Strain-Dependent Activation and Inhibition of Human Immunodeficiency Virus Entry by a Specific PF-68742 Stereoisomer.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.

Acetylcholine promotes binding of α-conotoxin MII at α3 ß2 nicotinic acetylcholine receptors.

α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3ß2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and ß2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3ß2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs.

Computational evaluations of charge coupling and hydrogen bonding in the active site of a family 7 cellobiohydrolase.

Solution pH and the pKa values of ionizable residues are critical factors known to influence enzyme catalysis, structural stability, and dynamical fluctuations. Presented here is an exhaustive computational study utilizing long time constant pH molecular dynamics, pH replica exchange simulations, and kinetic modeling to evaluate pH-dependent conformations, charge dynamics, residue pKa values, and the catalytic activity-pH profile for cellobiohydrolase Cel7B from Melanocarpus albomyces . The predicted pKa values support the role of Glu212 as the catalytic nucleophile and Glu217 as the acid-base residue. The presence of a charge-correlated active site and an extensive hydrogen bonding network is found to be critical in enabling favorable residue orientations for catalysis and shuttling excess protons around the active site. Clusters of amino acids are identified that act in concert to effectively modulate the optimal pH for catalysis while elevating the overall catalytic rate with respect to a noncoupled system. The work presented here demonstrates the complex and critical role of coupled ionizable residues to the proper functioning of cellobiohydrolase Cel7B, functionally related glycosyl hydrolases, and enzymes in general. The simulations also support the use of the CpHMD for the accurate prediction of residue pKa values and to evaluate the impact of pH on protein structure and charge dynamics.

An ionic liquid dependent mechanism for base catalyzed ß-elimination reactions from QM/MM simulations.

Ionic liquids have been proposed to induce a mechanistic change in the reaction pathway for the fundamentally important base-induced ß-elimination class compared to conventional solvents. The role of the reaction medium in the elimination of 1,1,1-tribromo-2,2-bis(3,4-dimethoxyphenyl)ethane via two bases, piperidine and pyrrolidine, has been computationally investigated using methanol and the ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate and hexafluorophosphate [BMIM][BF(4)] and [BMIM][PF(6)], respectively. QM/MM Monte Carlo simulations utilizing free-energy perturbation theory found the ionic liquids did produce a reaction pathway change from an E1cB-like mechanism in methanol to a pure E2 route that is consistent with experimental observations. The origin of the ionic liquid effect has been found as: (1) a combination of favorable electrostatic interactions, for example, bromine-imidazolium ion, and (2) π-π interactions that enhance the coplanarity between aromatic rings maximizing the electronic effects exerted on the reaction route. Solute-solvent interaction energies have been analyzed and show that liquid clathrate solvation of the transition state is primarily responsible for the observed mechanistic changes. This work provides the first theoretical evidence of an ionic liquid dependent mechanism and elucidates the interplay between sterics and electrostatics crucial to understanding the effect of these unique solvents upon chemical reactions.

Identification of HIV inhibitors guided by free energy perturbation calculations.

Free energy perturbation (FEP) theory coupled to molecular dynamics (MD) or Monte Carlo (MC) statistical mechanics offers a theoretically precise method for determining the free energy differences of related biological inhibitors. Traditionally requiring extensive computational resources and expertise, it is only recently that its impact is being felt in drug discovery. A review of computer-aided anti-HIV efforts employing FEP calculations is provided here that describes early and recent successes in the design of human immunodeficiency virus type 1 (HIV-1) protease and non-nucleoside reverse transcriptase inhibitors. In addition, our ongoing work developing and optimizing leads for small molecule inhibitors of cyclophilin A (CypA) is highlighted as an update on the current capabilities of the field. CypA has been shown to aid HIV-1 replication by catalyzing the cis/trans isomerization of a conserved Gly-Pro motif in the Nterminal domain of HIV-1 capsid (CA) protein. In the absence of a functional CypA, e.g., by the addition of an inhibitor such as cyclosporine A (CsA), HIV-1 has reduced infectivity. Our simulations of acylurea-based and 1-indanylketone-based CypA inhibitors have determined that their nanomolar and micromolar binding affinities, respectively, are tied to their ability to stabilize Arg55 and Asn102. A structurally novel 1-(2,6-dichlorobenzamido) indole core was proposed to maximize these interactions. FEP-guided optimization, experimental synthesis, and biological testing of lead compounds for toxicity and inhibition of wild-type HIV-1 and CA mutants have demonstrated a dose-dependent inhibition of HIV-1 infection in two cell lines. While the inhibition is modest compared to CsA, the results are encouraging.

Optimal scaling factors for CM1 and CM3 atomic charges in RM1-based aqueous simulations.

Scaling factors for atomic charges derived from the RM1 semiempirical quantum mechanical wavefunction in conjunction with CM1 and CM3 charge models have been optimized by minimizing errors in absolute free energies of hydration, ΔG(hyd) , for a set of 40 molecules. Monte Carlo statistical mechanics simulations and free energy perturbation theory were used to annihilate the solutes in gas and in a box of TIP4P water molecules. Lennard-Jones parameters from the optimized potentials for liquid simulations-all atom (OPLS-AA) force field were utilized for the organic compounds. Optimal charge scaling factors have been determined as 1.11 and 1.14 for the CM1R and CM3R methods, respectively, and the corresponding unsigned average errors in ΔG(hyd) relative to experiment were 2.05 and 1.89 kcal/mol. Computed errors in aniline and two derivatives were particularly large for RM1 and their removal from the data set lowered the overall errors to 1.61 and 1.75 kcal/mol for CM1R and CM3R. Comparisons are made to the AM1 method which yielded total errors in ΔG(hyd) of 1.50 and 1.64 kcal/mol for CM1A*1.14 and CM3A*1.15, respectively. This work is motivated by the need for a highly efficient yet accurate quantum mechanical (QM) method to study condensed-phase and enzymatic chemical reactions via mixed QM and molecular mechanical (QM/MM) simulations. As an initial test, the Menshutkin reaction between NH(3) and CH(3) Cl in water was computed using a RM1/TIP4P-Ew/CM3R procedure and the resultant ΔG(‡) , ΔG(rxn) , and geometries were in reasonable accord with other computational methods; however, some potentially serious shortcomings in RM1 are discussed.

Computational insight into small molecule inhibition of cyclophilins.

Cyclophilins (Cyp) are a family of cellular enzymes possessing peptidyl-prolyl isomerase activity, which catalyze the cis-trans interconversion of proline-containing peptide bonds. The two most abundant family members, CypA and CypB, have been identified as valid drug targets for a wide range of diseases, including HCV, HIV, and multiple cancers. However, the development of small molecule inhibitors that possess nM potency and high specificity for a particular Cyp is difficult given the complete conservation of all active site residues between the enzymes. Monte Carlo statistical sampling coupled to free energy perturbation theory (MC/FEP) calculations have been carried out to elucidate the origin of the experimentally observed nM inhibition of CypA by acylurea-based derivatives and the >200-fold in vitro selectivity between CypA and CypB from aryl 1-indanylketone-based µM inhibitors. The computed free-energies of binding were in close accord with those derived from experiments. Binding affinity values for the inhibitors were determined to be dependent upon the stabilization strength of the nonbonded interactions provided toward two catalytic residues: Arg55 and Asn102 in CypA and the analogous Arg63 and Asn110 residues in CypB. Fine-tuning of the hydrophobic interactions allowed for enhanced potency among derivatives. The aryl 1-indanylketones are predicted to differentiate between the cyclophilins by using distinct binding motifs that exploit subtle differences in the active site arrangements. Ideas for the development of new selective compounds with the potential for advancement to low-nanomolar inhibition are presented.

Development of OPLS-AA Force Field Parameters for 68 Unique Ionic Liquids.

OPLS-AA force field parameters have been developed and validated for use in the simulation of 68 unique combinations of room temperature ionic liquids featuring 1-alkyl-3-methylimidazolium [RMIM] (R = Me, Et, Bu, Hex, Oct), N-alkylpyridinium [RPyr], and choline cations, along with Cl(-), PF6(-), BF4(-), NO3(-), AlCl4(-), Al2Cl7(-), TfO(-), saccharinate, and acesulfamate anions. The new parameters were fit to conformational profiles from gas-phase ab initio calculations at the LMP2/cc-pVTZ(-f)//HF/6-31G(d) theory level and compared to experimental condensed-phase structural and thermodynamic data. Monte Carlo simulations of the ionic liquids gave relative deviations from experimental densities of ca. 1-3% at 25 °C for most combinations and also yielded close agreement over a temperature range of 5 to 90 °C. Predicted heats of vaporization compared well with available experimental data and estimates. Transferability of the new parameters to multiple alkyl side-chain lengths for [RMIM] and [RPyr] was determined to give excellent agreement with charges and torsion potentials developed specific to desired alkyl lengths in 35 separate ionic liquid simulations. As further validation of the newly developed parameters, the Kemp elimination reaction of benzisoxazole via piperidine was computed in 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] using mixed quantum and molecular mechanics (QM/MM) simulations and was found to give close agreement with the experimental free energy of activation.