Efficacy and mechanism of action of cipargamin as an antibabesial drug candidate

Babesiosis is a disease brought on by intraerythrocytic parasites of the genusBabesia. Current chemotherapies are accompanied by side effects and parasite relapse. Therefore, it is crucial to develop highly effective drugs againstBabesia. Cipargamin (CIP) has shown inhibition against apicomplexan parasites, mainlyPlasmodiumandToxoplasma. This study evaluated the growth-inhibiting properties of CIP againstBabesiaspp. and investigated the mechanism of CIP onB. gibsoni. The half inhibitory concentration (IC₅₀) values of CIP against thein vitrogrowth ofB. bovisandB. gibsoniwere 20.2 ± 1.4 nM and 69.4 ± 2.2 nM, respectively. CIP significantly inhibited the growth ofB. microtiandB. rodhaini in vivo.Resistance was conferred by L921V and L921I mutations in BgATP4, which reduced the sensitivity to CIP by 6.1- and 12.8-fold. The inhibitory potency of CIP against BgATP4-associated ATPase activity was moderately reduced in mutant strains, with a 1.3-fold and 2.4-fold decrease in BgATP4^L921Vand BgATP4^L921I, respectively, compared to that of BgATP4^WT. Anin silicoinvestigation revealed reductions in affinity for CIP binding to BgATP4^L921Vand BgATP4^L921Icompared to BgATP4^WT. Resistant strains showed no significant cross-resistance to atovaquone (ATO) or tafenoquine succinate (TQ), with less than a onefold change in IC₅₀values. Combining CIP with TQ effectively eliminatedB. microtiinfection in SCID mice with no relapse, and parasite DNA was not detected by qPCR within 90 days post-infection. Our findings reveal the efficacy of CIP as an anti-babesial agent, its limitations as a monotherapy due to resistance development, and the potential of combination therapy with TQ to overcome said resistance and achieve complete parasite clearance.