17-DMAG inhibits the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice

Abstract

Heat shock protein 90 (Hsp90) is a chaperone protein that stabilizes cells during stress or non-stress responses. Previous reports have shown that Hsp90 is a potential drug target to suppress the multiplication of several protozoan parasites. In this study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an Hsp90 inhibitor, was evaluated for its inhibitory effect on five in vitro cultures of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, and on the multiplication of a B. microti-infected mouse model. 17-DMAG showed the inhibitory effect in all of the species tested. The half maximum inhibition concentration (IC₅₀) of 17-DMAG on B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was 77.6 ± 2.9, 62.4 ± 1.9, 183.8 ± 3.2, 88.5 ± 9.6, and 307.7 ± 7.2 nM, respectively. The toxicity assay on MDBK and NIH/3T3 cell lines showed that 17-DMAG affected the viability of cells with an IC₅₀ of 15.5 ± 4 and 8.8 ± 2 μM, respectively. Since the IC₅₀s were much lower on the parasites than on the host cell lines, the selectivity index were high for all tested species. Furthermore, the two-drug combination of 17-DMAG with diminazene aceturate (DA) and atovaquone (AV) showed synergism or addition on in vitro cultures of Babesia and Theileria parasites. In the mouse model, 17-DMAG at a concentration of 30 mg/kg BW effectively inhibited the multiplication of B. microti. Moreover, if combined with DA or AV, 17-DMAG showed a comparable inhibition at the half dose. Taken together, these results indicate that 17-DMAG is a potent drug for treating piroplamosis. The data warrant further evaluation of 17-DMAG as an antibabesial drug and as an option in combination with atovaquone for the treatment of human babesiosis.

Keywords: 17-DMAG; Babesia; Chemotherapeutic; Hsp90 inhibitor; Theileria.

Graphical abstract

Fig. 1

Dose response curve of 17-DMAG on the in vitro cultures. The parasitemia of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi after exposure to various concentrations of 17-DMAG for 96 h as determined by SG-based fluorescence assay. The percentage of inhibition is expressed as the percentage of inhibited parasites compared with the positive control (untreated wells) after subtracting the negative control (uninfected RBC). The data were the mean and S.D. from triplicate experiments.

Fig. 2

Polygonogram of the degree of synergism. The polygonogram of synergism between 17-DMAG, diminazene aceturate (DA), and atovaquone (AV) based on the weighted average of combination index values (CIs) at IC₅₀, IC₇₅, IC₉₀, and IC₉₅ using the formula ((1 x IC₅₀) + (2 x IC₇₅) + (3 x IC₉₀) + (4 x IC₉₅))/10 (Chou, 2006). The degree of synergism was determined based on the following CI value: < 0.90 (synergism), 0.90–1.10 (additive), and >1.10 (antagonism).

Fig. 3

Effect of drug treatment on the growth of Babesia microti in mice. (A) Parasitemia after treatment with 17-DMAG, diminazene aceturate (DA), and atovaquone (AV) at a concentration of 30, 25, and 20 mg/kg body weight, respectively. (B) Parasitemia after treatment with the combination of 17-DMAG and DA at 15 mg/kg BW each, 17-DMAG with AV at 15 and 10 mg/kg BW, respectively, and a combination of DA and AV at 15 and 10 mg/kg BW, respectively. Drug administrations were started from day 4 until day 8 post infection (left right arrow). The data were the mean and S.D. from two separate experiments (in total of 10 mice per group).

Fig. 4

Body weight and hematology profiles of mice on day 10 post infection. The observation of mouse body weight (A) and red blood cell count (B) of mice on day 10 post infection. All data were the mean and S.D. from 10 mice in each group as a comparison with DMSO control and healthy control. The significant differences are marked with * if P is less than 0.05, ** if P is less than 0.01, and *** if P is less than 0.001.