[Show abstract][Hide abstract] ABSTRACT: Hybrids of vinca alkaloids and phomopsin A, linked by a glycine pattern, have been synthetized in one or two steps, by an original insertion reaction. These compounds have been elaborated in order to interact with both “vinca site” and “peptide site” of the vinca domain in tubulin. Two out of three hybrids are potent inhibitors of microtubules assembly and they present good cytotoxicity against different cell lines. Molecular modelling studies show they could bind, within the vinca domain, in similar spatial regions as that of vinca and phomopsin thanks to the flexibility provided by the glycine linker used to elaborate these hybrids.
[Show abstract][Hide abstract] ABSTRACT: We present a new protocol aimed at the structure-based design of drug-like molecules using a fragment approach. It starts from a suitably placed and well-defined "base fragment" and then uses an incremental construction algorithm and a scoring function to grow the molecule into prioritized candidates. The selection of the most promising solutions for synthesis and validation is guided by the optimization of the calculated ligand efficiency indices known as binding efficiency index (BEI) and surface efficiency index (SEI), which allow the user to navigate proficiently in chemico-biological space. A test case for the protocol is exemplified here using published data for inhibitors of protein kinase B, aka AKT, a key enzyme in several signal transduction pathways. Our procedure was able to identify the main features responsible for the binding of inhibitors and guided the selection process towards molecules that included or resembled those shown as the most active in the original studies.
[Show abstract][Hide abstract] ABSTRACT: Hybrids of vinca alkaloids and phomopsin A have been elaborated with the aim of interfering with both the "vinca site" and the "peptide site" of the so-called vinca domain in tubulin. They were synthesized by an efficient one-pot procedure that links directly the octahydrophomopsin lateral chain to the velbenamine moiety of 7'-homo-anhydrovinblastine. In their modeled complexes with tubulin, these hybrids were found to superimpose nicely on the tubulin-bound structures of vinblastine and phomopsin A. This good matching can account for the fact that two of them are very potent inhibitors of microtubule assembly and display good cytotoxicity against four cancer cell lines.
Journal of Medicinal Chemistry 05/2014; · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of novel thienopyrimidin-4-amines have been synthesized and evaluated as phosphodiesterase (PDE) inhibitors. A rationale for the observed selectivity against PDE7 has been obtained from molecular modelling studies on the most active compounds.
[Show abstract][Hide abstract] ABSTRACT: Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.
[Show abstract][Hide abstract] ABSTRACT: The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 Å resolution; it exhibits an αβ-hydrolase fold, consisting of a central β-sheet surrounded by α-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides.
PLoS ONE 03/2014; 9(3):e92257. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.
PLoS ONE 02/2014; 9(2):e89526. · 3.53 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are four orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogs has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.
ACS Chemical Biology 02/2014; · 5.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The type II dehydroquinase (DHQ2), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism these bacterial enzymes are reported. Asp89*/Asp88* from a symmetry-related neighboring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, while for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. Molecular dynamics simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine and after a large conformational change of a loop containing two conserved arginines (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role of these residues. The new insights can be used to advantage in the structure-based design of novel inhibitors.
[Show abstract][Hide abstract] ABSTRACT: ALFA is a fast computational tool for the conformational analysis of small molecules that uses a custom-made iterative algorithm to provide a set of representative conformers in an attempt to reproduce the diversity of states in which small molecules can exist, either isolated in solution or bound to a target. The results shown in this work prove that ALFA is fast enough to be integrated into massive high-throughput virtual screening protocols with the aim of incorporating ligand flexibility and also that ALFA reproduces crystallographic X-ray structures of bound ligands with great accuracy. Furthermore, the application includes a graphical user interface that allows its use through the popular molecular graphics program PyMOL to make it accessible to non-expert users. ALFA is distributed free of charge upon request from the authors.
Journal of Chemical Information and Modeling 01/2014; · 4.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that cells deficient in the Fanconi anemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anticancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand crosslink (ICL). Now we expand our observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells.
The sensitivity of FA-competent and FA-deficient transformed and untransformed cells to mitomycin C (MMC) and to three DBATs, trabectedin, Zalypsis and PM01183, was first assessed. Additionally, the functional interaction of these drugs with the FA pathway was comparatively investigated.
While untransformed FA-deficient hematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells showed the expected hypersensitivity to MMC. Furthermore, while MMC always activated the FA pathway, DBATs inhibited FA pathway in the cancer cell lines tested and this enhanced their response to MMC.
Taken together, our data show that although DBATs may functionally interact with DNA like agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors, rather than activators. Moreover, this effect was most significant in a variety of cancer cells. We propose that the inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of "fanconizing" cancer cells in order to make them more sensitive to other antitumor drugs.
British Journal of Pharmacology 08/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some computational methods currently exist that are employed to infer protein targets of small molecules and can therefore be used to find new targets for existing drugs, with the goals of repositioning the molecule for a different therapeutic purpose or explaining off-target effects due to multiple targeting. Inherent limitations, however, arise from the fact that chemical analogy is calculated on the basis of common frameworks or scaffolds and also because target information is neglected. The method we present addresses these issues by taking into account 3D information from both the ligand and the target.
ElectroShape is an established method for ultra-fast comparison of the shapes and charge distributions of ligands that is validated here for prediction of on-target activities, off-target profiles and adverse effects of drugs and drug-like molecules mined from the DrugBank database.
The method is shown to predict polypharmacology profiles and relate targets from two complementary viewpoints (ligand- and target-based networks).
The open-access web tool presented here (http://ub.cbm.uam.es/chemogenomics/) allows interactive navigation in a unified 'pharmacological space' from the viewpoints of both ligands and targets. It also enables prediction of pharmacological profiles, including likely side effects, for new compounds. We hope this web interface will help many pharmacologists to become aware of this new paradigm (up to now mostly used in the realm of the so-called "chemical biology") and encourage its use with a view to revealing 'hidden' relationships between new and existing compounds and pharmacologically relevant targets.
British Journal of Pharmacology 07/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sixteen new 7' homo-anhydrovinblastine derivatives were prepared in one or two steps from vinorelbine by means of an original and regiospecific rearrangement and subsequent diastereoselective reduction. This strategy has allowed fast access to a family of vinca alkaloid derivatives with an enlarged and functionalized ring C'. Their synthesis and biological evaluation are reported. One compound (compound 35) is 1.7 times more active than vinorelbine as a tubulin assembly inhibitor. Moreover, some of these compounds are highly cytotoxic and two of them are more potent than vinorelbine on HCT116 and K562 cell lines. Molecular modeling studies, carried out with two of the new vinca derivatives, provide useful hints about how a given functionalization introduced at positions 7' and 8' of the C' ring results in improved binding interactions between one of the new derivatives and the interdimer interface when compared to the parent compound vinblastine.
Journal of Medicinal Chemistry 07/2013; · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding at the interface: We tested the inhibitory activity of a set of peptide sequences derived from an α-helix of the dimeric trypanothione reductase from Leishmania infantum. Replacement of a glutamic acid residue with a lysine promoted monomer dissociation and enzyme inhibition.
[Show abstract][Hide abstract] ABSTRACT: Two series of 5'-triphenylmethyl (trityl)-substituted thymidine derivatives were synthesized and tested against Leishmania infantum axenic promastigotes and amastigotes. Several of these compounds show significant antileishmanial activity, with IC50 values in the low micromolar range. Among these, 3'-O-(isoleucylisoleucyl)-5'-O-(3,3,3-triphenylpropanoyl)thymidine displays particularly good activity against intracellular amastigotes. Assays performed to characterize the nature of parasite cell death in the presence of the tritylthymidines indicated significant alterations in mitochondrial transmembrane potential, an increase in superoxide concentrations, and also significant decreases in DNA degradation during the cell death process. Results point to the mitochondrial nuclease LiEndoG as a target for the action of this family of compounds.
[Show abstract][Hide abstract] ABSTRACT: Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of β-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds () proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand-receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications.
[Show abstract][Hide abstract] ABSTRACT: Herein we report comparative binding energy (COMBINE) analyses to derive quantitative structure-activity relationship (QSAR) models that help rationalize the determinants of binding affinity for inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway. Independent COMBINE models were derived for Helicobacter pylori and Mycobacterium tuberculosis DHQ2, which is an essential enzyme in both these pathogenic bacteria that has no counterpart in human cells. These studies quantify the importance of the hydrogen bonding interactions between the ligands and the water molecule involved in the DHQ2 reaction mechanism. They also highlight important differences in the ligand interactions with the interface pocket close to the active site that could provide guides for future inhibitor design.
[Show abstract][Hide abstract] ABSTRACT: During the last 30 years, significant progress has been made in the development of novel antiviral drugs, mainly crystallizing in the establishment of potent antiretroviral therapies and the approval of drugs inhibiting hepatitis C virus replication. Although major targets of antiviral intervention involve intracellular processes required for the synthesis of viral proteins and nucleic acids, a number of inhibitors blocking virus assembly, budding, maturation, entry or uncoating act on virions or viral capsids. In this review, we focus on the drug discovery process while presenting the currently used methodologies to identify novel antiviral drugs by using a computer-based approach. We provide examples illustrating structure-based antiviral drug development, specifically neuraminidase inhibitors against influenza virus (e.g. oseltamivir and zanamivir) and human immunodeficiency virus type 1 protease inhibitors (i.e. the development of darunavir from early peptidomimetic compounds such as saquinavir). A number of drugs in preclinical development acting against picornaviruses, hepatitis B virus and human immunodeficiency virus and their mechanism of action are presented to show how viral capsids can be exploited as targets of antiviral therapy.
[Show abstract][Hide abstract] ABSTRACT: Molecular modeling and computer simulation techniques have matured significantly in recent years and proved their value in the study of drug-DNA, drug-DNA-protein, drug-protein and protein-protein interactions. Evolution in this area has gone hand-in-hand with an increased availability of structural data on biological macromolecules, major advances in molecular mechanics force fields and considerable improvements in computer technologies, most significantly processing speeds, multiprocessor programming and data-storage capacity. The information derived from molecular simulations of drug-receptor complexes can be used to extract structural and energetic information that is usually beyond current experimental possibilities, provide independent accounts of experimentally observed behavior, help in the interpretation of biochemical or pharmacological results, and open new avenues for research by posing novel relevant questions that can guide the design of new experiments. As drug-screening tools, ligand- and fragment-docking platforms stand out as powerful techniques that can provide candidate molecules for hit and lead development. This review provides an overall perspective of the main methods and focuses on some selected applications to both classical and novel anticancer targets.