Structural, molecular and enzymatic analysis of Bos taurus enolase towards structure-based drug design

Tropical theileriosis is one of the among most common vector-born diseases caused by Theileria annulata parasites and causes serious economic losses in livestock production on a global scale. Theileria annulata is an obligate intracellular protozoan parasite that is transmitted by Hyalomma tick vectors and causes disease in ruminant animals, especially Bos taurus and Bos indicus. The success rates of tropical theileriosis treatment are low and resistance to buparvaquona has increased in the last decades. Recent studies have shown that Theileria annulata reveals resistance to Buparvaquona. For this reason, new treatment methods and drug studies should be increased in the combat against tropical theileriosis. Structrue-based drug design studies were studied for the first time against Theileria annulata enolase in a thesis study performed at research group in 2017 and coumarin derivatives were screened for their inhibitory activity on T. annulata enolase is an crucial enzyme in the glycolysis pathway of T. annulata essential energy metabolism. Of the 38 coumarin derivatives studied those who have potent inhibitory activity (IC50<15 μM) were reported as K8 (4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one), K9 (4-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one), K21 (4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2-one) and K23 (3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one). In order for an active substance to be used as a drug, the compound must inhibit the parasite target molecule but not the equivalent molecule in the host organism. In the direction of the structure-based drug design studies; structural, molecular and enzymatic analyzes of the enolase, which is the counterpart of Bos taurus, were performed in this thesis study. The gene encoding Bos taurus enolase (BtEno3) was cloned, expressed and the protein was successfully purified by affinity chromatography with 95% purity. The effect of temperature and pH changes on enzyme activity was analyzed and optimum conditions were determined as 40 °C, pH 6,5. Biochemical properties of enolase have been characterized for the first time in the literature, the kinetic parameters of the enzyme, Vmax and Km values, were calculated as 0,01305 μmol/min and 0.514 mM, respectively. In addition, in order to be used in silico analysis, 3D structure of Bos taurus enolase was built by homology modeling and coumarins and substrate were docked into enolase by molecular docking methods. Interactions in the enzyme-substrate, enzyme-coumarin and enzyme-coumarin-substrate complexes were analyzed by simulating molecular dynamics for 100 ns. Interactions between enolase-substrate have been reported to be the same as experimentally determined interactions in yeast. Estimated free binding energies of the substrate and coumarin derivatives to the enzyme were calculated by MM-GB(PB)SA methods. The effects of K8, K9, K21 and K23 coumarin derivatives, which highly inhibit enolase of T. annulata parasite, on the activity of Bos taurus enolase were studied in vitro and it was determined that these coumarin derivatives inhibited Bos taurus enolase at lower concentrations compared to T. annulata enolase (at 100 μM concentration of K8 %36,87, at 100 μM concentration of K9 %8,13 inhibition, K21 IC50: 77,69 μM and K23 IC50:84,42 μM). When the in vitro and in silico studies were evaluated together, K8, K9, K21 and K23 derivatives were reported to inhibit the parasite host Bos taurus enolase at higher coumarin derivative concentrations than the parasite enolase. The inhibition rates of these coumarin derivatives can be sorted as K21> K23> K8> K9. The conformational changes in the active site of the enzyme, the enzyme-substrate and enzyme-coumarin interactions were analyzed in silico after coumarin derivatives were docked to BtEno3. It has been determined that coumarins do not bind to the active site of the enzyme, not form tight bonds in the pockets and did not cause any conformation changes in the active site. It was revealed in silico studies that there is no lack of coordination in enzyme-substrate interaction. In the thesis study, in vitro and in silico studies showed that K8 and K9 coumarin derivatives inhibit parasite enolase much more effectively than host Bos taurus enolase. The proposed ligand will enlight the cell culture and pre-clinical studies to develop new drug candidates for the theileriosis treatment.