops in conferring opioid subtype selectivity is proposed. It is suggested that the extracellular loop regions (which represent the region of highest sequence variability among opioid subtypes) interact with opioid ligands in a primarily non‐specific fashion. Although these interactions are non‐specific, they appear to play a discriminatory role in ligand binding and, in certain cases, prevent particular ligands from binding among receptor subtypes. We propose that selectivity may be imparted through a mechanism of exclusion, rather than specific pharmacophore recognition within the extracellular loops and N‐terminal domain. This hypothesis is supported by a careful analysis of the binding profiles of several selective and non‐selective ligands to a variety of chimeric mutants. These results, when combined with results taken from single‐point mutation experiments point to the existence of a high affinity binding pocket within the transmembrane region which may be common among the opioid subtypes.

Stereochemical Requirements for Receptor Recognition of the μ-Opioid Peptide Endomorphin-1

A series of diastereoisomers of endomorphin-1 (EM1, Tyr(1)-Pro(2)-Trp(3)-Phe(4)-NH(2)) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe(4)]EM1 possessed 1/10 the potency of EM1, while potencies of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 were 50- and 100-fold lower, respectively. Drastic loss of activity occurred in the [D-Pro(2)]EM1 peptide. The structural determinants for the inactivity and reduced potency of the diastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro(2)]EM1 using NOE-derived distance constraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D-Pro(2)]EM1 was explained by structural comparison with EM1 (, FEBS Lett. 439:13-20). The two peptides showed an opposite orientation of the Trp(3) residue with respect to Tyr(1), thus suggesting a role of Pro(2) as a stereochemical spacer in orienting Trp(3) and Phe(4) toward regions suitable for mu-receptor interaction. The agonist activity of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 was attributed to their ability to adopt low-energy conformations that mimic those of EM1. The requirements for mu-receptor activation were examined further by comparing EM1 with the mu-peptide [D-Ala(2), MePhe(4), Gly-ol]-enkephalin (DAMGO). Conformations of DAMGO with a Tyr(1)-MePhe(4) phenyl ring separation of approximately 12 A were found to mimic Tyr(1)-Phe(4) of EM1, thus suggesting overlapping binding modes between these two peptides.

Application of the message-address concept to the docking of naltrexone and selective naltrexone-derived opioid antagonists into opioid receptor models

A binding site model for the opioid family of G-protein coupled receptors (GPCRs) is proposed based on the message-address concept of ligand recognition. Using ligand docking studies of the universal opioid antagonist, naltrexone, the structural basis for ‘message’ recognition is explored across all three receptor types, μ, δ, and κ. The binding mode proposed and basis for selectivity are also rationalized using the naltrexone-derived ligands, naltrindole (NTI) and norbinaltorphimine (nor BNI). These ligands are docked to the receptor according to the common naltrexone core or message. The resulting orientation places key ‘address’ elements in close proximity to amino acid residues critical to selectivity among receptor types. Selectivity is explained by sequence differences in the μ, δ, and κ receptors at these recognition points. Support for the model is derived from site directed mutagenesis studies and ligand binding data for the opioid receptors and other related GPCRs.

Conformational analysis of the endogenous μ‐opioid agonist endomorphin‐1 using NMR spectroscopy and molecular modeling

Endomorphin‐1 (Tyr‐Pro‐Trp‐Phe‐NH2) is a highly selective and potent agonist of the μ‐opioid receptor. To identify structural attributes unique to this opioid peptide and potential sites of recognition, a conformational analysis has been performed using multidimensional NMR and molecular modeling techniques. The spectroscopic results, derived from experiments in both DMSO and water, indicate that endomorphin‐1 exists in the cis‐ and trans‐configuration with respect to the Pro‐omega bond in approximately 25% and 75% populations, respectively. In DMSO, the cis‐configuration adopts a compact sandwich conformation in which the Tyr and Trp aromatic rings pack against the proline ring, whereas the trans‐configuration adopts an extended conformation. Although non‐random structure was not observed in water, condensed phase molecular dynamics calculations indicate that trans‐isomers dominate the population in this higher dielectric medium. Structural comparison of the cis‐ and trans‐configurations with morphine and selective μ‐peptide ligands PL‐017 and d‐TIPP, as well as the δ‐selective peptide ligands TIPP (δ‐antagonist, μ‐agonist) and DPDPE were also performed and suggest the trans‐isomer is likely the bioactive form. A hypothesis is proposed to explain μ‐ and δ‐selectivity based on the presence of spatially distinct selectivity pockets among these ligands.