Stanislav Gobec

University of Ljubljana, Lubliano, Ljubljana, Slovenia

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Publications (137)354.32 Total impact

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    ABSTRACT: Although a recent study of Debord et al. in Biochimie (2014; 97:72-77) described the thermodynamics of the catalysed hydrolysis of phenyl acetate by human paraoxonase-1, the mechanistic details along the reaction route of this enzyme remain unclear. Therefore, we briefly present the solvent kinetic isotope effects on the phenyl acetate esterase activity of paraoxonase-1 and its inhibition with the phenyl methylphosphonate anion, which is a stable isosteric analogue that mimics the high-energy tetrahedral intermediate on the hydroxide-promoted hydrolysis pathway. The data show normal isotope effects, while proton inventory analysis indicates that two protons contribute to the kinetic isotope effect. Coherently, moderate competitive inhibition with the phenyl methylphosphonate anion reveals that the rate-limiting transition state suboptimally resembles the tetrahedral intermediate. The implications of these findings can be attributed to two possible reaction mechanisms that might occur during the paraoxonase-1–catalysed hydrolysis of phenyl acetate.
    Biochimie 11/2014; · 3.14 Impact Factor
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    ABSTRACT: Butyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally, compound 1 inhibited amyloid β1-42 peptide self-induced aggregation into fibrils (by 61.7% at 10 µM), and protected cultured SH-SY5Y cells against amyloid-β-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease.
    Journal of medicinal chemistry. 09/2014;
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    ABSTRACT: 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.
    Bioorganic & medicinal chemistry. 06/2014;
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    ABSTRACT: In this study, we present the synthesis, biological characterization, and first crystal structure of an organometallic-clioquinol complex. Combining ruthenium with the established apoptotic agent and 8-hydroxyquinoline derivative, clioquinol, resulted in a complex that induces caspase-dependent cell death in leukaemia cells. This activity is copper independent and is improved compared to the parent compound, clioquinol. The study of the mode of action reveals that this clioquinol-ruthenium complex does not intercalate between DNA base pairs. Additionally, this clioquinol-ruthenium complex shows proteasome-independent inhibition of the NFκB signalling pathway, with no effects on cell-cycle distribution. These data suggest a mechanism of action that involves a target profile that is different from that for clioquinol alone.
    Dalton Transactions 04/2014; · 4.10 Impact Factor
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    ABSTRACT: 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 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 the 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 a hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities how computational tools can be proficiently used at all stages of the drug discovery process.
    Journal of Chemical Information and Modeling 04/2014; · 4.30 Impact Factor
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    ABSTRACT: The widespread emergence of resistant bacterial strains is becoming a serious threat to public health. This thus signifies the need for the development of new antibacterial agents with novel mechanisms of action. Continuous efforts in the design of novel antibacterials remain one of the biggest challenges in drug development. In this respect, the Mur enzymes, MurA-F, that are involved in the formation of UDP-N-acetylmuramyl-pentapeptide can be genuinely considered as promising antibacterial targets. This review provides an in-depth insight into the recent developments in the field of inhibitors of the MurA-F enzymes. Special attention is also given to compounds that act as multiple inhibitors of two, three or more of the Mur enzymes. Moreover, the reasons for the lack of preclinically successful inhibitors and the challenges to overcome these hurdles in the next years are also debated.
    Bioorganic Chemistry 04/2014; · 1.73 Impact Factor
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    ABSTRACT: Predicting the endocrine disruption potential of compounds is a daunting, but essential task. Here we report a new tool for this purpose that we termed Endocrine Disruptome. It is a free and simple to use web service that runs on an open source platform called DoTS (Docking interface for Target Systems). The molecular docking is handled via AutoDock Vina. Compounds are docked to 18 integrated and well validated crystal structures of 14 different human nuclear receptors: androgen receptor, estrogen receptors α and β, glucocorticoid receptor, liver X receptors α and β, mineralocorticoid receptor, peroxisome proliferator activated receptor α, β/δ and γ, progesterone receptor, retinoid X receptor α, thyroid receptor α and β. Endocrine Disruptome is free of charge and available at http://endocrinedisruptome.ki.si.
    Journal of Chemical Information and Modeling 03/2014; · 4.30 Impact Factor
  • Urban Košak, Boris Brus, Stanislav Gobec
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    ABSTRACT: Two convenient procedures are developed for the synthesis of protected piperidin-4-ylmethanamine derivatives from isonipecotamide (I) and isonipecotic acid (VI).
    Tetrahedron Letters 03/2014; 55(12):2037–2039. · 2.40 Impact Factor
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    ABSTRACT: MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis.
    European Journal of Medicinal Chemistry 02/2014; 73:83–96. · 3.43 Impact Factor
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    ABSTRACT: A simple five-step synthesis of fully substituted (4RS,5RS)-4-aminopyrazolidin-3-ones as analogs of D-cycloserine was developed. It comprises a two-step preparation of 5-substituted (4RS,5RS)-4-(benzyloxycarbonylamino)pyrazolidin-3-ones, reductive alkylation at N(1), alkylation of the amidic N(2) with alkyl halides, and simultaneous hydrogenolytic deprotection/reductive alkylation of the primary NH2 group. The synthesis enables an easy stepwise functionalization of the pyrazolidin-3-one core with only two types of common reagents, aldehydes (or ketones) and alkyl halides. The structures of products were elucidated by NMR spectroscopy and X-ray diffraction.
    Helvetica Chimica Acta 02/2014; 97(2). · 1.38 Impact Factor
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    ABSTRACT: We describe a ligand-based approach towards compounds with more specific targeting for Burkitt's lymphoma. Using three-dimensional ligand-based similarity searches and a previously described hit compound, we have identified six compounds that are chemically different but with similar spatial conformations. Biological evaluation revealed that one compound has better growth inhibition and improved selectivity towards Burkitt's lymphoma cells than the query compound. However, initial mechanism-of-action studies show a different target profile in comparison with the previous hit compound, which does not involve the inhibition of the proteasome or the NFκB pathway. The data from this study provide a solid basis for further efforts in the search for selective agents against Burkitt's lymphoma.
    Molecules (Basel, Switzerland). 01/2014; 19(11):19209-19.
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    ABSTRACT: CYP53A15, from the sorghum pathogen Cochliobolus lunatus is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. In our work we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus, and two other fungi, Aspergillus niger and Pleurotus ostreatus. In order to elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development. This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 12/2013; · 2.20 Impact Factor
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    ABSTRACT: Aims: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. Methods and Results: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. Conclusion: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. Significance and Impact of the Study: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.
    Journal of Applied Microbiology 12/2013; [Epub ahead of print](xx):1-12.. · 2.20 Impact Factor
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    ABSTRACT: Abstract The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.
    Biomolecular concepts 12/2013; 4(6):539-56.
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    ABSTRACT: Toll-like receptor 4 (TLR4) in complex with its accessory protein MD-2 represents an emerging target for the treatment of severe sepsis and neuropathic pain. We performed structure-based and ligand-based virtual screening targeting the TLR4-MD-2 interface. Three in silico hit compounds showed promising TLR4 antagonistic activities with micromolar IC50 values. These compounds also suppressed cytokine secretion by human peripheral blood mononuclear cells. The specific affinity of the most potent hit was confirmed by surface plasmon resonance direct-binding experiments. The results of our study represent a very promising starting point for the development of potent small-molecule antagonists of TLR4.
    European Journal of Medicinal Chemistry 10/2013; 70C:393-399. · 3.43 Impact Factor
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    ABSTRACT: Formation of the peptidoglycan stem pentapeptide requires the insertion of both L or D amino acids by the ATP dependent ligase enzymes MurC, D, E and F. The stereo chemical control of the third position amino acid in the pentapeptide, is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance and pathogenesis. Here we determine the X-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) at 1.8 angstrom resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In-vivo analysis and metabolomic data reveals that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus directed antimicrobials based, on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: The synthesis of U-shaped conformationally constrained analogues of peptides based on the 3-amino-2-oxo-1,5-diazabicyclo[3.3.0]octane-8-carboxylic acid scaffold was developed. [3+2] Cycloadditions of (1Z,4R*,5R*)-1-arylmethylidene-4-benzyloxycarbonylamino-3-oxo-5-phenylpyrazolidin-1-ium-2-ides to tert-butyl acrylate and tert-butyl methacrylate gave the corresponding racemic cycloadducts, in most cases as mixtures of isomers, which were separated by preparative chromatography. Selective deprotection of the C- and the N-terminal of these heterocyclic dipeptides followed by coupling with (S)-alanine derivatives and chromatographic separation furnished the non-racemic tripeptides as target compounds. The structures of racemic cycloadducts and non-racemic final products were determined by NMR spectroscopy and X-ray diffraction. The synthesized compounds were also evaluated for inhibition of MurD ligase and d-alanine:d-alanine ligase.
    Tetrahedron 08/2013; 69(32):6648-6665. · 2.80 Impact Factor
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    ABSTRACT: Abstract Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc-l-Ala-γ-d-Glu-meso-DAP (or l-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.
    European Journal of Medicinal Chemistry 08/2013; 66:32-45. · 3.43 Impact Factor
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    ABSTRACT: The ATP-dependent UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase MurF catalyses the last step in the cytoplasmic phase of peptidoglycan biosynthesis, which is critical in the formation of the bacterial cell wall and in the recycling of peptidoglycan intermediates. In this study, the crystallization of MurF from the Gram-negative pathogen Pseudomonas aeruginosa in the presence of its UDP-MurNAc-tripeptide substrate is reported. The crystals belonged to space group P212121, with unit-cell parameters a = 57.81, b = 87.29, c = 92.61 Å, and data were collected to 1.92 Å resolution, allowing study of the enzyme in the substrate-liganded form for the first time.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2013; 69(Pt 5):503-5. · 0.55 Impact Factor
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    ABSTRACT: Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H2O2 oxidative stress.
    Fungal Biology 05/2013; 117(5):368-379. · 2.08 Impact Factor

Publication Stats

865 Citations
354.32 Total Impact Points

Institutions

  • 1998–2014
    • University of Ljubljana
      • • Faculty of Pharmacy
      • • Faculty of Medicine
      • • Institute of Microbiology and Immunology
      Lubliano, Ljubljana, Slovenia
  • 2013
    • Blood transfusion centre of Slovenia - Zavod Republike Slovenije za transfuzijsko medicino
      Lubliano, Ljubljana, Slovenia
  • 2012
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2008–2012
    • Université Paris-Sud 11
      • Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (IBBMC)
      Orsay, Île-de-France, France
  • 2009–2011
    • National Institute of Chemistry
      Lubliano, Ljubljana, Slovenia
  • 2007
    • Sandoz
      Bâle, Basel-City, Switzerland