Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.     

Discovering New Classes of <Emphasis Type="Italic">Brugia malayi</Emphasis> Asparaginyl-tRNA Synthetase Inhibitors and Relating Specificity to Conformational Change

SLIDE software, which models the flexibility of protein and ligand side chains while docking, was used to screen several large databases to identify inhibitors of Brugia malayi asparaginyl-tRNA synthetase (AsnRS), a target for anti-parasitic drug design. Seven classes of compounds identified by SLIDE were confirmed as micromolar inhibitors of the enzyme. Analogs of one of these classes of inhibitors, the long side-chain variolins, cannot bind to the adenosyl pocket of the closed conformation of AsnRS due to steric clashes, though the short side-chain variolins identified by SLIDE␣apparently bind isosterically with adenosine. We hypothesized that an open conformation of the motif 2 loop also permits the long side-chain variolins to bind in the adenosine pocket and that their selectivity for Brugia relative to human AsnRS can be explained by differences in the sequence and conformation of this loop. Loop flexibility sampling using Rigidity Optimized Conformational Kinetics (ROCK) confirms this possibility, while scoring of the relative affinities of the different ligands by SLIDE correlates well with the compounds’ ranks in inhibition assays. Combining ROCK and SLIDE provides a promising approach for exploiting conformational flexibility in structure-based screening and design of species selective inhibitors.     

Automated Docking and the Search for HIV Protease Inhibitors

Abstract

This article will discuss the motivations, technologies, and future directions of computational automated docking in the context of the structure-based rational design of HIV-1 protease inhibitors. Docking simulations are widely used for screening of compound libraries to identify new drug leads, employing a simple model for rapid testing of thousands of compounds. Docking simulations are also useful for lead enhancement, using more detailed models to analyze the atomic interactions between inhibitors and target macromolecules. Major advances have been reported in the development of empirical force fields, which now allow assessment of relative binding strength and drug specificity, and extensions of automated docking techniques allow de novo drug design.     

A multilayer screening approach toward the discovery of novel Pf-DHFR inhibitors

A small yet diverse xanthone library was build and computationally docked against wild type Pf-DHFR by Molegro Virtual Docker (MolDock). For analysis of results an integrated approach based on re-ranking, scaling (based on heavy atom counts), pose clustering and visual inspection was implemented. Standard methods such as self-docking (for docking), EF analysis, average rank determinations (for size normalization), and cluster quality indices (for pose clustering) were used for validation of results. Three compounds X5, X113A and X164B displayed contact footprints similar to the known inhibitors with good scores. Finally, 16 compounds were extracted from ZINC data base by similarity based screening, docking score and drug/lead likeness. Out of these 16 compounds, 11 displayed very close contact footprints to experimentally known inhibitors, indicating there potential utility in further drug discovery efforts.     

Structure-based 3D QSAR and design of novel acetylcholinesterase inhibitors

The paper describes the construction, validation and application of a structure-based 3D QSAR model of novel acetylcholinesterase (AChE) inhibitors. Initial use was made of four X-ray structures of AChE complexed with small, non-specific inhibitors to create a model of the binding of recently developed aminopyridazine derivatives. Combined automated and manual docking methods were applied to dock the co-crystallized inhibitors into the binding pocket. Validation of the modelling process was achieved by comparing the predicted enzyme-bound conformation with the known conformation in the X-ray structure. The successful prediction of the binding conformation of the known inhibitors gave confidence that we could use our model to evaluate the binding conformation of the aminopyridazine compounds. The alignment of 42 aminopyridazine compounds derived by the docking procedure was taken as the basis for a 3D QSAR analysis applying the GRID/GOLPE method. A model of high quality was obtained using the GRID water probe, as confirmed by the cross-validation method (q² _LOO=0.937, q² _{L50% O}=0.910). The validated model, together with the information obtained from the calculated AChE-inhibitor complexes, were considered for the design of novel compounds. Seven designed inhibitors which were synthesized and tested were shown to be highly active. After performing our modelling study the X-ray structure of AChE complexed with donepezil, an inhibitor structurally related to the developed aminopyirdazines, has been made available. The good agreement found between the predicted binding conformation of the aminopyridazines and the one observed for donepezil in the crystal structure further supports our developed model.     

<Emphasis Type="Italic">In-silico</Emphasis> Screening using Flexible Ligand Binding Pockets: A Molecular Dynamics-based Approach

Summary In-silico screening of flexible ligands against flexible ligand binding pockets (LBP) is an emerging approach in structure-based drug discovery. Here, we describe a molecular dynamics (MD) based docking approach to investigate the influence on the high-throughput in-silico screening of small molecules against flexible ligand binding pockets. In our approach, an ensemble of 51 energetically favorable structures of the LBP of human estrogen receptor α (hERα) were collected from 3 ns MD simulations. In-silico screening of 3500 endocrine disrupting compounds against these flexible ligand binding pockets resulted in thousands of ER–ligand complexes of which 582 compounds were unique. Detailed analysis of MD generated structures showed that only 17 of the LBP residues significantly contribute to the overall binding pocket flexibility. Using the flexible LBP conformations generated, we have identified 32 compounds that bind better to the flexible ligand-binding pockets compared to the crystal structure. These compounds, though chemically divergent, are structurally similar to the natural hormone. Our MD-based approach in conjunction with grid–based distributed computing could be applied routinely for in-silico screening of large databases against any given target.     

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH⁺ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC₅₀ against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH⁺-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.     

An integrated in silico screening strategy for identifying promising disruptors of p53-MDM2 interaction

The p53 protein, also called guardian of the genome, plays a critical role in the cell cycle regulation and apoptosis. This protein is frequently inactivated in several types of human cancer by abnormally high levels of its negative regulator, mouse double minute 2 (MDM2). As a result, restoration of p53 function by inhibiting p53-MDM2 protein–protein interaction has been pursued as a compelling strategy for cancer therapy. To date, a limited number of small-molecules have been reported as effective p53−MDM2 inhibitors. X-ray structures of MDM2 in complex with some ligands are available in Protein Data Bank and herein, these data have been exploited to efficiently identify new p53-MDM2 interaction antagonists through a hierarchical virtual screening strategy. For this purpose, the first step was aimed at compiling a focused library of 686,630 structurally suitable compounds, from PubChem database, similar to two known effective inhibitors, Nutlin-3a and DP222669. These compounds were subjected to the subsequent structure-based approaches (quantum polarized ligand docking and molecular dynamics simulation) to select potential compounds with highest binding affinity for MDM2 protein. Additionally, ligand binding energy, ADMET properties and PAINS analysis were also considered as filtering criteria for selecting the most promising drug-like molecules. On the basis of these analyses, three top-ranked hit molecules, CID_118439641, CID_60452010 and CID_3106907, were found to have acceptable pharmacokinetics properties along with superior in silico inhibitory ability towards the p53-MDM2 interaction compared to known inhibitors. Molecular docking and molecular dynamics results well confirmed the interactions of the final selected compounds with critical residues within p53 binding site on the MDM2 hydrophobic clefts with satisfactory thermodynamics stability. Consequently, the new final scaffolds identified by the presented computational approach could offer a set of guidelines for designing promising anti-cancer agents targeting p53-MDM2 interaction.     

Design,Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile Inhibitors and Activity‐Based Probes as Promising Anti‐Trypanosoma brucei Agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small‐molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain‐like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile‐containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target‐based screening) and trypanocidal activity (i.e., whole‐organism‐based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity‐based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease‐relevant bloodstream form (BSF) and the insect‐residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti‐trypanosome agents, which possess better activities than existing drugs. The activity‐based probes generated from this study could also serve as valuable tools for parasite‐based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile‐containing compounds as potential anti‐parasitic agents.     

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-β-D-glucoside, emodin-8-O-β-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.     

Minor antifungal aromatic glycosides from the roots of Gentiana rigescens （Gentianaceae）

Two new phenolic glycosides,2,3-dihydroxybenzoic acid methyl ester 3-O-β-D-glucopyranosyl-(1-6)-β-D-glucopyranoside(1) and 2,5-dihydroxylbenzofuran 5-O-β-D-xylopyranosyl-(1-6)-O-β-D-glucopyranoside(2),were isolated as the minor chemical constituents from the roots of Gentiana rigescens,along with 15 known compounds.Their structures were elucidated by detailed spectroscopic analysis,including 1D,2D NMR and chemical method.All of these compounds were isolated for the first time from the title plant.Moreove...     

Pharmacophore modelling,atom-based 3D-QSAR generation and virtual screening of molecules projected for mPGES-1 inhibitory activity

COX-2 inhibitors exhibit anticancer effects in various cancer models but due to the adverse side effects associated with these inhibitors, targeting molecules downstream of COX-2 (such as mPGES-1) has been suggested. Even after calls for mPGES-1 inhibitor design, to date there are only a few published inhibitors targeting the enzyme and displaying anticancer activity. In the present study, we have deployed both ligand and structure-based drug design approaches to hunt novel drug-like candidates as mPGES-1 inhibitors. Fifty-four compounds with tested mPGES-1 inhibitory value were used to develop a model with four pharmacophoric features. 3D-QSAR studies were undertaken to check the robustness of the model. Statistical parameters such as r² = 0.9924, q² = 0.5761 and F test = 1139.7 indicated significant predictive ability of the proposed model. Our QSAR model exhibits sites where a hydrogen bond donor, hydrophobic group and the aromatic ring can be substituted so as to enhance the efficacy of the inhibitor. Furthermore, we used our validated pharmacophore model as a three-dimensional query to screen the FDA-approved Lopac database. Finally, five compounds were selected as potent mPGES-1 inhibitors on the basis of their docking energy and pharmacokinetic properties such as ADME and Lipinski rule of five.     

Evaluation of drug candidature: In silico ADMET,binding interactions with CDK7 and normal cell line studies of potentially anti-breast cancer enamidines

We have recently explored novel class of potentially anti-breast cancer active enamidines in which four molecules 4a-c and 4h showed higher anticancer activity compared to standard drug doxorubicin. As a part of extension of this work, we have further evaluated in silico cheminformatic studies on bioactivity prediction of synthesized series of enamidines using mole information. The normal cell line study of four lead compounds 4a-c and 4h against African green monkey kidney vero strain further revealed that the compounds complemented good selectivity in inhibition of cancer cells. The in silico bioactivity and molecular docking studies also revealed that the compounds have significant interactions with the drug targets. The results reveal that enamidine moieties are vital for anti-breast cancer activity as they possess excellent drug-like characteristics, being potentially good inhibitors of cyclin dependent kinases7 (CDK7).     

In vitro selection of self-cleaving DNAs

Background: Ribozymes catalyze an important set of chemical transformations in metabolism, and ‘engineered’ ribozymes have been made that catalyze a variety of additional reactions. The possibility that catalytic DNAs or ‘deoxyribozymes’ can be made has only recently been addressed. Specifically, it is unclear whether the absence of the 2′ hydroxyl renders DNA incapable of exhibiting efficient enzyme-like activity, making it impossible to discover natural or create artificial DNA biocatalysts.Results: We report the isolation by in vitro selection of two distinct classes of self-cleaving DNAs from a pool of random-sequence oligonucleotides. Individual catalysts from ‘class I’ require both Cu²⁺ and ascorbate to mediate oxidative self-cleavage. Individual catalysts from class II use Cu²⁺ as the sole cofactor. Further optimization of a class II individual by in vitro selection yielded new catalytic DNAs that facilitate Cu²⁺-dependent self-cleavage with rate enhancements exceeding 1000 000-fold relative to the uncatalyzed rate of DNA cleavage.Conclusions: Despite the absence of 2′ hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.     

Structure elucidation of two tryptophan-derived, high affinity Ah receptor ligands

Background: Environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other structurally related ‘environmental hormones’, exert their harmful biological effects through the Ah receptor signaling pathway. Several naturally occurring substances also bind to this receptor, but its natural role is still obscure. Tryptophan derivatives of the indolo[3,2-b]carbazole type, earlier suggested by us to be endogenous ligands for the receptor, should be a powerful tool in understanding receptor function. We therefore: set out to determine their identity.Results: The two tryptophan-derived Ah receptor ligands have been chemically analyzed and characterized by means of mass spectrometry, ¹H NMR and ¹³C NMR. UV, infra-red and fluorescence spectra were also recorded. All data are in accordance with the two compounds being closely related indolo[3,2-b]carbazole derivatives. Evidence is presented that compound A (MW = 312) is the symmetrical 6,12-diformylindolo[3,2-b]carbazole, and compound B (MW = 284) is the monosubstituted 6-formylindolo[3,2-b]carbazole.Conclusions: The elucidation of the structures of the two high affinity Ah receptor ligands 6,12-diformylindolo[3,2-b]carbazole and 6-formylindolo[3,2-b]carbazole provides the necessary basis for further mechanistic studies of this important group of compounds, and will help in determining the natural role of the Ah receptor.     

Structure-guided fragment-based<Emphasis Type="Italic"> in silico</Emphasis> drug design of dengue protease inhibitors

An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC₅₀ = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC₅₀ = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.     

Sphingosine kinase 1 (SK1) allosteric inhibitors that target the dimerization site

The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1.     

Ab initio Computational Modeling of Glyphosate Analogs: Conformational Perspective

A historical perspective on the application of conformational analysis to structure-based ligand design approach is presented. The application of isodensity molecular electrostatic potential surfaces with the conformational energy surfaces (CES) have allowed us to reach pertinent conclusions for aiding synthetic and biochemical studies. Here we illustrate such an application on the modeling of the potent analogs of an important, environmentally stringent herbicidal compound glyphosate by constructing conformational energy surfaces. The systems were modeled by substituting F, Cl, and NH— OH moiety to the position of pharmacophoric nitrogen center in glyphosate structure. All the calculations were thoroughly performed with ab initio MO theory at Hartree–Fock method using 3-21G(d) basis functions. On the basis of the results, we identified the bioactive conformations for N-fluoro-glyphosate, N-chloro-glyphosate, and N-hydroxyamino-glyphosate as (−38^∘, 77^∘), (−61^∘, 111^∘), and (−167^∘, −169^∘), respectively. Geometry optimization of certain selected conformations of these compounds using hybrid DFT method with 6–31+G(d) basis functions provides nearly equal values of φ and ψ. Moreover, the results indicate that the global minimum structures of N-fluoro and N-chloro analogs of glyphosate show cyclic conformation whereas the N-hydroxyamino-glyphosate global minimum structure shows spyrocyclic and zig-zag conformation. Also, the predicted bioactive conformation of N-hydroxyamino analog optimally overlaps with glyphosate backbone in EPSPS complex with 0.1 Å RMSD value. However, the other two compounds slightly deviate from the backbone of glyphosate with RMSD of 0.92 Å for N-fluoro-glyphosate and 0.83 Å for N-chloro-glyphosate. The linear N-hydroxyamino-glyphosate exhibits relatively more number of intermolecular hydrogen bond interactions as compared to the other two analogs. Further, comparison of CES of previously studied glyphosate analogs such as N-hydroxy-glyphosate (2.2 μM) and N-amino-glyphosate (0.61 μM) with the present systems reveals the order of activity as: N-hydroxyamino-glyphosate > N-fluoro-glyphosate > N-chloro-glyphosate based on CES flexibility. Also, the calculated heats of formation of N-fluoro-glyphosate, N-chloro-glyphosate, and N-hydroxyamino-glyphosate are −288, −209, and −288 kcal/mol, respectively, which clearly indicate that the N-hydroxyamino and N-fluoro analogs of glyphosate are thermodynamically more stable than N-amino-glyphosate (−278 kcal/mol).     

Design,synthesis, and anti- Trypanosoma cruzi evaluation of a new class of cell-growth inhibitors structurally related to Fenoxycarb

Several compounds, structurally related to the insect-growth regulator Fenoxycarb ( 1 ), were designed and synthesized. These compounds were tested as growth inhibitors of Trypanosoma cruzi cells (epimastigotes). Compounds 6, 16, 18 , and 22 were very active against T. cruzi making them promising good candidates either for blood-bank sterilization of Chagas'-disease surveillance, while compounds 11 , 12 , 13 , and 19 showed a moderate degree of activity.