Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on guinea-pig tracheal smooth muscle bradykinin receptors

It is speculated that bradykinin may play an important role in asthma. Thus, bradykinin receptor antagonists may have therapeutic potential against asthma. Orally active bradykinin antagonists would be more desirable for the treatment of the disease. In the present study, we examined the effects of a novel, potent, selective, and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2 ,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamin ocarbonyl)cinnamylamide hydrochloride), on guinea-pig tracheal smooth muscle bradykinin receptors. FR167344 inhibited [3H]bradykinin binding to bradykinin receptors in epithelium-denuded guinea-pig tracheal membrane with an IC50 of 2.1 nM and a Ki of 0.44 nM. This compound also inhibited bradykinin-induced contraction of epithelium-denuded guinea-pig trachea with a pK(B) of 10.8, but had no effect on carbachol-induced contraction of the trachea even at 10(-6) M. These results indicate that FR167344 has the specific antagonistic activity against guinea-pig tracheal smooth muscle bradykinin receptors.     

In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

3D-QSAR studies of heterocyclic quinones with inhibitory activity on vascular smooth muscle cell proliferation using pharmacophore-based alignment

The abnormal proliferation and migration of vascular smooth muscle cells (SMCs) play an important role in the pathology of coronary artery atherosclerosis and restenosis following angioplasty. It was reported that some heterocyclic quinone derivatives such as 6-arylamino-quinoxaline-5,8-diones and 6-arylamino-1H-benzo[d]imidazole-4,7-diones have inhibitory activity on rat aortic smooth muscle cell (RAoSMC) proliferation. To understand the structural basis for antiproliferative activity to design more potent agents, we generated pharmacophore models of representative molecules with high activity using Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database (GALAHAD) and aligned a series of compounds to the selected pharmacophore model, then performed three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). Good cross-validated correlations were obtained with CoMFA (resulting in q(2) of 0.734 and r(2) of 0.947) and CoMSIA (resulting in q(2) of 0.736 and r(2) of 0.913). The IC(50) values of the heterocyclic quinone derivatives on RAoSMC exhibited a strong correlation with steric and hydrophobic fields of the 3D structure of the molecules, resulting in the reliable prediction of inhibitory activity of the series of compounds.     

Molecular modeling, design, synthesis, and biological evaluation of novel 3',4'-dicamphanoyl-(+)-cis-khellactone (DCK) analogs as potent anti-HIV agents

Our current studies aimed at developing new potential anti-AIDS drug candidates have focused on the design and synthesis of new DCK analogs with improved molecular water solubility. Based on the structures and biodata of previous DCK analogs, 3D-QSAR studies have been performed which resulted in two reliable computational models, CoMFA and CoMSIA, with r(2) values of 0.995 and 0.987, and q(2) values of 0.662 and 0.657, respectively. In accord with these 3D-QSAR models, 15 new DCK analogs with polar functional groups at the 3-position were subsequently designed, synthesized, and evaluated against HIV-1 replication in H9 and MT4 cell lines. New DCK analogs 3b, 3c, 4b, 4c, 6a, 7c, and 9a showed promising potency with EC(50) values ranging from 0.09 to 0.0002 microM in both assays. Meanwhile, these promising compounds also showed a wide range of predicted logP values from 0.90 to 5.19, which increased the probability of identifying anti-HIV drug candidates from this class of compounds for clinical trials. Furthermore, both experimental and predicted values matched well, corroborating the reliability of the established 3D-QSAR models.     

Validation of a histamine H3 receptor model through structure-activity relationships for classical H3 antagonists

Histamine H(3) receptor is a G protein-coupled receptor whose activation inhibits the synthesis and release of histamine and other neurotransmitters from nerve endings and is involved in the modulation of different central nervous system functions. H(3) antagonists have been proposed for their potential usefulness in diseases characterized by impaired neurotransmission and they have demonstrated beneficial effects on learning and food intake in animal models. In the present work, a 3D model of the rat histamine H(3) receptor, built by comparative modeling from the crystallographic coordinates of bovine rhodopsin, is presented with the discussion of its ability to predict the potency of known and new H(3) antagonists. A putative binding site for classical, imidazole-derived H(3) antagonists was identified by molecular docking. Comparison with a known pharmacophore model and the binding affinity of a new rigid H(3) antagonist (compound 1, pK(i)=8.02) allowed the characterization of a binding scheme which could also account for the different affinities observed in a recently reported series of potent H(3) antagonists, characterized by a 2-aminobenzimidazole moiety. Molecular dynamics simulations were employed to assess the stability and reliability of the proposed binding mode. Two new conformationally constrained benzimidazole derivatives were prepared and their binding affinity was tested on rat brain membranes; compound 9, designed to reproduce the conformation of a known potent H(3) antagonist, showed higher potency than compound 8, as expected from the binding scheme hypothesized.     

Role of nitric oxide in beta3-adrenoceptor activation on basal tone of internal anal sphincter

Effects of activation of beta3-adrenoceptor (beta3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective beta3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a beta3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l-N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS [1-[2-(trifluoromethylphenyl)imidazole]] had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by beta3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by beta3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.     

Perchlorate binding to cytochrome c: a magnetic and optical study

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy. 2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4(-) concentration below 0.1 M. 3. Competition experiments between perchlorate and chloride show that ClO4(-) binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4(-) in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice. 4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high-spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form. The effects of perchlorate on cytochrome c are due to specific binding of this ion.     

Ion binding to cytochrome c studied by nuclear magnetic quadrupole relaxation.

The enhancement of the 35Cl- transverse relaxation rate on binding of chloride ions to oxidized and reduced cytochrome c has been studied under conditions of variable sodium chloride concentration, temperature, pH, sodium phosphate, iron hexacyanide, and sodium cyanide concentration. The results revealed the presence of a strong binding site(s) for chloride in both oxidized and reduced cyt c, with a higher affinity in ferrocytochrome c. Competition experiments suggest that these sites also bind iron hexacyanide and phosphate. Cyanide binding to the iron in ferricytochrome c at alkaline and neutral pH was shown to decrease the binding of chloride. The pH dependence of the 35Cl- relaxation rate has been fitted by using literature pK values for ionizable groups. No indications of Na+ binding to oxidized and reduced cytochrome c have been observed by using 23Na+ NMR. Our results suggest that chloride is bound near the exposed heme edge and that the surface structure or dynamics in this region are different in the two oxidation states.     

Oxidation of phenethylamine derivatives by cytochrome P450 2D6: the issue of substrate protonation in binding and catalysis.

Cytochrome P450 (P450) 2D6 oxidizes a wide variety of drugs typically at a distance of 5-7 A from a basic nitrogen on the substrate. To investigate the determinants of P450 2D6 catalysis, we analyzed the binding and oxidation of phenethylamine substrates. P450 2D6 discriminated between the various phenethylamines, as evidenced by binding and steady-state results. Whereas the spectral binding affinity for 3-methoxyphenethylamine and 4-methoxyphenethylamine was similar, the affinity for 4-hydroxyphenethylamine was 12-fold weaker than for 3-hydroxyphenethylamine at pH 7.4. The binding of 3,4-dihydroxyphenethylamine was equally poor. These equilibrium dissociation constants were based on the observation of both type I and type II perturbation difference spectra; the former involves displacement of the proximal H(2)O ligand, yielding an iron spin state change, and the latter requires nitrogen ligation to the heme iron. One explanation for the observed type II binding spectra is the presence of both protonated and unprotonated forms of these compounds. To address this possibility, the K(S) values for 3-methoxyphenethylamine and 4-methoxyphenethylamine were determined as a function of pH. Two apparent pK(a) values were determined, which corresponded to a P450 2D6 residue involved in binding and to a lowered pK(a) of a substrate amine group upon binding P450 2D6. The apparent pK(a) of the enzyme residue (6.6) is much higher than the expected pK(a) of Asp301, which has been hypothesized to play a role in binding. Interestingly, the apparent pK(a) for the methoxyphenethylamine derivatives decreased by as much as 2 pH units upon binding to P450 2D6. 3-Methoxyphenethylamine and 4-methoxyphenethylamine underwent sequential oxidations with O-demethylation and subsequent ring hydroxylation to form 3,4-dihydroxyphenethylamine (dopamine). At higher substrate concentrations, the second oxidation was inhibited. This result can be explained by the increasing concentration of the inhibitory unprotonated substrate. Nevertheless, the rates of methoxyphenethylamine oxidations are the highest reported for P450 2D6 substrates.     

Curves of ligand binding. The use of hyperbolic functions for expressing titration curves

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/ radical2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK(1) and pK(2) for the first and second molecules of ligand are called pK*+/-logm, so that K*(2)=K(1)K(2) and m(2)=K(1)/K(2), then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*+/-log(1/2)[m+ radical(m(2)-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*+/-log(1/2)[m+ radical(m(2)-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m>/=2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.     

Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid

Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.     

3D-QSAR studies on fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors by CoMFA and CoMSIA

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses using CoMFA and CoMSIA methods were conducted on a series of fluoropyrrolidine amides as dipeptidyl peptidase IV (DP-IV) inhibitors. The selected ligands were docked into the binding site of the 3D model of DP-IV using the GOLD software, and the possible interaction models between DP-IV and the inhibitors were obtained. Based on the binding conformations of these fluoropyrrolidine amides and their alignment inside the binding pocket of DP-IV, predictive 3D-QSAR models were established by CoMFA and CoMSIA analyses, which had conventional r ² and cross-validated coefficient values () up to 0.982 and 0.555 for CoMFA and 0.953 and 0.613 for CoMSIA, respectively. The predictive ability of these models was validated by six compounds that were in the testing set. Structure-based investigations and the final 3D-QSAR results provide the guide for designing new potent inhibitors.     

Homology modeling of the human 5-HT1A, 5-HT2A, D1, and D2 receptors: model refinement with molecular dynamics simulations and docking evaluation

5-HT(1A) serotonin and D1 dopamine receptor agonists have been postulated to be able to improve negative and cognitive impairment symptoms of schizophrenia, while partial agonists and antagonists of the D2 and 5-HT(2A) receptors have been reported to be effective in reducing positive symptoms. There is therefore a need for well-defined homology models for the design of more selective antipsychotic agents, since no three-dimensional (3D) crystal structures of these receptors are currently available. In this study, homology models were built based on the high-resolution crystal structure of the β(2)-adrenergic receptor (2RH1) and further refined via molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer system with the GROMOS96 53A6 united atom force field. Docking evaluations with representative agonists and antagonists using AutoDock 4.2 revealed binding modes in agreement with experimentally determined site-directed mutagenesis data and significant correlations between the computed and experimental pK (i) values. The models are also able to distinguish between antipsychotic agents with different selectivities and binding affinities for the four receptors, as well as to differentiate active compounds from decoys. Hence, these human 5-HT(1A), 5-HT(2A), D1 and D2 receptor homology models are capable of predicting the activities of novel ligands, and can be used as 3D templates for antipsychotic drug design and discovery.     

3D-QSAR and molecular docking studies of benzaldehyde thiosemicarbazone, benzaldehyde, benzoic acid, and their derivatives as phenoloxidase inhibitors

Phenoloxidase (PO), also known as tyrosinase, is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. Inhibition of PO may provide a basis for novel environmentally friendly insecticides. In the present study, we determined the inhibitory activities and IC50 values of 57 compounds belonging to the benzaldehyde thiosemicarbazone, benzaldehyde, and benzoic acid families against phenoloxidase from Pieris rapae (Lepidoptera) larvae. In addition, the inhibitory kinetics of 4-butylbenzaldehyde thiosemicarbazone against PO was measured in air-saturated solutions for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA). The results indicated that the compound is a reversible noncompetitive inhibitor. The bioactivity results were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using two molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). After carrying out superimposition using common substructure-based alignment, robust and predictive 3D-QSAR models were obtained from CoMFA (q2=0.926, r2=0.986) and CoMSIA (q2=0.933, r2=0.984) with six optimum components. The 3D-QSAR model built here will provide hints for the design of novel PO inhibitors. The molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX). The best scored candidates were docked flexibly, and the interaction between the representative compound 4-butylbenzaldehyde thiosemicarbazone and the active site was elucidated in detail.     

Assessing the quality of the homology-modeled 3D structures from electrostatic standpoint: test on bacterial nucleoside monophosphate kinase families

In this study, we address the issue of performing meaningful pK(a) calculations using homology modeled three-dimensional (3D) structures and analyze the possibility of using the calculated pK(a) values to detect structural defects in the models. For this purpose, the 3D structure of each member of five large protein families of a bacterial nucleoside monophosphate kinases (NMPK) have been modeled by means of homology-based approach. Further, we performed pK(a) calculations for the each model and for the template X-ray structures. Each bacterial NMPK family used in the study comprised on average 100 members providing a pool of sequences and 3D models large enough for reliable statistical analysis. It was shown that pK(a) values of titratable groups, which are highly conserved within a family, tend to be conserved among the models too. We demonstrated that homology modeled structures with sequence identity larger than 35% and gap percentile smaller than 10% can be used for meaningful pK(a) calculations. In addition, it was found that some highly conserved titratable groups either exhibit large pK(a) fluctuations among the models or have pK(a) values shifted by several pH units with respect to the pK(a) calculated for the X-ray structure. We demonstrated that such case usually indicates structural errors associated with the model. Thus, we argue that pK(a) calculations can be used for assessing the quality of the 3D models by monitoring fluctuations of the pK(a) values for highly conserved titratable residues within large sets of homologous proteins.     

A comparative molecular field and comparative molecular similarity indices analyses (CoMFA and CoMSIA) of N-phenyl-N'-(2-chloroethyl)ureas targeting the colchicine-binding site as anticancer agents

To decipher the mechanism underlying the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas (CEU) to the colchicine-binding site on beta(II)-tubulin and to design new and selective antimitotic drugs, we developed 3D quantitative structure-activity relationships (3D-QSAR) models using CoMFA and CoMSIA analyses. The present study correlates the cell growth inhibition activities of 56 structurally related CEU derivatives to several physicochemical parameters representing steric, electrostatic, and hydrophobic fields. Both CoMFA and CoMSIA models using two different optimum numbers of components (ONC) 10 and 4, respectively, gave good internal predictions and their cross-validated r2 values were between 0.639 and 0.743. These comprehensive CoMFA and CoMSIA models are useful in understanding the structure-activity relationships of CEU. The two models were compared to the X-ray crystal structure of the complex of tubulin-colchicine and analyzed for similarities between the two modes of analysis. These models will inspire the design of new CEU derivatives with enhanced inhibition of tumor cell growth and targeting specificity of beta(II)-tubulin and the cytoskeleton.     

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation.     

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.     

Receptor based 3D-QSAR to identify putative binders of Mycobacterium tuberculosis Enoyl acyl carrier protein reductase

In the current study, the applicability and scope of 3D-QSAR models (CoMFA and CoMSIA) to complement virtual screening using 3D pharmacophore and molecular docking is examined and applied to identify potential hits against Mycobacterium tuberculosis Enoyl acyl carrier protein reductase (MtENR). Initially CoMFA and CoMSIA models were developed using series of structurally related arylamides as MtENR inhibitors. Docking studies were employed to position the inhibitors into MtENR active site to derive receptor based 3D-QSAR models. Both CoMFA and CoMSIA yielded significant cross validated q² values of 0.663 and 0.639 and r² values of 0.989 and 0.963, respectively. The statistically significant models were validated by a test set of eight compounds with predictive r² value of 0.882 and 0.875 for CoMFA and CoMSIA. The contour maps from 3D-QSAR models in combination with docked binding structures help to better interpret the structure activity relationship. Integrated with CoMFA and CoMSIA predictive models structure based (3D-pharmacophore and molecular docking) virtual screening have been employed to explore potential hits against MtENR. A representative set of 20 compounds with high predicted IC₅₀ values were sorted out in the present study.     

Investigations of the myoglobin cavity mutant H93G with unnatural imidazole proximal ligands as a modular peroxide O-O bond cleavage model system

A general inability to elucidate extensive variations in the electronic characteristics of proximal heme iron ligands in heme proteins has hampered efforts to obtain a clear understanding of the role of the proximal heme iron ligand in the activation of oxygen and peroxide. The disadvantage of the frequently applied site-directed mutagenesis technique is that it is limited by the range of natural ligands available within the genetic code. The myoglobin cavity mutant H93G [Barrick, D. (1994) Biochemistry 33, 6546-6554] has its proximal histidine ligand replaced with glycine, a mutation which leaves an open cavity capable of accommodating a variety of unnatural potential proximal ligands. We have carried out investigations of the effect of changing the electron donor characteristics of a variety of substituted imidazole proximal ligands on the rate of formation of myoglobin compound II and identified a correlation between the substituted imidazole N-3 pK(a) (which provides a measure of the electron donor ability of N-3) and the apparent rate of formation of compound II. A similar rate dependence correlation is not observed upon binding of azide. This finding indicates that O-O bond cleavage and not the preceding peroxide binding step is being influenced by the electron donor characteristics of the substituted imidazole ligands. The proximal ligand effects are clearly visible, but their overall magnitude is quite low (1.7-fold increase in the O-O bond cleavage rate per pK(a) unit). This appears to provide support for recent commentaries which concluded that the partial ionization of the proximal histidine ligand in typical heme peroxidases may not be enough of an influence to provide a mechanistically critical push effect [Poulos, T. L. (1996) JBIC, J. Biol. Inorg. Chem. 1, 356-359]. Further attempts were made to define the mechanism of the influence of N-3 pK(a) on O-O bond cleavage by using peracetic acid and cumene hydroperoxide as mechanistic probes. The observation of heme destruction in these reactions indicates that displacement of the proximal imidazole ligands by peracetic acid or cumene hydroperoxide has occurred. A combination mutation (H64D/H93G) was prepared with the objective of observing compound I of H64D/H93G with substituted imidazoles as proximal ligands upon reaction with H(2)O(2). This double mutant was found to simultaneously bind imidazole to both axial positions, an arrangement which prevents a reaction with H(2)O(2).