DOI: 10.5061/dryad.8kprr4z02
title: Efficacy and mechanism of action of cipargamin as an antibabesial drug candidate

description: Babesiosis is a disease brought on by intraerythrocytic parasites of the
 genus Babesia. Current chemotherapies are accompanied by side effects and
 parasite relapse. Therefore, it is crucial to develop highly effective
 drugs against Babesia. Cipargamin (CIP) has shown inhibition against
 apicomplexan parasites, mainly Plasmodium and Toxoplasma. This study
 evaluated the growth-inhibiting properties of CIP against Babesia spp. and
 investigated the mechanism of CIP on B. gibsoni. The half inhibitory
 concentration (IC50) values of CIP against the in vitro growth of B. bovis
 and B. gibsoni were 20.2 ± 1.4 nM and 69.4 ± 2.2 nM, respectively. CIP
 significantly inhibited the growth of B. microti and B. 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
 BgATP4L921V and BgATP4L921I, respectively, compared to that of BgATP4WT.
 An in-silico investigation revealed reductions in affinity for CIP binding
 to BgATP4L921V and BgATP4L921I compared to BgATP4WT. Resistant strains
 showed no significant cross-resistance to atovaquone (ATO) or tafenoquine
 succinate (TQ), with less than a one-fold change in IC50 values. Combining
 CIP with TQ effectively eliminated B. microti infection 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.
description: **Project Title:** Efficacy and mechanism of action of cipargamin as an
 antibabesial drug candidate **Contact Information:**
 [lmm_2010@hotmail.com](mailto:lmm_2010@hotmail.com);
 [gen@obihiro.ac.jp](mailto:gen@obihiro.ac.jp) ## **Overview** This dataset
 accompanies the publication titled "Efficacy and mechanism of action
 of cipargamin as an antibabesial drug candidate." It includes data
 files corresponding to the figures and supplementary materials in the
 article. Each file is described below, along with variable definitions,
 experimental context, and processing methods. This README is designed to
 be a standalone guide, enabling users to interpret the data without
 referring to the original publication. ## **File Descriptions** Several
 .csv files are combined figures. Each subfigure is separated by empty
 rows. Subfigures are explained as needed. ### **Figure 1.csv** **1. Figure
 1A & B: Growth data** **Context:** Dose-dependent growth inhibition of
 CIP on *B. bovis* and *B. gibsoni in vitro*. **Variables:**
 Concentration_nM: Drug concentration in nanomolar (nM) *B.
 bovis*_growth_percent: Mean cell growth (%) of *B. bovis* across
 replicates *B. gibsoni*_growth_percent: Mean cell growth (%) of *B.
 gibsoni* across replicates **Notes:** IC50 values (half inhibitory
 concentration) were calculated from this data. Triplicate measurements
 were taken for each concentration. **2. Figure 1C: Inhibition data**
 **Context:** *In vivo* inhibition of *B. microti* proliferation in BALB/c
 mice by CIP and ATO + AZI. **Variables:** Day: Days post-infection
 Parasitemia (%): Parasitemia in blood (%) for each mouse (n=6 per group)
 Treatment_Group: CIP 20 mg/kg, ATO + AZI 20 mg/kg, or vehicle **Notes:**
 Data are presented as individual mouse values. CIP showed significant
 inhibition compared to ATO + AZI and vehicle groups. **3. Figure 1D:
 hematocrit data** **Context:** Hematocrit (HCT) values in *B. microti*
 infected BALB/c mice treated with CIP, ATO + AZI, or vehicle.
 **Variables:** Day: Timepoint of measurement (Day 0 to Day 32) HCT (%):
 The mean hematocrit value (%) for each group of mice Treatment_Group: CIP
 20 mg/kg, ATO + AZI 20 mg/kg, or vehicle **Notes:** HCT values were
 monitored to assess anemia due to parasite infection and treatment
 effects. Data indicate no significant anemia in drug-treated groups. **4.
 Figure 1E: Inhibition data** **Context:** *In vivo* inhibition of *B.
 rodhaini* proliferation in BALB/c mice by CIP and ATO + AZI.
 **Variables:** Day: Days post-infection Survival_Status: — = not
 applicable Parasitemia (%): Parasitemia in blood (%) for each mouse
 Treatment_Group: CIP 20 mg/kg, ATO + AZI 20 mg/kg, or vehicle **Notes:**
 CIP showed strong inhibition of *B. rodhaini* proliferation. ATO + AZI was
 less effective, and the vehicle group showed rapid disease progression.
 **5. Figure 1F: Survival data** **Context:** Survival rates of BALB/c mice
 treated with CIP, ATO + AZI, or vehicle following infection with *B.
 rodhaini*. **Variables:** Day: Timepoint of survival observation (Day 0 to
 Day 22) Survival_Status: ○ = alive, × = dead, — = not applicable
 Treatment_Group: CIP 20 mg/kg, ATO + AZI 20 mg/kg, or vehicle **Notes:**
 CIP-treated mice showed significantly higher survival rates compared to
 the other groups.   ### **Figure 2.csv** **1. Figure 2D: NGS variants**
 **Context:** Genes of high-frequency sequence variants detected by NGS.
 **Variables:** Strain: Name of the *Babesia gibsoni* strain (BgATP4WT,
 BgATP4L921V, or BgATP4L921I) Chr: Chromosome number pos: Genomic position
 of the mutation (position 2761 in this dataset) ref: Reference nucleotide
 at the mutation site cov: Sequencing coverage depth at this position A, C,
 G, T, N: Number of reads showing each nucleotide at the position Percent:
 Mutation frequency (percentage of reads showing the variant nucleotide)
 **Notes:** High-frequency sequence variants in the *Bgatp4* gene were
 confirmed in different *B. gibsoni *strains. All strains carry mutations
 at position 2761 on chromosome III: BgATP4WT: Reference nucleotide C
 (100%) BgATP4L921V: G variant (99.97%) BgATP4L921I: A variant (99.92%)
 Data was generated by next-generation sequencing (NGS) and validated for
 use in downstream functional analysis. **2. Figure 2E: Growth curve**
 **Context:** Dose-dependent *in vitro* growth curves of *B.
 gibsoni *strains expressing different ATP4 variants: BgATP4WT,
 BgATP4L921V, and BgATP4L921I. **Variables:** nM: CIP concentration in
 nanomolar BgATP4WT: % growth of BgATP4WT strain at each CIP concentration
 BgATP4L921V: % growth of BgATP4L921V strain at each CIP concentration
 BgATP4L921I: % growth of BgATP4L921I strain at each CIP concentration
 **Notes:** Growth inhibition is presented for increasing concentrations of
 CIP. The data represent means ± SD from one of three independent
 experiments. BgATP4L921V and BgATP4L921I strains showed reduced
 sensitivity to CIP compared to BgATP4WT strain.   ### **Figure 3.csv**
 **Context:** Mechanistic basis for resistance to CIP conferred by the
 L921V and L921I mutations in BgATP4. **Notes:** This figure presents the
 mechanistic analysis of CIP resistance in *B. gibsoni *strains expressing
 mutant ATP4 proteins (BgATP4L921V and BgATP4L921I). It includes
 morphological changes, ultrastructural analysis, intracellular sodium
 ([Na⁺]i) and proton (pHi) concentration changes, and ATPase activity of
 wild-type and mutant BgATP4 proteins in response to CIP. **1. Figure
 3B–Parasite size measurement** **Context:** Sizes of 100 parasites in
 untreated and CIP-treated groups measured with ImageJ software.
 **Variables:** Ctrl / CIP: Treatment condition (untreated vs. CIP-treated)
 Measurement (µm): Size measurements of individual parasites (n = 100 per
 group) **Notes:** Statistically significant differences were found between
 untreated and CIP-treated groups (t-test, ****, *P* &lt; 0.0001). Data
 represents results from a single experiment based on morphological
 observations from Figure 3A. **2. Figure 3D&E – [Na⁺]i concentration
 and pHi alkalinization in response to CIP in BgATP4WT Line** **Context:**
 Intracellular sodium concentration ([Na⁺]i) and pH (pHi) changes in
 BgATP4WT line after exposure to various concentrations of CIP.
 **Variables:** Time/min: Time points (0 to 20 min) CIP 0 nM / 1 nM / 20
 nM: Three different treatment conditions **Notes:** Data shows real-time
 [Na⁺]i and pHi changes after addition of 0, 1, or 20 nM CIP. Values
 represent mean ± SD from a representative experiment of six biological
 replicates. **3. Figure 3F&G – [Na⁺]i concentration and pHi changes in
 wild-type and resistant lines after 20 nM CIP** **Context:** Comparative
 analysis of [Na⁺]i concentration and pHi changes in three *B. gibsoni*
 lines after treatment with 20 nM CIP. **Variables:** BgATP4WT,
 BgATP4L921V, BgATP4L921I: Strain name \[Na⁺]i (mM): Measured intracellular
 sodium concentration pHi: Measured intracellular pH value **Notes:**
 BgATP4WT exhibited a higher [Na⁺]i and pHi increase than the mutant lines
 after CIP exposure. Indicates that resistance mutations affect the ion
 transport function of ATP4. **4. Figure 3H – ATPase activity**
 **Context:** ATPase activity of BgATP4WT, BgATP4L921V, and BgATP4L921I.
 **Variables:** BgATP4WT, BgATP4L921V, BgATP4L921I: Strain name ATPase
 Activity (nmol Pi/mg protein/min): Measured activity **Notes:** Mutant
 lines show significantly reduced ATPase activity compared to wild-type.
 **5. Figure 3I – Dose-dependent ATPase activity of BgATP4 variants**
 **Context:** Dose-dependent ATPase activity of BgATP4WT, BgATP4L921V, and
 BgATP4L921I *in vitro*. **Variables:** nM: CIP concentration **Notes:**
 Wild-type ATP4 showed greater inhibition at higher CIP concentrations
 compared to mutant variants. Each value represents the mean ± SD from
 three biological replicates.   ### **Figure 5.csv** Cross-resistance
 between atovaquone (ATO) and tafenoquine (TQ) is examined in resistant
 parasites, and the therapeutic efficacy of CIP and TQ combination is
 evaluated in a SCID mouse model infected with *Babesia microti*. **1.
 Figure 5A & B: Growth curve** **Context** Dose-dependent *in vitro*
 growth inhibition of *B. gibsoni* strains (BgATP4WT, BgATP4L921V,
 BgATP4L921I) by ATO and TQ. **Variables** nM: ATO and TQ concentration in
 nanomolar BgATP4WT/ BgATP4L921V/ BgATP4L921I: % growth of each strain at
 each drug concentration **Notes** Minimal changes in IC50 values
 (&lt;1-fold) suggest mild alterations in ATO and TQ susceptibility in
 resistant strains, without strong resistance or cross-resistance. Data
 represent means ± SD from six biological replicates. **2. Figure 5C:
 Parasitemia** **Context** *In vivo* inhibition of *B. microti*
 proliferation in SCID mice treated with CIP (20 mg/kg once daily for 7
 days), TQ (single 20 mg/kg dose), or CIP+TQ combination therapy.
 **Variables** Day: Days post-infection Vehicle / CIP / TQ / CIP+TQ:
 Parasitemia (%) in each treatment group based on five biological
 replicates **Notes** CIP alone showed significant suppression of
 parasitemia. TQ alone had moderate effect. CIP+TQ combination therapy
 showed the strongest inhibition, indicating potential synergistic effect.
 Data were collected from day 0 to day 90 post-infection. **3. Figure 5D:
 qPCR results** **Context** qPCR detection of *B. microti* DNA in blood
 samples from SCID mice at 90 days post-infection (DPI). **Variables** CIP
 / TQ / CIP+TQ / Vehicle: Cq values from qPCR analysis of parasite DNA
 **Notes** A cut-off Cq ≤ 35 was considered positive for *B. microti* DNA.
 Cq &gt; 35 or no amplification (NA) was considered negative. CIP+TQ group
 showed no detectable parasite DNA (NA), indicating potential curative
 effect.   ### **Figure\_1-figure\_supplement\_1.csv** **Supplemental
 figure 1A & B: MDCK & HFF cell viabilities** **Context**
 Cytotoxicity assay of CIP on Madin-Darby Canine Kidney (MDCK) cells and
 human foreskin fibroblasts (HFF). **Variables** μM: CIP concentration in
 micromolar (μM) Cell viability (%): Percentage of viable cells after CIP
 treatment relative to the untreated control **Notes** Cell viability was
 measured at 450 nm using the MTP-500 microplate reader. Each value
 represents the mean ± SD from three independent experiments, each
 performed in triplicate. CC50 (50% cytotoxic concentration) was calculated
 based on the viability data.   ### **Figure\_2-figure\_supplement\_1.txt:
 ATP4 multiple sequence alignment** **Context** This file contains the
 multiple sequence alignment (MSA) of ATP4 ortholog protein sequences from
 various species, including *Babesia gibsoni*, *Babesia bovis*, *Babesia
 microti*, *Toxoplasma gondii*, *Plasmodium falciparum*, and *Homo
 sapiens*. **Notes** Mutation site(s) identified in *B. gibsoni*, *P.
 falciparum*, and *T. gondii* are described in the main manuscript.   ###
 **Supplementary\_File\_1\_.txt: BgATP4 primers** **Context** This file
 provides a list of oligonucleotide primer pairs designed for amplification
 and mutation detection of the *Babesia gibsoni* *atp4* gene (*Bgatp4*).
 These primers were used in various molecular biology applications
 including PCR and gene sequencing. **Notes** Primer (5'–3'): DNA
 sequence of the primer in 5' to 3' direction Direction: F
 (forward), R (reverse)   ### **Supplementary\_File\_2.txt: BmATP4
 primers** **Context** This file provides a list of oligonucleotide primer
 pairs designed for amplification and mutation detection of the *Babesia
 microti* *atp4* gene (*Bmatp4*). These primers were used in various
 molecular biology applications including PCR and gene sequencing.
 **Notes** Primer (5'–3'): DNA sequence of the primer in 5'
 to 3' direction Direction: F (forward), R (reverse)   ###
 **Supplementary\_File\_3.txt: CIP docking interactions** **Context** This
 file contains the results of molecular docking simulations between CIP and
 three *Babesia gibsoni* ATP4 proteins (BgATP4): the wild-type (WT) and two
 mutant variants (L921V and L921I). **Notes** Each entry lists the residues
 interacting with CIP, the distance of interaction, and the type of
 interaction, including hydrogen bonds, halogen bonds, and hydrophobic
 interactions. The binding energy for each variant is provided in kcal/mol.
   ## **Software and Tools Used** Molecular docking: AlphaFold for
 prediction of BgATP4, PyMOL Molecular Graphic Models visualization: System
 and Discovery Studio Statistical analysis: GraphPad Prism v9 Image
 processing: ImageJ for measurement of parasites sizes, Jalview v2.11.3.2
 for sequence alignment   ## **License and Reuse** This dataset is released
 under the CC0 license. You are free to use, share, and modify it without
 restriction.   ## **Citation** 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,
 Xuenan Xuan, 2024. Efficacy and mechanism of action of cipargamin as an
 antibabesial drug candidate. eLife13:RP101128
 [https://doi.org/10.7554/eLife.101128.3](https://doi.org/10.7554/eLife.101128.3)   ## **Questions or Requests** For further clarification or additional data requests, please contact: [lmm_2010@hotmail.com](mailto:lmm_2010@hotmail.com); [gen@obihiro.ac.jp](mailto:gen@obihiro.ac.jp)