Ligand-induced conformational changes: improved predictions of ligand binding conformations and affinities

A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein tyrosine phosphatase 1B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal structure cocrystallized with a hexapeptide. The estimated binding energies for various docking modes as well as the RMS differences between the docked compounds and the crystallographic structure were calculated. In this scenario the estimated binding energies were not predictive inasmuch as docking modes with low estimated binding energies corresponded to relatively large RMS differences when aligned with the corresponding crystal structure. Secondly, the inhibitors were docked to their parent protein structures in which they were cocrystallized. In this case, there was a good correlation between low predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side chains exposed to the active site were considered in their allowed rotamer conformations and protein models containing all possible combinations of side-chain rotamers were generated. To evaluate which of these modeled active sites is the most likely binding site conformation for a certain inhibitor, the inhibitors were docked against all active site models. The receptor rotamer model corresponding to the lowest estimated binding energy is taken as the top candidate. Using this protocol, correct inhibitor binding modes could successfully be discriminated from proposed incorrect binding modes. Moreover, the ranking of the estimated ligand binding energies was in good agreement with experimentally observed binding affinities.     

Computer-aided analysis of the interactions of glutamine synthetase with its inhibitors

Mechanism of inhibition of glutamine synthetase (EC 6.3.1.2; GS) by phosphinothricin and its analogues was studied in some detail using molecular modeling methods. Among three possible conformations of phosphinothricin in the active site of GS, this compatible with binding mode of methionine sulfoximine, determined recently by crystallography, was found to be energetically favored. Basing on these results eleven inhibitors of GS were docked into its active site. Taking into consideration that phosphinothricin acts as suicide inhibitor, which is due to phosphorylation by the enzyme, seven of studied analogues were additionally analyzed in their phosphorylated forms. All the inhibitor-enzyme complexes were evaluated quantitatively by using eight scoring functions implemented in Insight and Sybyl program packages and significant correlation between the obtained scores and experimental pK(i) values was achieved. Computed surface charge distribution for five selected inhibitors in both free and phosphorylated forms and their comparison with electronic structure of enzymatic reaction transition state allowed us to determine important electronic features required to construct potent inhibitors of glutamine synthetase.     

Modeling of a new tubercular maltosyl transferase,GlgE, study of its binding sites and virtual screening

Recently maltosyl transferase of Mycobacterium tuberculosis (mtb GlgE) belonging to α-amylase family has been identified as a potential drug target. Despite its importance, its three dimensional (3D) structure is unavailable. In this study we have modeled its 3D homo-dimeric structure using its homologue in Streptomyces ceolicolor (stp GlgE) as the template. Its monomer consists of five domains and four inserts, out of which two inserts are unique to mtb GlgE. It also has three binding cavities. One primary (pbs) and two secondary (sbs1 and sbs2), with one unique insert appearing within sbs2. Investigation of its homo-dimeric model revealed the presence of a disulphide bridge between Cys-29 of both the chains which is absent in stp GlgE. Virtual screening with known substrates and substrate analogues of α-amylase family proteins indicated better binding of maltose to sbs1 than pbs. Among all computationally screened substrates 3-O-Alpha-d-Glucopyranosyl-d-Fructose (OTU) docked with best binding affinity to pbs. Interaction of known inhibitors of α-amylase family proteins from CHEMBL is also studied. This reveals for the first time the unique 3D structure of mtb GlgE and provides insights into its active sites and substrate binding affinities. This may help in developing new anti-tubercular drugs and its analogues.     

Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts.

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

Docking of sialic acid analogues against influenza A hemagglutinin: a correlational study between experimentally measured and computationally estimated affinities

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

Synthesis of 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose and their effects on lactose synthase

Syntheses are reported of 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose as potential inhibitors for lactose synthase [uridine 5′-(α-d-galactopyranosyl pyrophosphate):d-glucose 4-β-d-galactopyranosyltransferase, EC 2.4.1.22]. These syntheses involved SN2 displacement of the 4-methylsulfonyloxy group of methyl 2,3,6-tri-O-benzoyl-4-O-methylsulfonyl-α-d-galactopyranoside by iodide and azide ions. In both cases, inversion in configuration was observed. The resulting intermediates, methyl 2,3,6-tri-O-benzoyl-deoxy-4-iodo-α-d-glucopyranoside and methyl 4-azido-2,3,6-tri-O-benzoyl-deoxy-α-d-glucopyranoside, were obtained in crystalline form. Both 4-deoxy-d-xylo-hexose and 4-azido-4-deoxy-d-glucose were found to be inhibitors for lactose synthase in the presence of α-lactalbumin, but had no effect in the absence of α-lactalbumin. Both d-glucose analogues bind to the enzyme system far more weakly than d-glucose, suggesting that the recognition of the 4-OH group of the acceptor substrate is an important factor in binding.     

Synthesis,structure, and structure-activity relationships of divalent thrombin inhibitors containing an α-keto-amide transition-state mimetic

A new class of divalent thrombin inhibitors is described that contains an α-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the α-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their α-keto-amide counterparts. The most potent cv-keto-amide inhibitor, CVS995, with a K_i = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.     

Site-directed fragment-based generation of virtual sialic acid databases against influenza A hemagglutinin

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.

The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.     

Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes

Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.     

Binding of the chymotrypsin substrate,tryptophan methyl ester,by rat α-fetoprotein

We studied how tryptophan methyl ester and related compounds inhibit binding of estrone to rat α-fetoprotein and find that: (a) like chymotrypsin, α-fetoprotein binds tryptophan esters with higher affinity than tryptophan or its amides; (b) the affinity of α-fetoprotein for tryptophan methyl ester is 3.7 · 10^?4 M, which is close to the affinity of chymotrypsin (10^?4 M); (c) α-fetoprotein binding of tryptophan methyl ester is stereoselective and pH dependent. All of these observations suggest that there is a specific interaction between α-fetoprotein and the chymotrypsin substrate, tryptophan methyl ester, and that rat α-fetoprotein contains a site with some structural similarities to the catalytic site in chymotrypsin. Since we also find that tryptophan methyl ester is a competitive inhibitor of estrone binding to α-fetoprotein, it is possible that the protease substrate binding site on α-fetoprotein is spatially close to the estrone binding site.     

Applications of the ArgusLab4/AScore protocol in the structure-based binding affinity prediction of various inhibitors of group-1 and group-2 influenza virus neuraminidases (NAs)

Using the crystal structures of inhibitors bound to either group-2 or group-1 neuraminidases (NAs), AScore/ShapeDock (GaDock) scoring was shown to identify the binding modes in agreement with the experiment for all inhibitors docked in their own NA/inhibitor crystal structures. To investigate the effect of small changes in protein structure on predicted binding modes, in a set of 132 docking experiments (11 inhibitors docked in 12 group-2 NA structures), AScore/ShapeDock (GaDock) identified the correct binding modes of 116 complexes. In a total of 88 docking experiments (8 inhibitors docked in 11 group-1 NA structures), AScore/ShapeDock predicted 80 binding modes correctly. Flexible AScore/ShapeDock docking, as quite reproducible, is suggested to be convenient for designing novel H5N1 inhibitors.     

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.     

Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase.

Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular docking and structure-based virtual screening studies of potential drug target,CAAX prenyl proteases,of Leishmania donovani

Targeting CAAX prenyl proteases of Leishmania donovani can be a good approach towards developing a drug molecule against Leishmaniasis. We have modeled the structure of CAAX prenyl protease I and II of L. donovani, using homology modeling approach. The structures were further validated using Ramachandran plot and ProSA. Active site prediction has shown difference in the amino acid residues present at the active site of CAAX prenyl protease I and CAAX prenyl protease II. The electrostatic potential surface of the CAAX prenyl protease I and II has revealed that CAAX prenyl protease I has more electropositive and electronegative potentials as compared CAAX prenyl protease II suggesting significant difference in their activity. Molecular docking with known bisubstrate analog inhibitors of protein farnesyl transferase and peptidyl (acyloxy) methyl ketones reveals significant binding of these molecules with CAAX prenyl protease I, but comparatively less binding with CAAX prenyl protease II. New and potent inhibitors were also found using structure-based virtual screening. The best docked compounds obtained from virtual screening were subjected to induced fit docking to get best docked configurations. Prediction of drug-like characteristics has revealed that the best docked compounds are in line with Lipinski’s rule. Moreover, best docked protein–ligand complexes of CAAX prenyl protease I and II are found to be stable throughout 20 ns simulation. Overall, the study has identified potent drug molecules targeting CAAX prenyl protease I and II of L. donovani whose drug candidature can be verified further using biochemical and cellular studies.     

The crystal structure of the Leishmania major deoxyuridine triphosphate nucleotidohydrolase in complex with nucleotide analogues, dUMP, and deoxyuridine

Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.     

Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. II. Isomeric aza-deazaanalogues of adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.