In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum (LiNDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae, two ubiquinone-binding sites (UQI and UQII) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of LiNDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the LiNDH2 ubiquinone-binding site 1-UQI. Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum. The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIα targeting the ATP binding site.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of novel anticancer agents. Starting from our discovered 4-amino-1,3,5-triazine inhibitors of htIIα, we investigated a library of 2,4,6-trisubstituted-1,3,5-triazines for novel inhibitors that bind to the htIIα ATP binding site using a combination of structure-based and ligand-based pharmacophore models and molecular docking. 4,6-substituted-1,3,5-triazin-2(1H)-ones 8, 9 and 14 were identified as novel inhibitors with activity comparable to the established drug etoposide (1). Compound 8 inhibits the htIIα decatenation in a superior fashion to etoposide. Cleavage assays demonstrated that selected compounds 8 and 14 do not act as poisons and antagonize the poison effect of etoposide. Microscale thermophoresis (MST) confirmed binding of compound 8 to the htIIα ATPase domain and compound 14 effectively inhibits the htIIα mediated ATP hydrolysis. The molecular dynamics simulation study provides further insight into the molecular recognition. The 4,6-disubstituted-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the to the htIIα ATPase domain.

Structural elucidation of transmembrane transporter protein bilitranslocase: conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.

Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds.

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.

Inhibitor design strategy based on an enzyme structural flexibility: a case of bacterial MurD ligase.

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.

Cluster-based molecular docking study for in silico identification of novel 6-fluoroquinolones as potential inhibitors against Mycobacterium tuberculosis.

A classical protein sequence alignment and homology modeling strategy were used for building three Mycobacterium tuberculosis-DNA gyrase protein models using the available topoII-DNA-6FQ crystal structure complexes originating from different organisms. The recently determined M. tuberculosis-DNA gyrase apoprotein structures and topoII-DNA-6FQ complexes were used for defining the 6-fluoroquinolones (6-FQs) binding pockets. The quality of the generated models was initially validated by docking of the cocrystallized ligands into their binding site, and subsequently by quantitative evaluation of their discriminatory performances (identification of active/inactive 6-FQs) for a set of 145 6-FQs with known biological activity values. The M. tuberculosis-DNA gyrase model with the highest estimated discriminatory power was selected and used afterwards in an additional molecular docking experiment on a mixed combinatorial set of 427 drug-like 6-FQ analogs for which the biological activity values were predicted using a prebuilt counter-propagation artificial neural network model. A novel three-level Boolean-based [T/F (true/false)] clustering algorithm was used to assess the generated binding poses: Level 1 (geometry properties assessment), Level 2 (score-based clustering and selection of the (T)-signed highly scored Level 1 poses), and Level 3 (activity-based clustering and selection of the most "active" (T)-signed Level 2 hits). The frequency analysis of occurrence of the fragments attached at R(1) and R(7) position of the (T)-signed 6-FQs selected in Level 3 revealed several novel attractive fragments and confirmed some previous findings. We believe that this methodology could be successfully used in establishing novel possible structure-activity relationship recommendations in the 6-FQs optimization, which could be of great importance in the current antimycobacterial hit-to-lead processes.

Molecular dynamics simulation and linear interaction energy study of D-Glu-based inhibitors of the MurD ligase.

The biosynthetic pathway of the bacterial peptidoglycan, where MurD is an enzyme involved at the intracellular stage of its construction, represents a collection of highly selective macromolecular targets for novel antibacterial drug design. In this study as part of our investigation of the MurD bacterial target two recently discovered classes of the MurD ligase inhibitors were investigated resulting from the lead optimization phases of the N-sulfonamide D-Glu MurD inhibitors. Molecular dynamics simulations, based on novel structural data, in conjunction with the linear interaction energy (LIE) method suggested the transferability of our previously obtained LIE coefficients to further D-Glu based classes of MurD inhibitors. Analysis of the observed dynamical behavior of these compounds in the MurD active site was supported by static drug design techniques. These results complement the current knowledge of the MurD inhibitory mechanism and provide valuable support for the D-Glu paradigm of the inhibitor design.

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B.

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).

Target fishing and docking studies of the novel derivatives of aryl-aminopyridines with potential anticancer activity.

A set of 16 previously synthesized aryl-aminopyridine and aryl-aminoquinoline derivatives have been evaluated for cytotoxic activity against three cancer cell lines (human cervical cancer-HeLa; human chronic myeloid leukemia-K562; human melanoma-Fem-x) and two types of normal peripheral blood mononuclear cells, with and without phytohemaglutinin (PBMC-PHA; PBMC+PHA). Twelve of the studied compounds showed moderate cytotoxicity, with selectivity against K562 but not the remaining two cancer cell lines. Four compounds were not active in cytotoxicity assays, presumably due to high predicted lipophilicity and low solubility. To rationalize the observed cytotoxic effects, structure-based virtual screening was carried out against a pool of potential targets constructed using the inverse docking program Tarfisdock and bibliographical references. The putative targets were identified on the basis of the best correlation between docking scores and in vitro cytotoxicity. It is proposed that the mechanism of action of the studied aminopyridines involves the disruption of signaling pathways and cancer cell cycle through the inhibition of cyclin-dependent kinases and several tyrosine kinases, namely Bcr-Abl kinase and KIT receptor kinase. The obtained results can guide further structural modifications of the studied compounds aimed at developing selective agents targeting proteins involved in cancer cell survival and proliferation.

Quantitative structure-activity relationship study of antitubercular fluoroquinolones.

Quantitative structure-activity relationship study on three diverse sets of structurally similar fluoroquinolones was performed using a comprehensive set of molecular descriptors. Multiple linear regression technique was applied as a preprocessing tool to find the set of relevant descriptors (10) which are subsequently used in the artificial neural networks approach (non-linear procedure). The biological activity in the series (minimal inhibitory concentration (µg/mL) was treated as negative decade logarithm, pMIC). Using the non-linear technique counter propagation artificial neural networks, we obtained good predictive models. All models were validated using cross validation leave-one-out procedure. The results (the best models: Assay1, R = 0.8108; Assay2, R = 0.8454, and Assay3, R = 0.9212) obtained on external, previously excluded test datasets show the ability of these models in providing structure-activity relationship of fluoroquinolones. Thus, we demonstrated the advantage of non-linear approach in prediction of biological activity in these series. Furthermore, these validated models could be proficiently used for the design of novel structurally similar fluoroquinolone analogues with potentially higher activity.

In silico discovery of 2-amino-4-(2,4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B.

Cyclothialidines are a class of bacterial DNA gyrase B (GyrB) subunit inhibitors, targeting its ATP-binding site. Starting from the available structural information on cyclothialidine GR122222X (2), an in silico virtual screening campaign was designed combining molecular docking calculations with three-dimensional structure-based pharmacophore information. A novel class of 2-amino-4-(2,4-dihydroxyphenyl)thiazole based inhibitors (5-9) with low micromolar antigyrase activity was discovered.

Chemometrical Exploration of Combinatorially Generated Drug-like Space of 6-fluoroquinolone Analogs: A QSAR Study.

A classical virtual combinatorial chemistry approach (CombiChem) was applied for combinatorial generation of 5590 novel structurally-similar 6-fluoroquinolone analogs by using a virtual synthetic pathway with selected primary (43) and secondary amines (130). The obtained virtual combinatorial library was filtered using an in-house developed set of cheminformatics drug-likeness filters with pre-integrated Boolean options (TRUE/FALSE) for compounds reduction/selection. The retained number (304) of fluoroquinolone analogs (with TRUE outcome) defines the drug-like chemical space (CombiData). Quantitative structure-activity relationships (QSAR) study on these 304 virtually generated 6-fluoroquinolone analogs with unknown activity values was performed using a pre-built five-parameter multiple linear regression (MLR) model developed on a set of compounds with experimentally determined activity values (Rtr = 0.8417, Rtr-cv = 0.7884). The obtained activity values for the unknown compounds together with the model results were used to define the applicability domain (AD). The obtained AD offers a good graphical representation and establishment of structure-activity relationships (SAR) which could be used for design of new 6-fluoroquinolones with possible better activity.

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method.

MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase), a three-domain bacterial protein, catalyses a highly specific incorporation of D-glutamate to the cytoplasmic intermediate UDP-N-acetyl-muramoyl-L-alanine (UMA) utilizing ATP hydrolysis to ADP and P(i). This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl-phosphate intermediate, followed by a nucleophilic attack of D-glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP-N-acetyl-muramoyl-L-alanine-D-glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs.

Discovery of novel benzene 1,3-dicarboxylic acid inhibitors of bacterial MurD and MurE ligases by structure-based virtual screening approach.

The peptidoglycan biosynthetic pathway provides an array of potential targets for antibacterial drug design, attractive especially with respect to selective toxicity. Within this pathway, the members of the Mur ligase family are considered as promising emerging targets for novel antibacterial drug design. Based on the available MurD crystal structures co-crystallised with N-sulfonyl glutamic acid inhibitors, a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates-benzene 1,3-dicarboxylic acid derivatives-were identified and compounds 14 and 16 found to possess dual MurD and MurE inhibitory activity.

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase.

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD.

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design.

Discovery and development of ATPase inhibitors of DNA gyrase as antibacterial agents.

DNA gyrase is an attractive and well established target for the development of antibacterial agents. This bacterial enzyme, whose biological function is to control the topological state of DNA molecules, consists of two catalytic subunits; GyrA is responsible for DNA breakage and reunion, while the subunit GyrB contains the ATP-binding site. Coumarins and cyclothialidines are natural products that inhibit the ATPase activity of DNA gyrase by blocking the binding of ATP to subunit GyrB. The mechanism of action of these compounds was exhaustively characterized by biochemical methods and supported by protein crystallography. The abundance of crystallographic data on the N-terminal domain of GyrB in its complexes with various ligands has enabled the structure-based design of novel efficient chemotypes as inhibitors of the ATPase domain. This review summarizes the discovery of ATPase inhibitors of DNA gyrase B in the last decade and their development as potential antibacterial agents.

4-Substituted trinems as broad spectrum beta-lactamase inhibitors: structure-based design, synthesis, and biological activity.

A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary response to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic structural classes is the beta-lactams, in which the most common and the most efficient mechanism of bacterial resistance is the synthesis of beta-lactamases. Class C beta-lactamase enzymes are primarily cephalosporinases, mostly chromosomally encoded, and are inducible by exposure to some beta-lactam agents and resistant to inhibition by marketed beta-lactamase inhibitors. In an ongoing effort to alleviate this problem a series of novel 4-substituted trinems was designed and synthesized. Significant in vitro inhibitory activity was measured against the bacterial beta-lactamases of class C and additionally against class A. The lead compound LK-157 was shown to be a potent mechanism-based inactivator. Acylation of the active site Ser 64 of the class C enzyme beta-lactamase was observed in the solved crystal structures of two inhibitors complexes to AmpC enzyme from E. cloacae. Structure-activity relationships in the series reveal the importance of the trinem scaffold for inhibitory activity and the interesting potential of the series for further development.