Voltage-gated sodium channels are large transmembrane protein complexes responsible for ion current transmission in nerve, muscle and endocrine cells and cell systems. Defects in their expression and function are linked to multiple pathological conditions. Treatment of these conditions such as epilepsy, central nervous system diseases, heart complications and various pain states is still challenging due to nonselective action of used drugs on multiple voltage-gated sodium channels and subsequent adverse side effects. Development of selective modulators of specific voltage-gated sodium channel isoforms would thus be beneficial and would enable profound studies on the role of individual voltage-gated sodium channel isoform in patophysiology. Due to under-explored chemical space of marine natural products we selected alkaloids oroidin and clathrodin as starting point for the discovery of novel modulators of voltage-gated sodium channels. Compounds are composed of two terminal fragments, namely 2-aminoimidazole and pyrrole-2-carboxamide, interconnected by a central 3-aminopropene linker moiety. In the scope of our research hypothesis we postulated that terminal aminoimidazole represents key pharmacophoric element, hence we primarily exchanged the central linker moiety with a sterically rigidified piperazine structure by using ligand based drug design methodology. Our next goal was a systematic study of the influence of terminal fragment exchange on potency of synthesized compounds and involvement of postulated pharmacophoric element. All compounds were biologically evaluated on multiple voltage-gated sodium channel isoforms using patch clamp technique. On some of prepared compounds we measured modest potency on voltage-gated sodium channel 1.3 isoform and determined that distance between terminal heterocyclic nitrogens is a crucial parameter. We confirmed our pharmacophoric element assumption as replacement of terminal aminoimidazole led to loss of potency. The role of heterocyclic moiety opposite of aminoimidazole was also elaborated and presence of carbony group at the heterocyclic amide identified as necessary for potency. Replacement of pyrrolo-2-carboxyl fragment with larger indolo-2-carbonyl led to piperazine clathrodin analogue with an IC50 value of 19 μM, selective on 1.3 voltage-gated sodium channel isoform. Prepared library of piperazine oroidin (clathrodin) analogues is a suitable starting point for further research on 1.3 voltage-gated sodium channel selective modulators, especially in the light of emerging new channel structural data.
Secondary metabolites of sea sponges and their symbiotic organisms are involved in reproduction, signalling, regulation and defence roles in wholesome biological systems and they accordingly often display polypharmacological effects. Studied alkaloids oroidin and clathrodin from see sponges of the genus Agelas are reported in literature also as inhibitors of bacterial biofilm formation and antibacterial compounds. The latter observation is especially interesting in the light of emerging incidence of bacterial resistence and downfall of antibacterial therapy effectiveness. In the second part of research thesis we focused on antibacterial targets and identified structural similarity between oroidin lead compounds, prepared oroidin piperazine analogues and small molecule inhibitors of DNA gyrase B reported in literature. DNA Gyrase B is an essential bacterial enzyme responsible for the introduction of negativive supercoils or topological relaxation of DNA molecule during replication. It is composed of two subunits where subunit A performs operations on DNA molecule and subunit B hydrolises ATP supplying the required free energy. In spite of ATP as the substrate of DNA gyrase B, this enzyme subunit is a validated target for discovery of antibacterial compounds.
In contrast with key aminoimidazole fragment, that was essential for potency of piperazine oroidin (clathrodin) analogues on voltage-gated sodium channels, we focused on second pyrrole-2-carboxamide terminal fragment in the design of potential inhibitors of DNA gyrase B. The latter fragment was reported as a structure element that enables crucial interactions with amino acid residues in adenine pocket of ATP binding site on DNA gyrase B. Pyrrole-2-carboxamide or amide moiety is similarly connected to the second pharmacophoric element via a cyclic, aromatic, heteroaromatic, bicyclic, condensed bicyclic or other similar linker in small molecule DNA gyrase B inhibitors reported in literature. Aforementioned second pharmacophoric element is where our initially observed similarities end. Here a structural fragment incorporating a polar or preferably acidic group is imperative as it enables key interactions towards arginine residues at the ATP binding site entrance of DNA gyrase B. We employed our leads and their piperazine analogues in the in silico virtual screening campaign only to identify additional aminopiperidine and cyclohexane central linkers for the development of focused library of potential DNA gyrase B inhibitors. We synthesized more than 60 compounds of three structural types in order to evaluate the influence of central linker and suitability of various simple aliphatic and aromatic terminal fragments incorporating a carboxyl or ester group on the inhibitory potency of compounds on isolated DNA gyrase B from E. coli. We also investigated the effects of pyrrole-2-carboxamide / indole-2-carboxamide pharmacophore exchange. We identified and validated pyrrolo-aminopiperidine scaffold in combination with prepared compounds that displayed IC50 values in low micromolar range. Compound with pyrrolo-aminopiperidine scaffold and glutaryl terminal acidic fragment possessed an IC50 value of 480 nM. Secondly, we recognised a suitable 1,4-substituted cyclohexane central linker. Representative pyrrolo-1,4-trans-cyclohexane scaffold compound incorporating a terminal glycine fragment displayed an IC50 value of 15.1 μM on DNA gyrase B. With the biological evaluation data in hand, we used prepared compounds to study the possible binding modes. With in silico methodology of molecular docking and molecular dynamics the possible binding into ATP active site was confirmed. Furthermore, previously published binding mode of small molecule DNA gyrase B inhibitors was corroborated and our compounds were found capable of interacting with Asp73 (E. coli numbering) at the adenine binding pocket and arginine residues near the ATP binding site entrance. We also developed an in silico approach towards identification of key conserved water molecules in ATP binding site and substantiated previous reports on water-mediated hydrogen bonds between ligand and amino acid residues in adenine pocket of DNA gyrase B ATP binding site. Compounds of the pyrrolo-aminopiperidine structural class were also evaluated using surface plasmon resonance where binding to the G24 protein known as the smallest ATP binding fragment of DNA gyrase B. Most potent compounds displayed affinity to the G24 protein with the Kd values of 11–12 μM, a result in agreement with our IC50 experiments. Our research confirms preceding results and strucutral studies on DNA gyrase B inhibitors and enables a solid foundation for futere design and development of DNA gyrase B as potential antibacterial compounds. In the additional investigation, we selected a library of 1,3-tiazolidin-4-one compounds and with the help of pharmacoinformatics and in silico molecluar modelling proposed their mechanism of action on the bacterial enzyme undecaprenylpyrophosphate synthase. Compounds were biologically evaluated and minimal inhibitory concentrations were measured on 5 bacterial strains and C albicans yeast. The library displayed modest antibacterial activity where (Z)-4-((2-(4-methyl-piperidin-1-yl)-4-oxothiazol-5(4H)-ylidene)methyl)benzonitrile was the most potent with a MIC value of 10 μg/mL on P. aeruginosa bacterial strain.
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