Characterization of the Anopheles gambiae octopamine receptor and discovery of potential agonists and antagonists using a combined computational-experimental approach.

BACKGROUND: Octopamine receptors (OARs) perform key functions in the biological pathways of primarily invertebrates, making this class of G-protein coupled receptors (GPCRs) a potentially good target for insecticides. However, the lack of structural and experimental data for this insect-essential GPCR family has promoted the development of homology models that are good representations of their biological equivalents for in silico screening of small molecules. METHODS: Two Anopheles gambiae OARs were cloned, analysed and functionally characterized using a heterologous cell reporter system. Four antagonist- and four agonist-binding homology models were generated and virtually screened by docking against compounds obtained from the ZINC database. Resulting compounds from the virtual screen were tested experimentally using an in vitro reporter assay and in a mosquito larvicide bioassay. RESULTS: Six An. gambiae OAR/tyramine receptor genes were identified. Phylogenetic analysis revealed that the OAR (AGAP000045) that encodes two open reading frames is an α-adrenergic-like receptor. Both splice variants signal through cAMP and calcium. Mutagenesis analysis revealed that D100 in the TM3 region and S206 and S210 in the TM5 region are important to the activation of the GPCR. Some 2,150 compounds from the virtual screen were structurally analysed and 70 compounds were experimentally tested against AgOAR45B expressed in the GloResponse™CRE-luc2P HEK293 reporter cell line, revealing 21 antagonists, 17 weak antagonists, 2 agonists, and 5 weak agonists. CONCLUSION: Reported here is the functional characterization of two An. gambiae OARs and the discovery of new OAR agonists and antagonists based on virtual screening and molecular dynamics simulations. Four compounds were identified that had activity in a mosquito larva bioassay, three of which are imidazole derivatives. This combined computational and experimental approach is appropriate for the discovery of new and effective insecticides.

Docking applied to the prediction of the affinity of compounds to P-glycoprotein.

P-glycoprotein (P-gp) is involved in the transport of xenobiotic compounds and responsible for the decrease of the drug accumulation in multi-drug-resistant cells. In this investigation we compare several docking algorithms in order to find the conditions that are able to discriminate between P-gp binders and nonbinders. We built a comprehensive dataset of binders and nonbinders based on a careful analysis of the experimental data available in the literature, trying to overcome the discrepancy noticeable in the experimental results. We found that Autodock Vina flexible docking is the best choice for the tested options. The results will be useful to filter virtual screening results in the rational design of new drugs that are not expected to be expelled by P-gp.

Inhibition pattern of sulfamide-related compounds in binding to carbonic anhydrase isoforms I, II, VII, XII and XIV.

A set of sulfamides and sulfamates were synthesized and tested against several isoforms of carbonic anhydrase: CA I, CA II, CA VII, CA XII and CA XIV. The biological assays showed a broad range of inhibitory activity, and interesting results were found for several compounds in terms of activity (Ki <1µm) and selectivity: some aromatic sulfamides are active against CA I, CA II and/or CA VII; while they are less active in CA XII and CA XIV. On the other hand, bulky sulfamides are selective to CA VII. To understand the origin of the different inhibitory activity against each isozyme we used molecular modeling techniques such as docking and molecular dynamic simulations.

3D-QSAR design of novel antiepileptic sulfamides.

A three-dimensional quantitative structure-activity relationship method, the comparative molecular field analysis (CoMFA), was applied to design new anticonvulsant symmetric sulfamides. The training set (27 structures) was comprised by traditional and new-generation anticonvulsant (AC) ligands that exhibit a potent activity in MES test. Physicochemical determinants of binding, such as steric and electrostatic properties, were mapped onto the molecular structures of the set, in order to interpret graphically the CoMFA results in terms of field contribution maps. The 3D-QSAR models demonstrate a good ability to predict the activity of the designed compounds (r(2)=0.967, q(2)=0.756).

Characterization of the anticonvulsant profile of valpromide derivatives.

The antiepileptic activity of nine derivatives of valpromide is discussed. They comply with a pharmacophore model that establishes the essential structural and electronic features responsible for the protection against the MES test. The model results from the comparison of 17 structures, using density functional methodologies combined with an active analog approach. The derivatives of valpromide have been tested for anticonvulsant activity in mice. These compounds displayed a phenytoin-like profile, being active in the MES test and inactive in the PTZ test. 4-(Valproylamido)benzenesulfonamide is the most active compound, with an ED(50) of 53 micromol/kg and no neurotoxicity at doses up to 1000 micromol/kg. The pharmacological behavior of the drugs points to a sodium channel blocking effect as one of the associated mechanisms. This mechanism was tested positive for N-ethylvalpromide through its competition with the binding of [(3)H]batrachotoxin-A-20 alpha-benzoate to the voltage-dependent sodium channels from rat brain synaptosomes.

Theoretical characterization of stable eta1-N2O-, eta2-N2O-, eta1-N2-, and eta2-N2-bound species: intermediates in the addition reactions of nitrogen hydrides with the pentacyanonitrosylferrate(II) ion.

The addition of nitrogen hydrides (hydrazine, hydroxylamine, ammonia, azide) to the pentacyanonitrosylferrate(II) ion has been analyzed by means of density functional calculations, focusing on the identification of stable intermediates along the reaction paths. Initial reversible adduct formation and further decomposition lead to the eta(1)- and eta(2)-linkage isomers of N(2)O and N(2), depending on the nucleophile. The intermediates (adducts and gas-releasing precursors) have been characterized at the B3LYP/6-31G level of theory through the calculation of their structural and spectroscopic properties, modeling the solvent by means of a continuous approach. The eta(2)-N(2)O isomer is formed at an initial stage of adduct decompositions with the hydrazine and azide adducts. Further conversion to the eta(1)-N(2)O isomer is followed by Fe-N(2)O dissociation. Only the eta(1)-N(2)O isomer is predicted for the reaction with hydroxylamine, revealing a kinetically controlled N(2)O formation. eta(1)-N(2) and eta(2)-N(2) isomers are also predicted as stable species.

Kinetic, mechanistic, and DFT study of the electrophilic reactions of nitrosyl complexes with hydroxide.

We present a kinetic study of OH(-) additions to several nitrosyl complexes containing mainly ruthenium and different coligands (polypyridines, amines, pyridines, cyanides). According to a first-order rate law in each reactant, we propose a fast ion pair formation equilibrium, followed by addition of OH(-) to the [MX(5)NO](n) moieties, with formation of the [MX(5)NO(2)H](n(-1)) intermediates. Additional attack by a second OH(-) gives the final products, [MX(5)NO(2)]((n-2)). A linear plot was found for ln k(4) (the addition rate constant) against the redox potential for nitrosyl reduction, E(NO+/-NO), showing a free-energy relationship with a slope close to 20 V(-1), consistent with an associative mechanism. Theoretical DFT calculated descriptors, as the charge density in the [MNO] moieties and the LUMO energies, qualitatively correlate with the rate constants. A linear to bent transformation was calculated for the nitrosyl complexes, as they evolve to the angular MNO(2)H and MNO(2) complexes. The geometries were optimized for the different complexes and adduct-intermediates, showing significant changes in the relevant distances and angles upon OH(-) addition. IR vibrations and electronic transitions were also calculated. The complete reaction profile was studied for the nitroprusside ion, including the description of the transition state structure. Experimental activation parameters revealed that both the activation enthalpies and entropies increase when going from the negatively charged to the positively charged complexes. As the rate constants increase in the same direction, we conclude that the reactions are entropically driven, compensating, this function, the increase in the activation enthalpies. The latter trend can be explained by the energies involved in angular reorganization after OH(-) coordination, which are larger as the positive charge in the nitrosyl moiety becomes larger. The use of E(NO+/-NO) as a predictive tool for electrophilic reactivity could be extended to similar reactions implying other nucleophiles, such as amines and thiolates.

The electrophilic reactions of pentacyanonitrosylferrate(II) with hydrazine and substituted derivatives. Catalytic reduction of nitrite and theoretical prediction of eta(1)-, eta(2)-N(2)O bound intermediates.

The electrophilic reactivity of the pentacyanonitrosylferrate(II) ion, [Fe(CN)(5)NO](2)(-), toward hydrazine (Hz) and substituted hydrazines (MeHz, 1,1-Me(2)Hz, and 1,2-Me(2)Hz) has been studied by means of stoichiometric and kinetic experiments (pH 6-10). The reaction of Hz led to N(2)O and NH(3), with similar paths for MeHz and 1,1-Me(2)Hz, which form the corresponding amines. A parallel path has been found for MeHz, leading to N(2)O, N(2), and MeOH. The reaction of 1,2-Me(2)Hz follows a different route, characterized by azomethane formation (MeNNMe), full reduction of nitrosyl to NH(3), and intermediate detection of [Fe(CN)(5)NO](3)(-). In the above reactions, [Fe(CN)(5)H(2)O](3)(-) was always a product, allowing the system to proceed catalytically for nitrite reduction, an issue relevant in relation to the behavior of the nitrite and nitric oxide reductase enzymes. The mechanism comprises initial reversible adduct formation through the binding of the nucleophile to the N-atom of nitrosyl. The adducts decompose through OH(-) attack giving the final products, without intermediate detection. Rate constants for the adduct-formation steps (k = 0.43 M(-)(1) s(-)(1), 25 degrees C for Hz) decrease with methylation by about an order of magnitude. Among the different systems studied, one-, two-, and multielectron reductions of bound NO(+) are analyzed comparatively, with consideration of the role of NO, HNO (nitroxyl), and hydroxylamine as bound intermediates. A DFT study (B3LYP) of the reaction profile allows one to characterize intermediates in the potential hypersurface. These are the initial adducts, as well as their decomposition products, the eta(1)- and eta(2)-linkage isomers of N(2)O.