Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4,5`bithiazols inhibitors of gyrase B with a low micromolar inhibitory activity, by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques: Differential Scanning Fluorimetry (DSF), Surface Plasmon Resonance (SPR) and microscale thermophoresis (MST). The binding mode of the potent inhibitor was revealed by X-ray crystallography, confirming our initial in silico binding model.
COBISS.SI-ID: 4999450
In this study we presented 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 (Perdih et al. Proteins 2007 in Perdih et al.CTC 2012) 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 one compound 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 aminothiazole compound 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.
COBISS.SI-ID: 5462810
We were a collaborating partner with the research groups from the University of Delhi, India and Infectious Disease Research Institute (Seattle ZDA) in the development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The assay is optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Our furan-based benzene monocarboxylic acid derivatives were used in the validation of their newly developed one-pot assay that reconstructs the entire Mur pathway (from M. tuberculosis). These results also showed that furan-based derivatives are active on a variety of Mur ligases from different bacterial species and have potential for multispectral action.
COBISS.SI-ID: 6019098
Starting from our discovered 4-amino-1,3,5-triazine inhibitors of human topoisomerase II? we initiated an in silico design study of a focused library of 2,4,6-trisubstituted-1,3,5-triazines to optimize the series.6substituted-4-(benzylthio)-1,3,5-triazin-2(1H)-ones were identified as novel topoII? inhibitors. The most active compound was able to inhibit the human topo II?-mediated DNA decatenation and did not induce double-stranded breaks. Furthermore it inhibited the cleavage reaction induced by etoposide showing its inhibition step is in the early stages of topoII catalytic cycle. Binding studies using microscale thermophoresis (MST) established binding of 1,3,5-triazin-2(1H)-one compound to the htII? ATPase domain, confirming for the first time the binding of a monocyclic catalytic inhibitors to the htII? ATPase domain. The discovered 6-substituted-4-(benzylthio)-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the ATP binding site and have the potential for hit to lead development of a monocyclic series of catalytic inhibitors of human DNA topoisomerase II?.
COBISS.SI-ID: 3886961
Monoamine oxidases (MAOs) are flavoenzymes important in regulating amine neurotransmitter levels and are the central pharmacological targets in treating depression and Parkinson's disease. On the basis of quantum chemical calculations, we have proposed a new two-stephydride mechanism for the MAO-catalysed oxidative deamination of amines. In the rate-limiting first step, through its N5 atom, the flavin abstracts a hydride anion from the substrate alpha-carbon atom and forms a strong covalent adduct with the thus created cation. This is followed by flavin N1 deprotonation of the substrate amino group, facilitated with two active-site water molecules, to produce fully reduced flavin, FADH2, and neutral imine. We have demonstrated that our mechanism is in agreement with available experimental data and provided evidence against both traditional polar nucleophilic and single-electron radical pathways. These results provide valuable information for mechanistic studies on other flavoenzymes and for the design of new antidepressants and antiparkinsonian drugs.
COBISS.SI-ID: 5142810
We collected experimental kinetic rate constants for chemical processes responsible for the development and progress of neurodegeneration, focused on the enzymatic and non-enzymatic degradation of amine neurotransmitters and their reactive and neurotoxic metabolites. A gross scheme of neurodegeneration on the molecular level is based on two pathways. Firstly, reactive species oxidize heavy atom ions, which enhances the interaction with alpha synuclein, thus promoting its folding to the beta form and giving rise to insoluble amyloid plaques. The latter prevents the function of vesicular transport leading to gradual neuronal death. In the second pathway reactive oxygen species chemically decompose neuron membranes. The current analysis can be employed in developing strategies for the prevention and treatment of neurodegeneration, and, hopefully, aid in the building of an overall kinetic molecular model of neurodegeneration itself.
COBISS.SI-ID: 5723162
This work scrutinizes the Y326I point mutation effect on the kinetics of oxidative deamination of phenylethylamine (PEA) catalyzed by monoamine oxidase B (MAO B) enzyme. PEA is a neuromodulator capable of affecting plasticity of the brain and is an endogenous amphetamine responsible for mood enhancing effect caused by physical exercise. Comparison of reaction free energy profiles delivered by simulation of the reaction in the wild type MAO B and its Y326I mutant yields an increase of the barrier by 1.06 kcal/mol on mutation, corresponding to a roughly 6-fold decrease in the reaction rate. This is in excellent agreement with experimental kinetic studies.
COBISS.SI-ID: 6321434
This work addresses the selectivity of the two monoamine oxidase isoenzymes (MAO A and MAO B) towards adrealine substrate, by using state-of-the-art Empirical Valence Bond (EVB) simulations. We studied the chemical reactivity of the MAO A catalyzed decomposition of adrenaline and we obtained a value of activation free energy of 17.3 ± 0.4 kcal/mol. The corresponding value for MAO B is 15.7 ± 0.7 kcal/mol. Both values are in good agreement with the estimated experimental barriers of 16.6 and 16.0 kcal/mol for MAO A and MAO B, respectively. The fact that we reproduced the kinetic data and preferential catalytic effect of MAO B over MAO A gives additional support to the validity of the proposed hydride transfer mechanism. Furthermore, we demonstrate that adrenaline is preferably involved in the reaction in a neutral rather than in a protonated form due to considerably higher barriers computed for the protonated adrenaline substrate. The results are discussed in the context of chemical mechanism of MAO enzymes and possible applications of multiscale simulation to rationalize the effects of MAO activity on adrenaline level.
COBISS.SI-ID: 6282778
The idea that tunneling is enhanced by the compression of the donor–acceptor distance has attracted significant interest. In particular, recent studies argued that this proposal is consistent with pressure effects on enzymatic reactions, and that the observed pressure effects support the idea of vibrationally enhanced catalysis. However, a careful analysis of the current works reveals serious inconsistencies in the evidence presented to support these hypotheses. Apparently, tunneling decreases upon compression, and external pressure does not lead to the applicable compression of the free energy surface. Additionally, pressure experiments do not provide actual evidence for vibrationally enhanced catalysis. Finally, the temperature dependence of the entropy change inhydride transfer reactions is shown to reflect simple electrostatic effects.
COBISS.SI-ID: 4420378
The article describes the application of various modern computational biochemistry methods in order to improve the understanding of a relationship between the structure and function of large biological systems including ion channels, transporters, receptors and metabolic enzymes. A brief introduction to various computational techniques is presented. We gave focuses on the monoamine oxidase family of enzymes, which catalyze the degradation of amine neurotransmitters in various parts of the brain, the imbalance of which is associated with the development and progression of a range of neurodegenerative disorders. Inhibitors that act mainly on MAO A are used in the treatment of depression, due to their ability to raise serotonin concentrations, while MAO B inhibitors decrease dopamine degradation and improve motor control in patients with Parkinson disease. Our results give strong support that both MAO isoforms, A and B, operate through the hydride transfer mechanism, which is important information in aiding a rational design of more effective MAO inhibitors. Relevance of MAO catalyzed reactions and MAO inhibition in the context of neurodegeneration is also discussed.
COBISS.SI-ID: 5942554