Anti-piroplasmic Activity of Novobiocin as Heat Shock Protein 90 Inhibitor Against in-vitro Cultured Theileria Equi and Babesia Caballi Parasites

Background: Theileria equi and Babesia caballi are the causative agents for equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating clinical form of EP. Serious side effects and uncompleted clearance of infection is major drawback of ID. Heat-shock proteins (HSP) play a vital role in the life cycle of these haemoprtozoa by way of preventing alteration in protein conformation. These HSPs are activated during transfer of EP sporozoites from tick vector (poikilotherm) to natural host (homeotherm) and helped it for survival. In this present study we have targeted the heat shock protein 90 pathway of T. equi and B. caballi by its inhibitor drug - novobiocin. Methods: Dose-dependent ecacy of novobiocin drug was observed on growth of T. equi and B. caballi in in-vitro culture. Cell cytotoxicity on host peripheral mononuclear cells (PBMCs) was also checked with different concentration of novobiocin. It was also checked for its haemolytic activity on equine erythrocyte (RBCs) by standard technique. In-vivo organ toxicity of novobiocin was also assessed in mice model with identied methods. Results: IC 50 (50 % Inhibitory concentration) value of novobiocin against T. equi and B. caballi was 165 µM and 84.85 µM, respectively. Novobiocin signicantly arrested the in-vitro growth of T. equi and B. caballi parasites at respective 100 μM and 200 μM drug concentration. In-vitro treated parasites become dead with distorted nuclear material and showed no further viability. The drug was found safe on the equine PBMCs and RBCs cell line even at 1000 µM concentration and CC 50 (50 %, cytotoxicity concentration) values were 11.628 mM and 261.97 mM. A very high specic selective index (SSI) was also observed 70.47 and 1587 for respective equine PBMCs and RBCs. Organ specic biochemical markers and histopathological examination indicated no adverse

Theileria equi and Babesia caballi are haemoprotozoa responsible for causing equine piroplasmosis. The disease is primarily transmitted by tick of the genus Dermacentor, Hyalomma and Rhipicephalus [19]. Prevalence of equine piroplasmosis have been reported from many countries and usually coincide with distribution of tick-vectors [14,20,25,30]. International movement of piroplasm affected equids have been restricted as per trade guidelines of O ce International des epizooties (OIE) [13,15]. The currently available drugs are not suitable to completely clear the T. equi or B. caballi parasite from latently infected animals [2,12,28].
Most of the haemoprotozoan have to cope with repeated host body temperature uctuation after transmission of sporozoite from arthopod vector. Protozoa speci c heat shock proteins 90 (Hsp-90) get activated during such temperature change and plays a vital role in the life-cycle of parasite within host [1,22].In spite of fact that Hsp-90 has a critical role in its multiplication inside the equine RBCs, not much efforts have been exerted in pursuing researches against this drug target [6]. In this study we targeted the heat shock protein pathway of T. equi and B. caballi by its inhibitor novobiocin drug. Novobiocin is an antibiotic and inhibitor of chaperone-Hsp-90 [17]. Novobiocin derivates have shown a promising result as anticancer agents by inhibiting the function of this chaperone [3]. Novobiocin also has in-vitro growth inhibition e cacy against Plasmodium falciparum and inhibited 80% ATPase activity of the parasite at 30 µM concentration [24]. Keeping in view anti-bacterial, anti-cancerous and anti-plasmodial potential of novobiocin drug, this present study was planned for investigating its anti-T. equi/B. caballi activity and invitro/in-vivo cytotoxicity or organ toxicity.

Methods
The parasites and in-vitro cultivation Theileria equi and Babesia caballi parasites (Indian isolates) were cultured in horse red blood cells (RBCs) through continuous microaerophilic stationary-phase (MASP) culture system. The culture medium M 199 and RPMI 1640, (Sigma-Aldrich, India) were used for MASP culture of T. equi and B. caballi, respectively. These mediums were supplemented with 40% de brinated equine serum, antibiotic solution (containing 60 IU/ml penicillin and 60 mg/ml streptomycin) and 200 µM hypoxanthine solution. Theileria equi and B. caballi MASP cultures were maintained at a temperature of 37 °C with micro-aerophilic atmosphere of 5% CO 2 , 3% O 2 , and 95% N.
In-vitro growth inhibition assay Theileria equi and B. caballi parasitized RBCs were obtained from primary MASP cultures at 10% and 6% parasitaemia respectively. These parasitized RBC's (T. equi and B. caballi) were adjusted to 1% parasitemia by diluting with uninfected RBCs obtained from a healthy horse and used for in-vitro evaluation of drug. The parasite growth inhibition assay was performed in 48 well culture plates. Fifty microliter of T. equi or B. caballi parasite infected RBCs (at 1% parasitaemia) were dispensed per well (in triplicate) together with 500 µl of the culture complete medium containing the indicated drug concentrations. Novobiocin drug (cat no.: N1628-1G; Sigma-Aldrich, India) was tested at 1 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM concentrations against T. equi and 10 µM, 20 µM, 50 µM, 100 µM, 200 µM against B. caballi parasites. Cultures without novobiocin drug molecule and cultures containing only DMSO (0.005% and 0.5%) were also prepared and initiated in MASP system for control experiments. ID drug was diluted to 0.5 µg/ml, 1.0 µg/ml and 10.0 µg/ml concentration and kept as positive drug control experiment against T. equi and B. caballi parasites in MASP in-vitro culture system. These in-vitro cultures with or without drug molecules concentrations were incubated at 37°C in an atmosphere of 5% CO 2 , 3% O 2 , and 95% N, for a period of 96 hours (h). The overlaid culture medium was replaced daily with fresh medium containing indicated drug molecule concentration. IC 50 value was calculated by standard curve tting technique. [2].

In-vitro viability test
After 96 h of in-vitro treatment with novobiocin, 20 µL of drug-treated/un-treated parasitized RBCs were collected (at different concentration) and transferred to a fresh culture plate (48 wells) containing 30 µL of parasite-free fresh equine RBCs in 500 µl of growth medium (without any drug molecule). The overlaid growth medium was replaced after every 24 h for the next 72 h, and parasite recrudescence was determined by examining its stained blood smears [2].
In-vitro cytotoxicity assay on equine PBMCs Cytotoxicity of each concentration of drug molecules was analysed on peripheral blood mononuclear cells (PBMCs) by resazurin-based cell viability assay [8]. Brie y, freshly collected equine PBMCs were suspended in 1 ml complete growth medium consisting of RPMI-1640 supplemented with 2 mM Lglutamine, 60 µg/ml penicillin, 100 µg/ml streptomycin and 10% foetal bovine serum (Sigma Aldrich, India). Enriched PBMCs suspension with complete growth media was adjusted to a nal concentration of 3 × 10 7 cells/100 µL and distributed to each well (100 µL volume) in 96 well culture plate. Simultaneously, phytohaemagglutinin-A (PHA, at the concentration of 10 µg/ml) in 50 µL volume was also added to each of these well. The culture plate was incubated at 37 °C having 5% CO 2 in air for 48 h. After 48 h, PBMCs in cultured wells were treated with 100 µl volume of the different respective concentration of novobiocin drug molecule -1 µM, 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, 1000 µM, 2000 µM. The 96 well culture plate was again incubated (as above) for another 24 h, followed by addition of 25 µL of resazurin dye (150 µg/ml) and culture plate was kept in an incubator for another 4 h. The change of dye colour was monitored by measuring optical density (OD) at 570 nm and 650 nm. The effective OD value for each well was calculated by deducting OD 570 value from its respective OD 650 value. The IC 50 of each drug molecule on PBMCs was calculated from a regression equation based on the effective OD value, as mentioned above. Effect of drug molecules on PBMCs in terms of per cent viable cell population was determined as below: In-vitro drug haemolytic assay In-vitro drug haemolytic assay was performed as per standard protocol [23]. Freshly collected 5 ml equine whole blood was centrifuged at 1200 g for 10 min and nal pellet was washed three times with 1X PBS (Phosphate buffer saline) at 1200 g for 5 min after discarding the supernatant. Twenty microliters of RBCs suspension were added to each well of 96 well culture plate. Further, 180 µL of each of novobiocin concentration (1 µM, 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, 1000 µM, 2000 µM) prepared in solubilising buffer (10% dimethylformamide in PBS) was also added to each well. RBCs suspension (20 µL) was added to 180 µl of distilled water or solubilising buffer and taken as positive or negative control analysis. The 96 well plate was incubated at 37˚C for 90 min. Contents of each well after incubation were transferred into 2 ml micro-centrifuge tube, followed by centrifugation at 3000 g for 5 minutes. Supernatants were transferred to new 96 well plates and OD was measured at 543 nm in UV spectrophotometer. Percentage of haemolysis was determined as below: Speci c Selectivity Index (ssi) In the present study, the degree of selectivity of novobiocin drug molecule against T. equi or B. caballi in comparison to mammalian cells (equine PBMCs or RBCs) at respective IC 50 concentration was analysed as per standard method [16] using the below mentioned formula: In-vivo organ toxicity of novobiocin in mice The organ toxicity of novobiocin was analysed in six groups of mice (n = 6, in each group). Each ve group was administered (by intraperitoneal route) the drug at respective dose rate of 5, 10, 20, 50, 100 mg/kg body weight, whereas sixth group was kept as no-drug control and administered PBS only.
Mice in each group were observed for 14 days. Blood from these group of mice were collected at 0 h, 24 h and 72 h interval from retro-orbital sinus under general anaesthesia. Serum was isolated from these mice and processed for biochemical analysis with-respect-to changes in organ speci c biochemical markers. Mice were sacri ced on day 14 post-drug treatment and biopsy sample from vital organs were collected (liver, lung, heart, kidney and spleen) and processed for histopathological examination as per standard protocol.

Statistical analysis
Anti-piroplasmic activity of novobiocin drug molecules against T. equi and B. caballi was computed by Two Way ANOVA test followed by Bonferroni Post-hoc test (p < 0.05). The P values < 0.05 were considered as statistically signi cant differences between the novobiocin treated wells and control wells. Correlation between novobiocin concentration and its cytotoxicity and haemolytic activity was accessed by using Graphpad prism version 4.0 software (San Diego California, USA).

Results
In-vitro growth inhibitory e cacy of novobiocin against T. equi and B. caballi In-vitro growth inhibitory effects of different concentrations of novobiocin against T. equi and B. caballi were analysed by Bonferroni Post-hoc test (Two Way ANOVA) in-between treated and control groups ( Fig. 1A and 1C). Stained blood smears prepared from in-vitro drug treated/control wells over a period of 96 h were examined for morphological changes in parasite (T. equi/B. caballi) at different concentrations of novobiocin ( Fig. 1B and 1D). T. equi or B. caballi parasite with conspicuous outline cytoplasm membrane and nuclear material were indicative of live dividing parasites while, dead parasites usually were pyknotic with condense/distorted nuclear material. T. equi and B. caballi parasites were dead, showing dotshaped/distorted nuclear material, at 100 µM and 200 µM concentration of novobiocin at 96 h of in-vitro treatment. While, in control culture parasites were live, healthy and dividing throughout the 96 h of in-vitro period ( Fig. 1B and 1D).

In-vitro cytotoxicity of novobiocin on PBMCs
Various concentrations of novobiocin (ranging from 1 µM to 1000 µM) tested for cytotoxicity on equine PBMCs and less than 10% cytotoxicity was observed at highest concentration of novobiocin (1000 µM) ( Fig. 2A). The extrapolated cytotoxic concentration (CC 50 ) by regression analysis was 11628 µM. The speci c selective index (SSI) of novobiocin in this in-vitro study was 70.47, indicative of its safety on mammalian cell lines.

In-vitro haemolytic activity of novobiocin on equine erythrocytes
Haemolytic activity of novobiocin on equine RBCs was also assessed. Different concentrations of novobiocin (from 1 µM to 2000 µM) were analysed and 0.01% to 0.44% haemolysis was observed (Fig. 2B). CC 50 of novobiocin on horse erythrocyte was analysed by regression analysis and found to be 261973 µM. A very high SSI value (1587) was observed for horse erythrocyte which indicated its safety.

In-vivo toxicity of novobiocin in mice model
Organ toxicity of novobiocin was tested in different mice groups and biochemical parameters were analysed. A signi cant (p < 0.05) rise in SGOT and SGPT was observed in group I mice (dosage @ 100 mg/kg body weight) at 24 h interval of inoculation of novobiocin drug, which thereafter decreased to insigni cant level ( Fig. 3C and 3D). An insigni cant difference (p > 0.05) in kidney biochemical markers like creatinine, blood urea nitrogen ( Fig. 3G and 3H) and other parameters like bilirubin and total protein ( Fig. 3E and 3F) was observed at different time interval after inoculation of novobiocin in different group of mice. Tissue samples collected from different organs from these mice groups (I to VI) were subjected to histopathological examination. Hepatocytes in group I mice showed minimal diffuse cytoplasmic rarefaction and vacuolation as compared control group (VI) mice ( Fig. 3A and 3B). It is indicative of liver damage in this group of mice. No adverse histopathological changes were observed in any organ of mice of other groups. As such, 50 mg/kg dose rate of novobiocin may be considered as 'No Observed Adverse Effect Level' (NOAEL) concentration.

Discussion
Heat shock protein (Hsp) are multiunit chaperon complexes and help in proper folding of the target protein. Heat shock protein-90 (Hsp-90) is the most extensively studied and largest protein of this family. Hsp-90 is obligatory for maintaining integrity of number of client proteins involved in cellular and signal transduction processes [10,27]. Hsp-90 has four structural domains -N-terminal, charged linker region, middle domain and C-terminal [26]. N-terminal is the ATP-binding domain of the Hsp-90 and interacts with most of its inhibitors -geldanamycin [26].
Novobiocin is an amino-coumarin class of antibiotic and exhibits potent activity against Gram-positive bacteria. Novobiocin possess Hsp-90 C-terminal nucleotide-binding site activity and induced degradation of Hsp-90 dependant client protein ErbB2, mutant p53 in a concentration-dependent manner in cancer cell lines [18]. In this study we examined in-vitro growth inhibitory e cacy of novobiocin against T. equi and B. caballi parasites. Novobiocin signi cantly arrested the in-vitro growth of T. equi and B. caballi parasites leading to their death at respective 100 µM and 200 µM drug concentration. Novobiocin greatly reduced Plasmodium falciparum parasitaemia during rst cycle of growth (trophozoite to schizont) in in-vitro culture at 30 µM concentration [24]. Novobiocin eliminated P. falciparum parasite in in-vitro culture by inhibiting its ATPase activity. Novobiocin IC 50 value against T. equi and B. caballi was 165 µM and 84.85 µM in the present study. While other author [24] observed IC 50 value of 280 µM and 210 µM against respective FCC 1 (a chloroquine-susceptible) VNS (Viet Nam Smith, a chloroquine-resistant) strains of P.
falciparum. The IC 50 observed in our experiments against T. equi and B. caballi is approximately 1.48 to 2.88 times low as observed against P. falciparum, indicating its effectiveness. Further, novobiocin in-vitro treatment also altered morphology of T. equi and B. caballi parasites. After 96 h of in-vitro treatment (at 100 µM and 200 µM concentration) these parasites become dead with distorted nuclear material ( Fig. 1B and 1D). Theileria annulata (bovine) has four isoforms of Hsp-90 and respective genes were aligned with orthologues from other closely related apicomplexan parasites [11]. Phylogenic tree derived from this alignment indicated close relationship between the isoform within the Apicomplexa. Further, sequence alignment analysis of Hsp-90-like proteins across Theileria, Babesia, and Plasmodium species indicated conservation of C-terminal sequences motif. The inhibitory e cacy of novobiocin against T. equi and B. caballi in the present study may be attributed due to its anti-Hsp-90 properties as evidence in other Apicomplexa parasites.
Novobiocin drug was found safe on the horse PBMCs as well as erythrocytes and < 10% cytotoxicity was observed on PBMCs at highest concentration of 1000 µM or 2000 µM. The CC 50 value was 11.628 mM and 261.97 mM and respective SSI was 70.47 and 1587 for horse PBMCs and erythrocytes. Another group of researchers [7] tested novobiocin for in-vitro inhibition of Kaposi's sarcoma-associated herpesvirus (KSHV). A marked inhibition of KSHV was reported with SSI of 31.62 (and CC 50 of 871 µM), indicating its safety on lymphoma cell line (BCBL-1). In our experiment novobiocin was observed as safe drug on horse PBMCs and erythrocytes with very high CC 50 and SSI values.
Organ toxicity of novobiocin was also assessed in mice model at different dosage. Organ speci c biochemical markers and histopathological examination indicated no adverse effect of the drug at a dose rate of 50 mg/kg of body weight. Therapeutically, novobiocin was used primarily for infections due to penicillin-resistant Staphylococcus aureus or against pneumococcal pneumonia [4,5]. Oral dosage of novobiocin (@ 1-2 g/day) was well absorbed and therapeutically useful concentration (18.8 µg/ml following 0.5 g dose) was readily achieved in the bloodstream [29]. Novobiocin has been observed to be safe in non-infection clinical studies at high dosage (3-9 g/day, orally administration) while high plasma concentration was achieved (150 µM sustained for 24 h @ 5.5 g dosage) and no serious toxicities were observed [9,21]. Our study generated novobiocin organ toxicity data in mice and indicated its in-vivo safety.
It is crucial to continually look for target speci c novel anti-Theilerial/Babecidal compounds. In this study, we tested novobiocin drug, a Hsp-90 inhibitor and results suggested its anti-T. equi/B. caballi potential.

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Availability of data and materials
All supporting data of presenting manuscript was included within this article and its additional les. The data generated during present study is available from corresponding author for further request.

Competing interests
The authors declare that they have no con ict of interest.