Investigation on Theileria annulata enolase inhibition potential of coumarin derivatives to identify new drug candidates to be used in the treatment of theileriosis

Tropical theileriosis is a tick-borne disease caused by an apicomplexan parasite Theileria annulata. The disease has an economic importance due to the high rate of morbidity and mortality accompanied by the appearance of this parasite in all geographical regions of Turkey. Resistance against commonly used antitheilerial drug, buparvaquone, has already been reported and therefore, alternative drug candidates should be identified. In this way, enolase might be a druggable protein target because the enzyme is a multifunctional protein that catalyzes the conversion of 2-phosphoglyceric acid to phosphoenolpyruvate in glycolysis and was observed as a plasminogen receptor could interfere with the virulence of parasites. In this study, coumarin derivatives were selected as potential enzyme inhibitors for inhibition of T. annulata enolase. These compounds are naturally found in plants and have diverse biological activities. In addition, coumarins could be used as potential therapeutic drug candidates due to their low toxicity. The aim of this study was to assess inhibition potential of coumarin derivatives on T. annulata enolase (TaENO) activity, for the first time in literature. Firstly, the protein was expressed, and purified by affinity chromatography and then, enzyme stabilization studies were performed. Inhibitor screening analyses, determination of IC50 values and identification of inhibition mechanism were conducted for coumarin derivatives. Finally, protein was structurally analyzed by homology modelling and molecular docking in the presence and absence of inhibitor. Prior to inhibition studies, protein stabilization analyses of TaENO were performed and it was found that the enzyme remained stable with adding 6 M ethylene glycol at +4 °C. Inhibitor screening analyses were carried out using 38 coumarin derivatives on highly purified TaENO (>95%). Five coumarin derivatives [3-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (K5); 4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (K8); 4-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (K9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (K21) and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (K23)] showed high inhibitory effects (>90%) among 38 coumarin derivatives and IC50 values of these compunds were calculated. The lowest IC50 value were determined as 8,871 µM for K21 coumarin derivative. IC50 values for K5, K8, K9 and K23 were also found as 27,910 µM, 10,450 µM, 13,170 µM and 10,863 µM, respectively. Kinetic analyses were performed to determine inhibition mechanisms of four coumarin derivatives (K8, K9, K21 and K23) that had IC50 values of <15 μM. Our results indicated that these compounds inhibited the enzyme activity by uncompetitive inhibition. Inhibition potential of a commercial enolase inhibitor, ENOblock®, has been studied on rabbit muscle enolase in the literature was also evaluated, and it was found that the inhibitor possesses very low inhibitory effect on TaENO (15,04% inhibition at 100 µM). In addition, the binding of K21 coumarin derivative which showed the highest inhibition potential on TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Open and closed conformations of T. annulata enolase were modelled by homology modelling. These models showed the similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. Active site and potential drug binding regions of TaENO model were predicted by comparison with protein sequence analysis. Molecular docking analyses of the substrate (2-phosphoglyceric acid, 2-PG) and the four coumarin derivatives were also performed to predict possible binding poses on TaENO. Taken together, our results revealed that these coumarin derivatives (K8, K9, K21 and K23) are potent, non-substrate, uncompetitive inhibitors of TaENO. In silico analyses have also predicted that K8 and K21 coumarin derivatives which had high inhibitory effects on TaENO according to the results of inhibition analyses, were interacted with high affinity to the potential regions out of the active site. In this study, potential TaENO inhibitors were described for the first time in the literature by structure-based drug design approach. These inhibitors may be used to facilitate further in vitro and in vivo analysis towards developing new drug candidates for the treatment of drug resistant T. annulata infections.