Project Title: Efficacy and mechanism of action of cipargamin as an antibabesial drug candidate

Contact Information: lmm_2010@hotmail.com; 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 < 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 (<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 > 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

 

Questions or Requests

For further clarification or additional data requests, please contact: lmm_2010@hotmail.com; gen@obihiro.ac.jp