The compound FLLL-32 was obtained from (Merck KGaA 64271 Darmstadt, Germany) and dissolved in (0.1%) dimethyl sulfoxide (DMSO) for stock concentration and kept at −30°C until use. The antibabesial drug, DA (Ganaseg, Ciba-Geigy Japan Ltd., Tokyo, Japan) was employed as a control drug. DA, ID (Sigma-Aldrich, Tokyo, Japan), and MMV396693 (MolPort, Latvia) were used for the in vitro combination inhibition assay. The nucleic acid stain SYBR Green I (SGI) (Lonza, Rockland, United States; 10,000x) was stored at −20°C and thawed before use. A lysis solution comprising Tris (130 mM; pH 7.5), EDTA (10 mM), saponin (0.016%; W/V), and TritonX-100 (1.6%; V/V) had been prepared in advance and stored at 4°C. Both SGI and lysis buffer were used for inhibition assay either in vitro or in vivo.

Using a microaerophilic stationary-phase culture technique, B. bovis (Texas strain), B. bigemina (Argentina strain), B. divergens (German strain), B. Caballi (USDA strain), and Theileria equi (USDA strain) were grown and maintained in purified bovine or equine red blood cells (RBCs). Babesia caballi was cultured in RPMI 1640 medium, whereas B. bovis, B. bigemina, and T. equi were cultured in Medium 199 (both media were purchased from Sigma-Aldrich). For equine Babesia and Theileria parasites, 40% normal horse serum was added to the media, whereas media was supplemented with 40% normal bovine serum for bovine Babesia parasites, along with penicillin G at 60 units per mL, streptomycin at 60 mg/mL, and amphotericin B at 0.15 mg/mL (all from Sigma-Aldrich). Theileria equi cultures were supplied with 13.6 g of hypoxanthine (ICN Biomedicals, Inc., United States) per mL. All parasite cultures were grown at 37°C in a 5% CO₂, 5% O₂, and 90% N₂ environment.

The fluorescence assay using an SGI stain was employed to study the effect of FLLL-32 on Babesia/Theileria growth (Rizk et al., 2015; Rizk et al., 2016). Double 96-well plates (Nunc, Roskilde, Denmark) were utilized to culture bovine Babesia as well as equine Babesia and Theileria in the infected pRBCs with either media only (blank wells) or a medium containing 0.005–200 μM FLLL-32. Positive control cultures, on the other hand, were treated with DA concentration ranging from 0.25 to 10 μM. Negative experimental controls included wells containing only the pRBCs with media containing the used solvent (0.1% DMSO). The plates were then incubated for 4 days at 37°C, and the IC₅₀ values for FLLL-32 and DA were calculated on the 4th day based on growth inhibition in three separate experiments. On the fourth day of treatment, the viability assay was carried out by mixing 1.5 μL of the control or FLLL32-treated infected RBCs with 3.5 μL of parasite-free RBCs, suspending the mixture in fresh growth medium without the addition of drugs, and incubating the mixture at 37°C for the following 4 days without changing the medium (Rizk et al., 2016). The IC₅₀ values of FLLL32 were calculated using the non-linear regression curve fit in GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, United States) (Rizk et al., 2016; Rizk et al., 2017; Rizk et al., 2019; Rizk et al., 2021a; Rizk et al., 2021b; El-Sayed et al., 2023).

Combination therapies of FLLL-32 with the commonly used (DA, and ID), and the recently identified antibabesial drugs (MMV396693) were evaluated. The combination ratios ranged from 0.50 to 0.75 IC₅₀s of the selected drugs (Supplementary Table S1) were prepared as previously described (Rizk et al., 2023). A 96-well plate containing B. bovis, B. bigemina, B. caballi, and T. equi pRBCs was treated with a two-drug combination; FLLL-32+DA, FLLL-32+ID, and FLLL-32+MMV396693 at concentrations of 0.5 x IC₅₀ and 0.75 x IC₅₀ in triplicates.

All in vitro tests were conducted at 1% parasitemia, and either 2.5% hematocrit (HCT) for B. bigemina and B. bovis, or 5% HCT for B. divergens, B. caballi, and T. equi parasites (Rizk et al., 2017). Next, 100 μL lysis buffer mixed with a 2× SGI was added to each well in the 96-wells plates after 4 days of incubation. The mean fluorescence values were then plotted against the logarithm of drug concentrations. Each drug concentration was tested in triplicate in each experiment and values of fluorescence assays were calculated from three separate experiments. The parasite survival was evaluated using non-linear expression analyses (Schadich et al., 2022).

Reverse transcription-PCR (RT-PCR) was used to assess the effect of FLLL-32 treatment on the mRNA transcription of the target B. bovis genes including S-adenosylhomocysteine hydrolase (BbSAHH) and the Histone deacetylase (BbHDAC3) (El-Sayed et al., 2023). Babesia bovis was grown in bovine RBCs on 24-well culture plates (Nunc, Roskilde) as previously described and was treated for 8 h with FLLL-32 at the 99% inhibitory concentration (IC99) (18.94 μM) and 0.1% DMSO as a negative control. After that, pRBCs were collected and washed with phosphate buffer saline. Following the manufacturer’s guidelines, total RNA was extracted using a commercial RNeasy mini kit (QIAGEN, Germantown Rd, Germantown, MD, United States). A Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Tokyo, Japan) was used to measure the RNA concentration, and one Step RNA Kit (AMV) (Takara, Japan) was used for conducting the RT-PCR following the manufacturer’s instructions. The B. bovis profilin (BbPROF) gene is used as the reference control gene. The BbSAHH, BbHDAC3, and BbPROF genes were amplified using total RNA (150 ng) from the treatment cultures and the control. The specific forward and reverse primers used are listed in Table 1, and the PCR conditions were 30 min at 50°C for reverse transcription, followed by 2 min of 94°C denaturation, 30 cycles of 94°C for 30 s denaturation, 55°C for 30 s annealing for BbHDAC3, BbPROF, and 57°C for BbSAHH, 1 min of 72°C extension, and 5 min of 72°C final extension (Mukhjargal et al., 2012). After staining with ethidium bromide, the amplified products were electrophoresed on 2.0% (w/v) agarose gels and visualized with a UV transilluminator (Nippon Gene, Tokyo, Japan). Gel electrophoresis bands were analyzed using ImageJ software (Schadich et al., 2012; El-Sayed et al., 2023).

Four groups of BALB/c mice (n = 5 per group) aged 8 weeks (CLEA, Tokyo, Japan) were injected intraperitoneally with 1 × 10⁷ B. microti (Munich strain) iRBCs except for the mice in the first group which remained uninfected and served as a negative control. When infected mice demonstrated 1% parasitemia, mice in two experimental groups were given daily injections of tested drugs (FLLL-32 and DA) for 5 days whereas, one group was non-treated and served as a control group. One group was treated with FLLL-32 intraperitoneal at a dosage of 50 mg/kg while, the other group was given DA intraperitoneal at a dosage of 25 mg/kg (positive control). A venous tail blood sample (2.5 μL) was collected from each mouse and transferred to a 96-well plate with RPMI 1640 Medium previously mixed with 50 μl of lysis solution. Following that, 50 μL of lysis buffer with 2x SGI nucleic acid stain was mixed into each well. Eventually, the plate was incubated in the dark for 1 h. The inhibitory effects of FLLL-32 and DA on the growth of B. microti were evaluated using a fluorescence spectrophotometer every 48 h until 30 days post-inoculation. Following the completion of the study, all of the mice were euthanized humanely via inhalation of the chemical chloroform, which was followed by neck dislocation (physical euthanasia).

The obtained data were analyzed using GraphPad Prism. Differences between the control and treated groups were determined by one-way analysis of variance (ANOVA) and unpaired t-tests. The statistical significance was defined as p-value < 0.05. The statistically significant differences between the drug-treated and positive-control groups were used in the viability test as an indication of parasite regrowth (Rizk et al., 2016).

All experimental protocols in this work were approved by the Animal Care and Use Committee at Obihiro University of Agriculture and Veterinary Medicine (Approval No. 27-65). All experiments were carried out following the Fundamental Guidelines for the Proper Conduct of Animal Experiment and Related Activities at Academic Research Institutions issued by Japan’s Ministry of Education, Culture, Sports, Science, and Technology. The pathogen experiment’s IDs were as follows: Babesia microti: 20170905; equine piroplasm parasites: 201910-2; and bovine Babesia: 201708-4.

FLLL-32 treatments of 0.005-, 5, and 50 μM, respectively, significantly inhibited (p < 0.05) the in vitro growth of B. bovis, B. bigemina, and B. divergens (Figure 1). Meanwhile, 50, and 5 μM FLLL-32 treatments significantly inhibited (p < 0.05) the growth of B. caballi, and T. Equi, respectively (Figure 2). FLLL-32 exhibited the highest inhibitory effects on B. bovis growth, with an IC₅₀ value of 9.57 ± 1.18 µM (Table 2). The estimated IC₅₀ for B. divergens, B. bigemina, B. caballi, and T. equi were 26.46 ± 3.67, 28.14 ± 4.38, 28.95 ± 3.13, and 30.42 ± 1.54 µM, respectively (Table 1). After that, the regrowth of the parasites was assessed after stopping the treatment using the viability test. The results demonstrated that all tested parasites did not regrow at a dosage of ≥50 μM FLLL-32 (Supplementary Table S2). These findings suggest that FLLL-32 inhibits B. bovis growth more effectively than other bovine and equine piroplasmids in vitro.

To evaluate whether the in vitro inhibitory efficacy of FLLL-32 will increase when used in combination therapy, a combination consisting of FLLL-32 with either DA, ID, or MMV396693 was used. FLLL32 at a concentration of 0.75 x IC₅₀ in combination with 0.5 x IC₅₀ and 0.75 x IC₅₀ DA showed additive effects against B. bovis on the fourth day of treatment (Table 3). FLLL-32 and MMV396693 combination at a concentration of 0.75 x IC₅₀ exhibited an additive effect on B. bigemina (Table 4), meanwhile, the same combination showed synergistic interaction on the growth of B. Caballi (Table 5). FLLL-32 and ID combination at a concentration of 0.75xIC₅₀ showed an additive effect on B. caballi (Table 5). Similarly, this drug combination (0.75 x IC₅₀ FLLL32 and 0.75 x IC₅₀ ID) showed synergistic interaction on the growth of T. equi (Table 6).

In the B. bovis culture, at the IC₉₉ concentration, the FLLL-32 doesn’t affect the transcription of the BbHDAC3 gene in 8 h treatments as it was shown by comparison of the level of mRNA transcript of this gene in treated cells with those of controls (cells treated with 0.1% DMSO) (Figure 3). Interestingly, in these treatments, the expression of the other B. bovis gene, BbSAHH in the cells treated by FLLL-32 was completely inhibited while its level in controls was not changed (Figure 3). The expression level of BbPROF gene did not differ between treated cells and controls (data not shown).

Parasitemia levels were significantly reduced (p < 0.05) in mice treated with FLLL-32 from day10 post infection (pi) to reach zero level at day 16 pi in comparison with the non-treated control group (Figure 4). Treatment with 50 mg/kg FLLL-32 resulted in 35% inhibition at day 10 p.i. (peak of parasitemia) (Figure 4). Peak fluorescence values in the treated groups with FLLL-32 50 mg/kg reached an average of 1213 at day 12 pi. Fluorescence readings were significantly reduced (p < 0.05) in mice treated with FLLL-32 from days 10–26 p.i. When compared to positive control mice (infected nontreated) which is similar to DA-treated mice (Figure 4). The obtained results suggested the hopeful antibabesial efficacy of FLLL-32 in an infected experimental animal model.