The lead compound is a compound that has appropriate pharmacological activity and is the basic molecule for further development of the active substance. At the beginning, the search for the lead compounds of new targets is performed by high-throughput screening. Later, the potency is confirmed by biophysical methods, also with isothermal titration calorimetry. Parallel to the potency, it is necessary to evaluate the physicochemical properties of the compounds when we are searching and optimizing the lead compound. One of the basic parameter is solubility, which has a major impact on other parameters. The development of new antibacterial drugs is essential due to bacteria mutations. An important target for antibacterial activity is the enzyme gyrase in the group of topoisomerases, which catalyses topological changes in the DNA of bacteria. Tetramer consists of two gyrase A and two gyrase B. We determined kinetic and thermodynamic solubility of the inhibitors of gyrase B. For evaluation of kinetic solubility, we dissolved the compound in dimethyl sulphoxide, then we mixed it with the phosphate buffer, shook this suspension at 25 °C for 24 hours, centrifuged and determined the concentration of the supernatant. For thermodynamic solubility, we dissolved the excess of the compound in the phosphate buffer, shook again for 24 hours at 25 °C and after centrifugation we determined the concentration of the compound in the supernatant. For determination of the concentration, we used high and ultra-high performance liquid chromatography. Very poor solubility (less than 10 μM) of all inhibitors were determined because compounds have lipophilic groups such as pyrrole, chlorine, bromine, methyl, benzyloxy and pyridine. As a result, all compounds have high lipophilic properties, manifesting in a longer retention time, a higher retention factor and a higher calculated logarithm of the partition coefficient (above 2,71). By adding more hydrophilic groups such as urea acid, oxalic acid, acetamide, we can reduce lipophilicity and slightly increase solubility, but unfortunately not enough. In the second part, we performed test titrations by isothermal titration calorimetry of calcium chloride with ethylenediaminetetraacetic acid. Then we performed titrations of IVN-36 and KOB-23 with gyrase B. Unfortunately, at the studied concentrations there were no responses. We can conclude that the binding of IVN-36 and KOB-23 to gyrase B is entropy-guided. The binding of such compounds is non-specific and has in vivo lower efficacy and more side effects due to binding to plasma proteins and other targets. All the studied inhibitors have high lipophilicity and poor solubility, so they are more likely for non-specific binding. Therefore, the analysed inhibitors are not good lead compounds.
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