Design and characterization of a heterocyclic electrophilic fragment library for the discovery of cysteine-targeted covalent inhibitors.

A fragment library of electrophilic small heterocycles was characterized through cysteine-reactivity and aqueous stability tests that suggested their potential as covalent warheads. The analysis of theoretical and experimental descriptors revealed correlations between the electronic properties of the heterocyclic cores and their reactivity against GSH that are helpful in identifying suitable fragments for cysteines with specific nucleophilicity. The most important advantage of these fragments is that they show only minimal structural differences from non-electrophilic counterparts. Therefore, they could be used effectively in the design of targeted covalent inhibitors with minimal influence on key non-covalent interactions.

A virtual high throughput screen for high affinity cytochrome P450cam substrates. Implications for in silico prediction of drug metabolism.

Structure-based virtual screening techniques require reliable scoring functions to discriminate potential substrates effectively. In this study we compared the performance of GOLD, PMF, DOCK and FlexX scoring functions in FlexX flexible docking to cytochrome P450cam binding site. Crystal structures of protein-substrate complexes were most effectively reproduced by the FlexX/PMF method. On the other hand, the FlexX/GOLD approach provided the best correlation between experimental binding constants and predicted scores. Binding modes selected by the FlexX/PMF approach were rescored by GOLD to obtain a reliable measure of binding energetics. The effectiveness of the FlexX/PMF/GOLD method was demonstrated by the correct classification of 32 out of the 33 experimentally studied compounds and also in a virtual HTS test on a library of 10,000 compounds. Although almost all the available functions were developed to be general, our study on cytochrome P450cam substrates suggests that careful selection or even tailoring the scoring function might increase the prediction power of virtual screens significantly. The FlexX/PMF/GOLD methodology was tested on cytochrome P450 3A4 substrates and inhibitors. This preliminary study revealed that the combined function was able to recognise 334 out of the 345 compounds bound to 3A4.

Distinct behavior of mutant triosephosphate isomerase in hemolysate and in isolated form: molecular basis of enzyme deficiency.

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and "stickiness" of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed.

High-throughput prediction of blood-brain partitioning: a thermodynamic approach.

A high-throughput in silico screening tool for potentially CNS active compounds was developed on the basis of the correlation of solvation free energies and blood-brain partitioning (log(cbrain/cblood) = log BB) data available from experimental sources. Utilizing a thermodynamic approach, solvation free energies were calculated by the fast and efficient generalized Born/surface area continuum solvation model, which enabled us to evaluate more than 10 compounds/min. Our training set involved a structurally diverse set of 55 compounds and yielded a function of log BB = 0.035Gsolv + 0.2592 (r = 0.85, standard error 0.37). Calculation of solvation free energies for 8700 CNS active compounds (CIPSLINE database) revealed that Gsolv is higher than -50 kJ/mol for the 96% of these compounds which can be used as suitable criteria for the identification of compounds preferable for CNS penetration.

A neural network based virtual high throughput screening test for the prediction of CNS activity.

A virtual high throughput screening test to identify potentially CNS-active drugs has been developed. Discrimination was based on the knowledge available in databases containing CNS-active (Cipsline from Prous Science) and inactive compounds (Chemical Directory from Sigma-Aldrich). Molecular structures were represented using 2D Unit y fingerprints and a feedforward neural network was trained to classify molecules regarding their CNS activity. The parameterized network was validated by reclassification of the training set elements, by the classification of a test set preselected from the Prous database, and also by the prediction of activity for known CNS drugs not used in the training set but available in the Medchem database (Daylight). These tests revealed that our neural net recognized at least 89% of CNS-active compounds and would be suitable for use in our virtual screening protocol.

Construction of a 3D model of oligopeptidase B, a potential processing enzyme in prokaryotes.

A three dimensional structural model of oligopeptidase B (OpB) was constructed by homology modeling. High resolution X-ray structure of prolyl oligopeptidase (PEP), the only protein with sequential and functional homology was used as a template. Initial models of OpB were built by the MODELLER and were analysed by the PROCHECK programs. The best quality model was chosen for further refinement by two different techniques--either constrained molecular dynamics simulations or simulated annealing calculations starting from 500 K. The overall quality of each of the refined models was evaluated and the simulated annealing procedure found to be more effective. The refined model was analysed by different protein analysis programs including PROCHECK for the evaluation of the Ramachandran plot quality, PROSA for testing interaction energies and WHATIF for the calculation of packing quality. This structure was found to be satisfactory and also stable at room temperature as demonstrated by a 300 ps long unconstrained molecular dynamics simulation. Calculation of molecular electrostatic potentials revealed that the binding site of OpB is more negative than that of PEP, in accordance with the experimentally observed selectivity of OpB towards proteolysis at dibasic sites. A recently developed Monte Carlo docking method was used provide a structural rationale for the affinity differences measured between Z-Arg and Z-Arg-Arg substrates.

Metalloporphyrin catalyzed oxidation of N-hydroxyguanidines: a biomimetic model for the H2O2-dependent activity of nitric oxide synthase.

A chemical model for the H2O2 promoted oxidation by nitric oxide synthase (NOS) has been developed. Biomimetic oxidations were carried out using H2O2 and tetrakis(perfluorophenyl)porphyrinato-iron(III) chloride (FeTPPF20) as a catalyst. Similarly to NOS our model system produces Ndelta-cyanoornithine, citrulline and NO from NOHA and did not oxidize arginine itself. Based on these results we propose a peroxide shunt to be involved in the catalytic cycle of NOS. To the best of our knowledge this is the first chemical system that semiquantitatively mimics NOS activity.

Cytochrome P450 catalyzed nitric oxide synthesis: a theoretical study.

Similar to nitric oxide synthase (NOS) cytochrome P450 isoforms (e.g. 3A and 4E) can produce nitric oxide from arginine. Although the active site of both proteins contains a protoporphyrin IX unit having an axial cystein ligand, their effectiveness in the synthesis of NO differs significantly. Now the molecular basis of this functional difference was investigated. A homology model for cytochrome P450 3A4 was refined and compared to the X-ray structure of iNOS. We found the active site of iNOS to be more readily accessible for the substrate than that of P450. Docking calculations were performed using the Monte Carlo conformational analysis technique on all internal and external degrees of freedom of arginine and active site residues as well. The lowest energy conformation of the cytochrome P450 3A4-substrate complex was compared to the high resolution X-ray structure of the iNOS-arginine complex. Comparison of substrate orientations revealed that arginine binds in a similar conformation in both enzymes. In contrast to iNOS we found, however, that in P450 partially negative propionate side chains of protoporphyrin IX are located on the opposite side of the heme plane. As a result of this and the absence of other negatively charged residues the distal (substrate binding) side of P450 should be less negative than that of NOS and therefore its affinity toward the partially positive arginine is reduced. Comparison of molecular electrostatic potentials calculated within the active site of the proteins supports this proposal. Reduced affinity in combination with limited substrate access might be responsible for the less effective NO synthesis of cytochrome P450 observed experimentally.

[Role of macromolecular simulations in structural biochemistry]. / Makromolekuláris szimulációk alkalmazási lehetóségei a szerkezeti biokémiában.

In addition to experimental studies, macromolecular simulations were found to be useful in the structural investigation of proteins and protein-ligand complexes. Computational techniques involving molecular mechanics, molecular dynamics and conformational analysis are widely used to predict 3D protein structures and to explore structure-function relationships. The effectiveness of these methods is presented on representative examples including the homology modelling of a processing enzyme, oligopeptidase B and the investigation of nitric oxide binding to metmyoglobin. Our results demonstrated that macromolecular simulations in combination with experimental methods should be considered as an important tool for structure-based drug design.

Chemical models of cytochrome P450 catalyzed insecticide metabolism. Application to the oxidative metabolism of carbamate insecticides.

Cytochrome P450 (CP450) catalyzed oxidative metabolism of carbofuran (1), carbaryl (2), and pirimicarb (3) has been modeled using biomimetic oxidations catalyzed by iron(III) tetraarylporphyrins. Oxidation products of 1 were identified by comparison of HPLC retention times measured under standardized conditions for metabolites synthesized and characterized by (1)H and (13)C NMR spectroscopy. Comparison of product distributions to in vivo metabolic profiles revealed that the H(2)O(2)/meso-tetrakis(pentafluorophenyl)porphyrin iron(III) chloride [Fe(TF(20)PP)] system mimics the action of insect CP450s against carbofuran. The effectiveness of this system was further demonstrated by the biomimetic oxidation of other carbamate insecticides (2 and 3) monitored by HPLC/electrospray MS. The predictive power of this biomimetic model was compared to that of knowledge-based expert systems. Although similar models were recently applied in pharmaceutical research, the usefulness of this approach has first been demonstrated for the prediction of metabolic profiles of agrochemicals.

Role of proximal His93 in nitric oxide binding to metmyoglobin. Application of continuum solvation in Monte Carlo protein simulations.

Monte Carlo protein simulations with continuum solvation were used to explore the conformational mobility of NO within the active site of metmyoglobin. To the best of our knowledge this is the first application of a continuum solvation model for exploring protein binding sites. The usefulness of the Monte Carlo conformational analysis was demonstrated in comparative molecular dynamics simulations. Analysis of conformer populations revealed that Monte Carlo conformational analysis is more effective in mapping the relevant region of the potential surface. Distribution of low-energy conformers obtained for the metmyoglobin-NO complex was found to depend on the orientation of proximal His93. Different charge distributions corresponding to the two experimentally verified possible torsions of this proximal residue result in strong binding of NO or its release to a nearby hydrophobic trap. Conformer populations obtained by Monte Carlo conformational analysis were grouped into three main families: one, with the NO directly bound to the iron, appears when the CA-CB-CG-CD2 torsion of His93 is at its ligand binding value (-113 degrees); and two conformers exist where NO is trapped in a nearby hydrophobic pocket, the same cavity that was determined to be the geminate trap of CO in ferrous Mb as a result of the torsional flip of His93 to its ligand releasing state (-125 degrees). Based on this analysis, we suggest that the electrostatic rearrangement coupled to the conformational fluctuation of the proximal His leads to the geminate trapping of the ligand. Conformational rearrangement of the proximal side would provide the possibility of rebinding of the ligand to Fe.

Cytochrome P450 catalysed insecticide metabolism: chemical and theoretical models.

Increased rate of cytochrome P450 catalysed metabolism is a characteristic factor in insecticide resistance. In addition to in vivo and in vitro methods, models of structural and theoretical biochemistry as well as bioorganic studies were found to be useful for understanding this increased metabolic activity on a molecular basis. Metabolic profiles can be reproduced using P450 mimic synthetic porphyrins and predicted by computer assisted biomolecular modelling. Insight into the mechanism of metabolic reactions might lead to specific P450 inhibitors reducing metabolic resistance and the environmental risk of high doses of pesticides.

[Application of theoretical and synthetic models to cytochrome P450 catalysed metabolic reactions]. / Elméleti és preparatív enzimmodellek alkalmazása citokrom P450 katalizált metabolitikus reakciók vizsgálatára.

In addition to in vivo and in vitro methods, models of structural and theoretical biochemistry as well as bioorganic studies were found to be useful for understanding metabolic activity on a molecular basis. Metabolism of an AChE inhibitor, carbofuran was studied. Product selectivity observed in vitro and in vivo was explained by theoretical methods. Metabolic profiles can be reproduced using P450 mimic synthetic porphyrins. Insight into the mechanism of metabolic reactions might be applied for the identification of metabolically sensitive substructures as well as for the prediction of metabolic profiles.

On the conformation of tiazofurin analogues.

Tiazofurin, an important inhibitor of inosine 5'-monophosphate dehydrogenase, has been argued to possess a restricted glycosylic bond due to an energetically favorable intramolecular (1-4) electrostatic interaction between the partial positive sulfur and the negative oxygen of the ribose. This rigidity has been appointed as a plausible cause that leads to activity in the sulfur containing compounds as opposed to the inactive oxazofurin-like analogues (i.e. S is replaced by an oxygen) that lack this favorable interaction. We reinvestigated this notion by using computational methods to report that although the above interaction (or its lack) is likely to contribute to the low-energy conformation of these classes of molecules, the flexibility of the glycosylic bond is ultimately determined by steric interaction of the heteroatoms with the C2'-H and O4' of the ribose. Application of this theory in the design of new analogues is presented as well.

Molecular similarity analysis on biologically active macrocyclic bis(bibenzyls).

Conformational analysis of marchantin A (1), a bis(diarylether) type, and riccardin A (2), a diarylether-biphenyl type macrocyclic bis(bibenzyl) was carried out by systematic unbounded multiple minimum search (SUMM). Mobility of the macrocyclic rings was analysed by variable temperature 1H-NMR study. Molecular similarity analysis was performed on the minimum energy conformers of 1 and 2 comparing their steric, electrostatic and hydrophobic properties. Correlation between complexation properties and calmodulin inhibitor activity was established. Differences in steric and electrostatic profiles may be responsible for the reduced Ca2+ affinity and activity of 2.

Nuclear magnetic resonance and molecular modeling study on mycophenolic acid: implications for binding to inosine monophosphate dehydrogenase.

The conformation of the sodium salt of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase (IMPD), derived from 1D DIFNOE and 2D ROESY experiments in water and molecular dynamics (MD) is described. The hexenoic acid side chain conformation consistent with the NMR data was similar to that seen in the X-ray structure of MPA. The solution conformation was applied in a molecular modeling study in order to explore the potential features of enzyme binding. Our results, based on striking similarities in molecular volume and electrostatic isopotential between MPA and cofactor NAD+, lead to the suggestion that MPA is capable of binding to the nicotinamide site of IMPD and mimicking the NAD+ inverse regulation of the enzyme. In addition, our proposed model is in good agreement with the observed high affinity of the dinucleotide analogues thiazole- and selenazole-4-carboxamide adenine dinucleotide to IMPD.

The biological activity of cyclic bis(bibenzyls): a rational approach.

The biological activities reported for marchantin A (1), a natural cyclic bis(bibenzyl), were studied in comparison with cepharanthine (2), a therapeutically useful bisbenzylisoquinoline alkaloid. Based on the examination of steric, electrostatic, and hydrophobic similarity, as well as on the comparison of biological activities, the similar therapeutic properties of 1 and 2 can be attributed to binding on a common receptor. The wide range of activity of 1 can be interpreted by a mechanism of action based on a calcium binding.