Anti-piroplasmic activity of novobiocin as heat shock protein 90 inhibitor against in vitro cultured Theileria equi and Babesia caballi parasites.

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating the clinical form of EP. Serious side effects and incomplete clearance of infection is a major drawback of ID. Heat-shock proteins (Hsp) play a vital role in the life cycle of these haemoprotozoans by preventing alteration in protein conformation. These Hsp are activated during transmission of EP sporozoites from the tick vector (poikilotherm) to the natural host (homeotherm) and facilitate parasite survival. In the present study, we targeted the heat shock protein 90 (Hsp-90) pathway of T. equi and B. caballi by using its inhibitor drug - novobiocin. Dose-dependent efficacy of novobiocin on the growth of T. equi and B. caballi was observed in in vitro culture. Additionally, we examined dose-dependent cell cytotoxicity on host peripheral mononuclear cells (PBMCs) and haemolytic activity on equine red blood cells (RBC). In vivo organ toxicity of novobiocin was also assessed in a mouse model. The IC50 (50 % inhibitory concentration) value of novobiocin against T. equi and B. caballi was 165 µM and 84.85 µM, respectively. Novobiocin significantly arrested the in vitro growth of T. equi and B. caballi parasites at 100 µM and 200 µM drug concentration, respectively. In vitro treated parasites had distorted nuclear material and showed no further viability. Based on the equine PBMCs and RBC, the drug was found to be safe even at 1000 µM concentration and the CC50 (50 % cytotoxicity concentration) values were 11.63 mM and 261.97 mM. Very high specific selective index (SSI) values (70.47 and 1587) were observed for equine PBMCs and RBC, respectively. Organ-specific biochemical markers and histopathological examination indicated no adverse effect of the drug at a dose rate of 50 mg kg body weight in the mouse model. The results demonstrate the growth inhibitory effect of novobiocin against T. equi and B. caballi parasites and its safety for host cell lines with very high SSI. Hence, it can be inferred that the Theileria/Babesia Hsp-90 family are potential drug targets worthy of further investigation.

In vitro growth inhibitory efficacy of some target specific novel drug molecules against Theileria equi.

The in vitro growth inhibitory efficacies of five drug molecules against Theileria equi were evaluated in in vitro cultured parasites. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in vitro culture system was used to assess the growth inhibitory effect of harmaline hydrochloride dihydrate (HHD), hexadecyltrimethylammonium bromide (HDTAB), hesparidin methyl chalcone (HMC), andrographolide and imidocarb dipropionate against T. equi. The 50% inhibitory concentration value of HHD, HDTAB, HMC, and imidocarb dipropionate for T. equi growth were 17.42 µM, 14.00 µM, 246.34 µM and 0.279 µM (equivalent to 0.139 µg/ml), respectively (P<0.05). The andrographolide was not effective in inhibiting in vitro growth of T. equi in the present study. Furthermore, the in vitro cytotoxicity of these five drugs was evaluated on horse PBMC. At 2000 µM concentration of HHD, HDTAB, HMC, andrographolide and imidocarb dipropionate were 8.34, 46.44, 58.53, 31.06, 15.14% cytotoxic on PBMC, respectively. Out of our four tested drug molecules, HHD was having low IC50 value along with least cytotoxicity, as compared to reference drug imidocarb dipropionate. The difference in IC50 value of HDTAB and HHD was significant, but HDTAB was moderately more cytotoxic on PBMC cell lines. HHD and HDTAB are selective inhibitor for heat shock protein 90 (Hsp90) and choline kinase pathway. It can be concluded that HHD and HDTAB are potential drug molecules against T. equi parasite by acting on Hsp90 and choline kinase pathway.