Microbiology and Infectious Disease

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

Hang Li
Shengwei Ji
Nanang R Ariefta
Eloiza May S Galon
Shimaa AES El-Sayed
Thom Do
Lijun Jia
Miako Sakaguchi
Masahito Asada
Yoshifumi Nishikawa
Xin Qin
Mingming Liu author has email address
Xuenan Xuan author has email address

National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Obihiro, Japan
Department of Veterinary Medicine, Agriculture College of Yanbian University, Yanji, China
College of Veterinary Medicine and Biomedical Sciences, Cavite State University, Indang, Philippines
Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Mansoura University, Mansoura Egypt
Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
School of Basic Medicine, Hubei University of Arts and Science, Xiangyang, China

https://doi.org/10.7554/eLife.101128.2

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Figures and data

CIP demonstrates potent inhibition on Babesia spp..
(A) and (B) Dose-dependent growth curve of CIP on B. bovis and B. gibsoni in vitro. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate. IC₅₀: the half maximal inhibitory concentration. (C) Inhibitory effects of CIP and atovaquone (ATO) plus azithromycin (AZI) on the proliferation of B. microti in BALB/c mice. (D) Hematocrit (HCT) values in mice treated with CIP or ATO plus AZI compared with vehicle-treated mice. (E) Inhibitory effects of CIP and ATO plus AZI on the proliferation of B. rodhaini in BALB/c mice. (F) Survival rates of CIP-treated, ATO plus AZI-treated, and vehicle-treated mice. The treatment time is shown by two-way arrows, and significant differences (P < 0.01) between the drug-treated groups and the vehicle-treated control group are indicated by asterisks. The data from one of six individual experiments are expressed as means ± SD. *, P ˂ 0.05; **, P ˂ 0.01.

Mutations in BgATP4 mediate CIP resistance.
(A-C) Representative sequencing chromatogram of wild-type and resistant parasites from CIP-treated B. gibsoni. The resistant parasite genomic DNA is extracted from blood samples after a 60 day-treatment. The BgATP4 gene was amplified and sequenced using the DNA. (D) Genes of high-frequency sequence variants detected by NGS. (E) Dose-dependent growth curve of BgATP4^WT, BgATP4^L921V, and BgATP4^L921I in vitro. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate.

Mechanistic basis for resistance to CIP conferred by the L921V and L921I mutations in BgATP4.
(A) Untreated and CIP-treated parasite morphology after incubation for 72 h. Scale bar: 5 µm. (B) Sizes of 100 parasites in two groups measured with ImageJ software in panels A. Statistically significant differences between the means of variables determined by t-test. ****, P ˂ 0.0001. (C) TEM of untreated and CIP-treated parasite. N, Nucleus; SB, spherical body; V, vacuole. Scale bar: 500 nm. (D) [Na⁺]_i concentrations after addition of CIP in BgATP4^WT line. Representative traces from the experiment that highlight the impact of adding 20 nM CIP (blue), 1 nM CIP (green), or 0 nM CIP (grey) on the concentration [Na⁺]_i of the BgATP4^WT line. (E) Alkalinization of pH_i in BgATP4^WT line upon addition of the ATP4 inhibitor. (F) Addition of 20 nM CIP to the wild-type and resistant parasite lines results in different [Na⁺]_i concentrations. (G) Addition of 20 nM CIP to the wild-type and resistant parasite lines results in different pH_i concentrations. Experiments were performed in technical duplicates for at least three biological repeats. (H) Data acquired in the low Na⁺ condition (containing only the 2 mM Na⁺ introduced upon addition of 1 mM Na₂ATP) was subtracted from data obtained in the high Na⁺ condition to determine the ATPase activity related to the BgATP4 proteins. The results are presented as the average of data from three independent tests. (I) Dose-dependent BgATP4-associated ATPase activity curve of BgATP4^WT, BgATP4^L921V, and BgATP4^L921I in vitro. ATPase activity was determined at pH 7.2 in the presence of 150 mM Na⁺ and 1mM Na₂ATP. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate. *, P ˂ 0.05; **, P ˂ 0.01.

Binding sites proximal to BgATP4 residue 921 predicted by molecular docking.
(A) The lowest energy poses for CIP were located in reference to the whole protein structure, docking against the WT (green), L921V (yellow), and L921I (pink) mutant BgATP4. The side chain of L921 is also shown in a red stick at its position. (B-D) The zoomed views of the binding locations of CIP.

Cross-resistance between ATO and TQ in resistant parasites and combination therapy based on CIP plus TQ.
(A) Dose-dependent growth curve of BgATP4^WT, BgATP4^L921V, and BgATP4^L921I by ATO treatment in vitro. (B) Dose-dependent growth curve of BgATP4^WT, BgATP4^L921V, and BgATP4^L921I by TQ treatment in vitro. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate. (C) Inhibitory effects of CIP plus TQ on the proliferation of B. microti in SCID mice. The data are presented as the means from one of five independent experiments. (D) Parasite DNA was detected by qPCR on genomic DNA extracted from blood collected from untreated and treated SCID mice infected with B. microti at 90 DPI. A dotted grey line across the graph represents the average cut-off Cq value. Cut-off Cq ≤ 35 was considered as positive, while Cq > 35 or no amplification was considered as negative. Each sample was analyzed in duplicate, and two independent experiments were conducted. NA indicates no amplification.

Cytotoxicity assay of CIP on the Madin-Darby canine kidney (MDCK) cells and human foreskin fibroblasts (HFF).
The MTP-500 microplate reader is utilized to detect the absorbance at 450 nm. The results are displayed as the mean ± standard deviation of three separate tests. CC₅₀: the 50% cytotoxic concentration.

Multiple sequence alignment of ATP4 in different species.
A yellow square and arrow denotes the BgATP4 mutation site discovered in this investigation; purple squares and arrows represent sites linked to P. falciparum CIP resistance, and a gray square and arrow represents sites associated with T. gondii.

Binding sites for CIP found by Gnina search across the entire surface of the protein.
(A-C) The binding space of WT, L921V, and L921I mutants in BgATP4 are labeled in green, yellow and pink, respectively.

Proposed mechanism of inhibition of CIP on wild-type and mutant parasite-infected erythrocytes.
CIP disrupts the BgATP4 function of wild-type parasites, which causes a net influx of Na⁺ and efflux of H⁺ from the parasite. The osmotic load imposed on the influx of Na⁺ further brings about parasite swelling and internal alkalinization, which are the main factors in Babesia death. Mutations in ATP4 minimize the susceptibility to ATP4 inhibitors by recovering H⁺ and Na⁺ balance, as indicated by the dotted arrows.

Primer sets of B. gibsoni ATP4

Primer sets of B. microti ATP4

Interactions of CIP from docking simulations

Interactions of CIP from docking simulations

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