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Biomedical subjects

F Gago

Publications and source records attributed to F Gago.

At least 19 recordsLinked to original sources
PubMed1999

Regulation of cyclooxygenase activity by metamizol.

The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxygenase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC50 of about 150 microg/ml. A similar IC50 value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings, molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were markedly different. The IC50 values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells (BAEC) cells and human platelets were 1730 +/- 150 microg/ml and 486 +/- 56 microg/ml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages and primary human leukocytes activated by lipopolysaccharide yielded IC50 values of 12 +/- 1.8 microg/ml and 21 +/- 2.9 microg/ml, respectively. These data indicate that the IC50 values obtained with purified enzymes or disrupted cells cannot always be extrapolated to the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs (NSAIDs) in intact cells. The data presented here also indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol.

Animals↗
PubMed1999

Solution structure determination by two-dimensional 1H NMR of omega-conotoxin MVIID, a calcium channel blocker peptide.

The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.

Calcium Channel Blockers↗
PubMed1999

Molecular model of the interaction between nimesulide and human cyclooxygenase-2.

The cyclooxygenase-2 (COX-2) isoenzyme is a key target for COX-2-selective non-steroidal anti-inflammatory drugs (NSAIDs). An important difference in binding of nimesulide compared with non-selective NSAIDs appears to involve the amino acid at position 523 of the enzyme. Replacement of valine with isoleucine at this position provides access to a binding site that is larger in COX-2 than in COX-1. Nimesulide appears to exploit this enlarged binding site for establishing a number of favourable contacts with the enzyme that lead to selective inhibition of COX-2. We made these conclusions from a three-dimensional molecular model of the active site of human COX-2, constructed using the X-ray coordinates of COX-1 from sheep seminal vesicles and COX-2 from mouse fibroblasts as templates, with the aid of sequence alignment methods and molecular modelling techniques. The resulting model was refined, and the active site was probed for regions of steric and electrostatic complementarity for ligand binding. Docking studies were then undertaken with many different nimesulide conformers, a family of which could establish very favourable interactions with the NSAID binding site of human COX-2 by exploiting the extra space made available by the isoleucine/valine replacement. The stability of the resulting complexes was studied by simulating molecular dynamics.

Cyclooxygenase 1↗
PubMed1999

Recognition elements that determine affinity and sequence-specific binding to DNA of 2QN, a biosynthetic bis-quinoline analogue of echinomycin.

Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

Anti-Bacterial Agents↗
PubMed1998

Abasic analogues of TSAO-T as the first sugar derivatives that specifically inhibit HIV-1 reverse transcriptase.

With the aim of assessing the role that the thymine base of TSAO-T may play in the interaction of TSAO compounds with HIV-1 reverse transcriptase (RT), we have designed, synthesized, and evaluated for their anti-HIV-1 activity a series of 3-spiro sugar derivatives substituted at the anomeric position with nonaromatic rings or with amine, amide, urea, or thiourea moieties that mimic parts or the whole thymine base of TSAO-T. Also, a dihydrouracil TSAO analogue and O-glycosyl 3-spiro sugar derivatives substituted at the anomeric position with methyloxy or benzyloxy groups have been prepared. Compounds substituted at the anomeric position with an azido, amino, or methoxy group, respectively, were devoid of marked antiviral activity (EC50: 10-200 microM). However, the substituted urea sugar derivatives led to an increase in antiviral potency (EC50: 0.35-4 microM), among them those urea derivatives that mimic most closely the intact TSAO-T molecule retained the highest antiviral activity. Also, the dihydrouracil TSAO derivative retained pronounced anti-HIV-1 activity. None of the compounds showed any anti-HIV-2 activity. The results described herein represent the first examples of sugar derivatives that interact in a specific manner with HIV-1 RT. Molecular modeling studies carried out with a prototype urea derivative indicate that a heteroaromatic ring is not an absolute requirement for a favorable interaction between TSAO-T and HIV-1 RT. Urea derivatives, which can mimic to a large extent both the shape and the electrostatic potential of a thymine ring, can effectively replace this nucleic acid base when incorporated into a TSAO molecular framework with only moderate loss of activity.

Animals↗
PubMed1998

Comparative binding energy analysis of HIV-1 protease inhibitors: incorporation of solvent effects and validation as a powerful tool in receptor-based drug design.

A comparative binding energy (COMBINE) analysis (Ortiz et al. J. Med. Chem. 1995, 38, 2681-2691) has been performed on a training set of 33 HIV-1 protease inhibitors, and the resulting regression models have been validated using an additional external set of 16 inhibitors. This data set was originally reported by Holloway et al. (J. Med. Chem. 1995, 38, 305-317), who showed the usefulness of molecular mechanics interaction energies for predicting the activity of novel HIV-1 protease inhibitors within the framework of the MM2X force field and linear regression techniques. We first used the AMBER force field on the same set of three-dimensional structures to check up on any possible force-field dependencies. In agreement with the previous findings, the calculated raw ligand-receptor interaction energies were highly correlated with the inhibitory activities (r2 = 0.81), and the linear regression model relating both magnitudes had an acceptable predictive ability both in internal validation tests (q2 = 0.79, SDEPcv = 0.61) and when applied to the external set of 16 different inhibitors (SDEPex = 1.08). When the interaction energies were further analyzed using the COMBINE formalism, the resulting PLS model showed improved fitting properties (r2 = 0.89) and provided better estimations for the activity of the compounds in the external data set (SDEPex = 0.83). Computation of the electrostatic part of the ligand-receptor interactions by numerically solving the Poisson-Boltzmann equation did not improve the quality of the linear regression model. On the contrary, incorporation of the solvent-screened residue-based electrostatic interactions and two additional descriptors representing the electrostatic energy contributions to the partial desolvation of both the ligands and the receptor resulted in a COMBINE model that achieved a remarkable predictive ability, as assessed by both internal (q2 = 0.73, SDEPcv = 0.69) and external validation tests (SDEPex = 0.59). Finally, when all the inhibitors studied were merged into a single expanded set, a new model was obtained that explained 91% of the variance in biological activity (r2 = 0.91), with very high predictive ability (q2 = 0.81, SDEPcv = 0.66). In addition, the COMBINE analysis provided valuable information about the relative importance of the contributions to the activity of individual residues that can be fruitfully used to design better inhibitors. All in all, COMBINE analysis is validated as a powerful methodology for predicting binding affinities and pharmacological activities of congeneric ligands that bind to a common receptor.

Drug Design↗
PubMed1998

Stacking interactions and intercalative DNA binding.

The DNA-binding properties of many ligands can be rationalized on the basis of their structural and electronic complementarity with the functional groups present in the minor and major grooves of particular DNA sequences. Specific hydrogen bonding patterns are particularly useful for the purpose of sequence recognition. Less obvious, however, is the influence of base composition on the conformational preferences of individual base steps and on the binding of intercalating moieties which become sandwiched between contiguous base pairs. Improved knowledge of stacking interactions may lead to a better understanding of the architecture and inherent flexibility of particular DNA sequences and may provide insight into the principles that dictate the structural changes and specificity patterns observed in the binding of some intercalating ligands to DNA.

DNA↗
PubMed1998

Regiospecific synthesis and anti-human immunodeficiency virus activity of novel 5-substituted N-alkylcarbamoyl and N,N-dialkylcarbamoyl 1,2,3-triazole-TSAO analogues.

Several 5-N-alkyl and 5-N,N-dialkylcarbamoyl substituted analogues of the anti-human immunodeficiency virus (HIV) type 1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-5- (N,N-dimethylcarbamoyl)-1,2,3-triazole]-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide) have been prepared and evaluated as inhibitors of HIV-1 replication. A new regiospecific synthetic procedure is described. The compounds were prepared by cycloaddition of the appropriate glycosylazide to 2-oxoalkylidentriphenyl-phosphoranes, followed by treatment with primary or secondary amines, to yield, exclusively, 5-substituted 1,2,3-triazole-TSAO analogues. Several 5-substituted 1,2,3-triazole-TSAO derivatives proved to be potent inhibitors of HIV-1 replication with higher antiviral selectivity than that of the parent TSAO prototype.

Anti-HIV Agents↗
PubMed1998

Novel 3'-spiro nucleoside analogues of TSAO-T. Part II. A comparative study based on NMR conformational analysis in solution and theoretical calculations.

The structures of two novel 3'-spiro nucleosides analogues of the potent human immunodeficiency virus type 1 (HIV-1) reverse trancriptase (RT) inhibitor TSAO-m3T, in solution, as derived from NMR spectroscopy are described. In these TSAO analogues the spiro amino oxathioledioxide moiety has been replaced by spiro amino oxazolone or spiro amino oxathiazoledioxide moieties. A comparative study based on theoretical calculations of the hydrophobicity, the solvation free energies and molecular electrostatic potentials (MEP) of the three compounds is also described. No significant conformational differences were detected in solution between TSAO-m3T and its analogues that might account for the differences observed in their inhibitory activity against HIV-1 RT. The calculated hydrophobicity (log P) values, dipole moments and the electrostatic contributions to the solvation free energies of the three spiro ring systems were also similar. However, the differences found in the calculated MEPs of the spiro systems between TSAO-m3T and its analogues suggest that the different electrostatic surroundings of the 4"-amino group of the spiro moiety in the analogues may be responsible for a detrimental electrostatic interaction of the spiro rings with the Glu-B138 of RT.

Anti-HIV Agents↗
PubMed1997

Assessment of solvation effects on calculated binding affinity differences: trypsin inhibition by flavonoids as a model system for congeneric series.

On the basis of molecular models of the interaction between trypsin and a series of seven structurally congeneric bioflavonoid inhibitors, the influence of solvation effects in the calculation of binding free energy differences in congeneric series has been assessed. The models were derived by making use of the X-ray crystal structure of bovine trypsin and the DOCK program, and the complementarity of the interactions between the functional groups of the docked molecules and the binding site region was corroborated independently with the GRID program. Interaction energies calculated for the complexes using molecular mechanics were found to correlate with the experimental inhibitory activities, although the quality of the correlation was dependent on the molecular modeling protocol. To understand why such correlations could be obtained in the absence of an explicit description of solvent effects, the in vitro activities were transformed into binding free energies, and continuum electrostatic theory was used to incorporate solvent effects by approximating them to the electrostatic contribution to the binding free energies. The results of our calculations show that, within this congeneric series, the cost in electrostatic free energy of desolvating both the enzyme binding site and the buried part of the inhibitors (delta Gdesolv) is roughly constant within the series. On the other hand, the electrostatic interaction energy (EeleLR) varies only slightly along the series in comparison with the van der Waals interaction (EVDWLR), and this variation is mostly solvent-independent, i.e., the reaction field energy of the solvent in the bound state (EsrfLR) makes almost a negligible contribution to the binding free energy differences. As a result, differences in binding free energy are dominated by the van der Waals term, while the electrostatic contribution is, to a good approximation, solvent-independent. A similar scenario may account for the good correlations frequently found between ligand activities and ligand-receptor interaction energies derived using plain molecular mechanics, although generality remains to be determined.

Animals↗
PubMed1997

Reliability of comparative molecular field analysis models: effects of data scaling and variable selection using a set of human synovial fluid phospholipase A2 inhibitors.

The effects of data pretreatment, data scaling, and variable selection on three-dimensional quantitative structure-activity relationships derived by comparative molecular field analysis (CoMFA) using the GRID energy function were studied in detail for a set of inhibitors of the human synovial fluid phospholipase A2 (HSF-PLA2). The quality of the models was evaluated for predictive power and ability to map the receptor binding site by (a) comparison of predicted and experimental activities using cross-validation and external validation sets and (b) comparison of the regions selected in space in the CoMFA models with a crystal structure of a HSF-PLA2-inhibitor complex, with optimized comparative binding energy analysis (COMBINE) models (Ortiz et al., 1995) and with structure-activity relationships derived previously for different sets of compounds. It is found that (1) data scaling and dielectric modeling strongly influence CoMFA results. Unscaled data and a uniform dielectric constant of 4 are well suited to GRID-CoMFA studies for the present compound set. (2) The GOLPE and Q2-GRS variable selection methods select variables in roughly the same regions in Cartesian space, but they produce different models in chemometric space and differ in their sensitivity to data scaling and pretreatment and their tendency to overfitting. (3) CoMFA models are consistent with COMBINE models in that they identify approximately the same intermolecular interactions as relevant for activity. Our study provides support for the qualitative receptor-mapping properties of CoMFA models and for the validity of variable selection when applied with care and also provides guidelines for how to evaluate the quality of CoMFA models.

Enzyme Inhibitors↗
PubMed1997

Simulation of alternative binding modes in a structure-based QSAR study of HIV-1 protease inhibitors.

We have used a published set of inhibitors of HIV-1 protease to build a COMBINE-type structure-based QSAR model with good predictive ability (r2 = 0.90, q2 = 0.69). Since the compounds in the training series exhibit most of their structural variability on one-half of the pseudosymmetrical binding cavity and only one binding orientation was explored for each molecule, the model describes mainly the effect of the structural changes on interactions involving only one-half of the binding cavity (pockets S1' and S2'). Thus, the model cannot be expected to give accurate predictions for new compounds exhibiting structural variation in both halves. The model does in fact show a tendency to underpredict slightly the biological activity of the molecules in the external test set. In an attempt to improve the quality of the model, both possible orientations of the ligands are now considered so that structural variation takes place in all binding pockets. One possibility would have been to build an additional set of complexes with the inhibitors docked in a reversed orientation. The alternative we have explored, however, consists of manipulating the data matrix describing the interaction energies so that each row is duplicated and the order of the variables in the duplicated rows is swapped between subunits. This simple approach has produced a new model that is similar in quality to the original model (r2 = 0.89, q2 = 0.64) but lacks the tendency to underpredict the activity of the compounds in the external set. Moreover, since equivalent residues are assigned equivalent weights, the model is insensitive to ligand orientation and is easier to interpret.

Artificial Intelligence↗
PubMed1997

Structure-affinity relationships for the binding of actinomycin D to DNA.

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin-d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino-purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure-affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in design of novel sequence-specific DNA-binding agents.

Anti-Bacterial Agents↗
PubMed1996

Molecular dynamics simulations of the bis-intercalated complexes of ditercalinium and Flexi-Di with the hexanucleotide d(GCGCGC)2: theoretical analysis of the interaction and rationale for the sequence binding specificity.

The X-ray crystal structures of the complexes of ditercalinium and Flexi-Di with d(CGCG)2 have been studied by computational chemistry methods in an attempt to rationalize their distinct structural features. In addition, the complexes of these two bisintercalating drugs with d(GCGCGC)2 have been modeled and subjected to 0.5 ns of molecular dynamics simulations in explicit solvent with the aim of evaluating the relative importance of hydrogen bonding and stacking interactions in the sequence binding specificity of these compounds. According to our calculations, the electrostatic term is attractive for the stacking interactions between the pyridocarbazole chromophores of these drugs and the base pairs that make up the sandwiched GpC step. On the contrary, this energy term is repulsive for the base pairs that make up the boundaries of the bisintercalation site. This differential electrostatic binding energy component, which is shown to have a strong orientational dependence, could lie at the origin of the observed binding preferences of these drugs. In addition, both the Lennard-Jones and the electrostatic energy terms contribute to stabilizing the underwound central GpC step. The attractive electrostatic interactions between the linkers and the major groove are in concert with the stacking specificities for the sandwiched GpC step, which is thus very effectively stapled by the drugs. The hydrogen-bonding potential of the linkers, however, appears to be reduced in an aqueous medium due to competing interactions with water. Binding of either ditercalinium or Flexi-Di to d(GCGCGC)2 appears to favor the A-type conformation that this DNA molecule most likely adopts in the free state. The possible relevance of these findings to the process of bis-intercalation and to the pharmacological action of these compounds is discussed.

Carbazoles↗
PubMed1995

Prediction of drug binding affinities by comparative binding energy analysis.

A new computational method for deducing quantitative structure-activity relationships (QSARs) using structural data from ligand-macromolecule complexes is presented. First, the ligand-macromolecule interaction energy is computed for a set of ligands using molecular mechanics calculations. Then, by selecting and scaling components of the ligand-macromolecule interaction energy that show good predictive ability, a regression equation is obtained in which activity is correlated with the interaction energies of parts of the ligands and key regions of the macromolecule. Application to the interaction of the human synovial fluid phospholipase A2 with 26 inhibitors indicates that the derived QSAR has good predictive ability and provides insight into the mechanism of enzyme inhibition. The method, which we term comparative binding energy (COMBINE) analysis, is expected to be applicable to ligand-receptor interactions in a range of contexts including rational drug design, host-guest systems, and protein engineering.

Binding Sites↗
PubMed1994

DNA sequence-specific reading by echinomycin: role of hydrogen bonding and stacking interactions.

The binding of echinomycin to DNA hexamers of the form GpApXpZpTpC, where the central XpZ step can be CpG, TpA, GpC, or ApT, has been studied by molecular modeling and molecular mechanics techniques. Interaction energies have also been calculated for the complexation of echinomycin with sequences containing the preferred central CpG step and different flanking base pairs. Besides, two more sets of sequences incorporating either 2,6-diaminopurine (DAP) or hypoxanthine in place of adenine or guanine, respectively, have been examined. The aim of this work was to evaluate the relative importance of hydrogen-bonding and stacking interactions in the association of echinomycin with DNA and further rationalize the experimental evidence. The results of these calculations are in consonance with available data from footprinting experiments and appear to support our previous hypothesis that, in addition to the crucial intermolecular hydrogen bonds in the central region, the stacking interactions involving the quinoxaline-2-carboxamide chromophores of the drug and the DNA base pairs play an important role in modulating the binding specificity of this bisintercalating antitumor antibiotic. This is most clearly seen when sequences with similar minor-groove environments are compared (e.g. CpI vs TpA or CpG vs TpDAP). The dipole moment of N-methylquinoxaline-2-carboxamide has been measured (mu = 4.15 +/- 0.03 D) and compares very well with the calculated value (mu = 4.14 D). The fact that G:C, I:C, A:T, and DAP:T base pairs are shown to be endowed with distinct van der Waals and electrostatic stacking properties with respect to this heteroaromatic ring system could have important implications for the design of novel DNA mono- and bis-intercalating agents.

2-Aminopurine↗
PubMed1994

Binding of echinomycin to d(GCGC)2 and d(CCGG)2: distinct stacking interactions dictate the sequence-dependent formation of Hoogsteen base pairs.

Molecular dynamics simulations have been used to explore the behavior of the complexes of echinomycin with the DNA tetramers d(GCGC)2 and d(CCGG)2 in which the terminal bases have been paired according to either a Hoogsteen or a Watson-Crick hydrogen bonding scheme. The energy of the four resulting complexes has been monitored along the dynamics trajectories and the interaction energy between echinomycin and DNA has been decomposed into contributions arising from the planar aromatic systems and the depsipeptide part of the antibiotic. Our calculations predict a large increase in overall stabilization upon protonation of the terminal cytosines and subsequent Hoogsteen pair formation in the complex of echinomycin with d(GCGC)2 but not with d(CCGG)2, in agreement with the experimental evidence [Gao and Patel, Quart. Rev. Biophys. 22, 93-138 (1989)]. The conformational preferences appear to arise mainly from differential stacking interactions in which the electrostatic component is shown to play a dominant role. Differences in hydrogen bonding patterns are also found among the complexes and these are compared in relation to available crystal structures. The binding of echinomycin to DNA appears as a complex process involving many interrelated variables.

Base Composition↗