Furamidine, a methyltransferase inhibitor, is a potential anti-Babesia spp. chemotherapeutic

Abstract

Epigenetic inhibitors targeting histone methyltransferases (HMTs) have been proven to be promising for blood protozoan treatment. However, little is known about the effects of HMT inhibitors on Babesia parasites. Here, in vitro and in vivo drug tests were performed to evaluate the efficacy of several compounds targeting various HMTs for Babesia treatment. Their cytotoxicity to MDOK cells was also assessed. Among these compounds, furamidine demonstrated outstanding activity in vitro at the nanomolar level (IC₅₀s of 0.03 ± 0.55, 0.02 ± 0.50, and0.02 ± 0.76 μM at 48, 72, and 96 h, respectively). Furthermore, the IC₅₀ of furamidine on MDOK cells was ~ 100 μM after 24 h, ~ 45 μM after 48 h and ~ 40 μM after 72 h. The therapeutic index of furamidine was greater than 1,500. In addition, furamidine effectively inhibited the growth of B. duncani and B. microti in hamsters and BALB/c mice. Furthermore, furamidine was demonstrated high in vivo safety. These findings suggest that furamidine could be an effective alternative drug for treating babesiosis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00436-025-08576-z.

Introduction

Babesiosis is an emerging, tick-borne, malaria-like disease caused by Apicomplexa parasites of the genus Babesia (Homer et al. 2000; Uilenberg 2006). Babesia, which is an obligate intraerythrocytic protozoan, is a widely distributed pathogen worldwide (Krause 2019; Kumar et al. 2021; Lim et al. 2020; Montero et al. 2023; Peniche-Lara et al. 2018). Currently, there are more than 100 Babesia species identified globally that can infect domestic and wild animals and humans (Ord and Lobo 2015; Uilenberg 1995; Vannier et al. 2015). Human babesiosis is mainly caused by five Babesia species including Babesia microti in Europe, USA, and Asia (Fang et al. 2015; Vannier and Krause 2012), Babesia divergens in Europe (Hildebrandt et al. 2021), Babesia crassa in Asia (Jia et al. 2018), Babesia duncani in USA and Canada (Vannier and Krause 2012), and Babesia motasi in Korea (Hong et al. 2019). Since the first case of human babesiosis was reported in 1957 (Skrabalo and Deanovic 1957), cases have been reported each year throughout the world (Fang et al. 2015; Hildebrandt et al. 2021; Kumar et al. 2021). Babesia microti is the most common cause of human babesiosis, with more than 2000 cases reported each year in USA and more than 100 patients reported in China (Kumar et al. 2021). In Europe, 1827 cases of human babesiosis caused by B. divergens were reported in 2020 (Hildebrandt et al. 2021). Furthermore, a study by Jinming Wang, et al. showed that the prevalence of B. divergens was 1.3% in Gansu Province, China (Wang et al. 2019). In addition, B. duncani, which was first reported in Washington in 1994 (Quick et al. 1993) and can cause fatal infections, has received much attention in recent years (Fang and Ben Mamoun 2024; Vydyam et al. 2024a; Vydyam et al. 2024b). The clinical manifestations of Babesia infection include pyrexia, malaise, headache, intravascular hemolysis, and even in severe cases, multiorgan failure and death (Huang et al. 2023; Yao et al. 2023) It can be mild in healthy individuals but severe or even life-threatening in immunocompromised and elderly people (Kjemtrup and Conrad 2000). In addition to tick-borne transmission, Babesia can be transmitted through blood transfusions, organ transplants or vertical transmission (Fox et al. 2006; Joseph et al. 2012). Currently, babesiosis has become an emerging socioeconomic challenge across the world.

Great efforts have been made to improve babesiosis management during the past several decades (Huang et al. 2023; Vydyam et al. 2024a; Vydyam et al. 2024b). However, the outcomes, to some extent, are usually discouraging. For example, atovaquone, which is a recommended drug for routine use in the treatment of babesiosis, often relapses following the end of administration (Vydyam et al. 2024a). In addition, Diminazene Aceturate (DA) is a veterinary drug routinely applied to eliminate Piroplasm parasites, but severe side effects limit its routine use in human babesiosis treatment despite its excellent therapeutic efficacy in animals (Mosqueda et al. 2012). Moreover, the emergence of resistant strains has brought new challenges to the treatment of babesiosis (Mosqueda et al. 2012). Therefore, there is an urgent need for effective and safe strategies to control this type of epidemic infectious disease.

Previous studies have demonstrated that histone modification plays a pivotal role in Apicomplexan parasites (Drummond et al. 2005; Li and Seto 2016). Histone methyltransferases (HMTs) are responsible for histone methylation on lysine or arginine residues (Jabeena and Rajavelu 2019; Saha 2020). HMT can add one, two, or three methyl groups to a lysine residue or one or two to an arginine residue. The activation or depression of gene expression is mainly associated with the number of methyl groups and the specific site of histone methylation (Fleck et al. 2021). Intriguingly, HMT is considered a novel potential target for the development of drugs for treating eukaryotic parasites (Malmquist et al. 2012; Ngwa et al. 2019; Rodrigues et al. 2002; Zuma et al. 2017). Several HMT inhibitors including UNC1999, LLY-507, GSK343, chaetocin, BIX-01294 and sinefungin had been evaluated against B. divergens, while, only BIX-01294 showed a moderate inhibitory activity against B. divergens in vitro (Vanheer and Kafsack 2021). In addition, furamidine, formerly known as DB75, which is a protein arginine methyltransferase inhibitor, could effectively inhibit B. divergens in vitro and B. microti in vivo (Nehrbass-Stuedli et al. 2011). However, little is known about the role of HMT inhibitors in the treatment of babesiosis caused by Babesia sp. Xinjiang or Babesia duncani.

Herein, we conducted in vitro and in vivo experiments to evaluate the effects of a set of HMT inhibitors on Babesia parasite proliferation. Among these chemical compounds, furamidine exhibited an outstanding anti-Babesia effect on the in vitro growth of BxjG5. Moreover, furamidine effectively inhibited the proliferation of B. duncani and B. microti in vivo; and prolonged the survival time of B. duncani-infected animals after lethal infection.

Materials and methods

Chemical compounds

Several epigenetic chemical compounds targeting different protein methyltransferases including furamidine, PFI-2 hydrochloride (PFI-2HCl), UNC1999, GSK3368715, amodiaquine, LLY-507, GSK343, chaetocin, BIX-01294 and sinefungin were evaluated for their ability to inhibit Babesia in vitro or/and in vivo. All epigenetic drugs were purchased from MedChemExpress LLC (MCE, NJ, USA). The basic features of the 10 compounds such as CAS number and target are listed in Table 1. As imidocarb dipropionate and DA are widely used to treat protozoa infections in small ruminant and ruminant, such as babesiosis, theileriosis and trypanosomiasis (Baneth 2018; da Silva Oliveira and de Freitas 2015; Grause et al. 2013; Obi et al. 2020), they were employed as positive controls for in vitro and in vivo Babesia treatment. Imidocarb dipropionate and DA were offered by MedChemExpress LLC (MCE, NJ, USA) and Lanzhou IS Abundant Pharmaceutical Co., Ltd (Lanzhou, China), respectively. Nine of the ten protein methyltransferase inhibitors and imidocarb dipropionate were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 50 mM, while sinefungin and DA were dissolved in sterilized water to final concentrations of 10 mM and 10 mg/ml, respectively. All dissolved solutions were stored at −20℃ until use.

Table 1.

Summary of the compounds used in this study

Compound	CAS No	Description
Furamidine	73819–26-8	Protein arginine methyltransferase 1 (PRMT1) inhibitor
BIX-01294	935693–62-2	G9a and GLP Histone Methyltransferase inhibitor
Sinefungin	58944–73-3	A SET7/9 inhibitor
PFI-2 hydrochloride	1627607–87-7	Cell-active inhibitor of the methyltransferase activity of SETD7
UNC-1999	1431612–23-5	A SAM-competitive, potent and selective inhibitor of EZH2/1
GSK3368715	2227587–26-8	Type I protein arginine methyltransferases inhibitor
Amodiaquine	86–42-0	Histamine N-methyl transferase inhibitor
LLY-507	1793053–37-8	Protein lysine methyltransferase SMYD2 inhibitor
GSK343	1346704–33-3	Histone lysine N-methyltransferase EZH2 inhibitor
Chaetocin	28097–03-2	A natural histone methyltransferase inhibitor
Imidocarb dipropionate	55750–06-6	A potent antiprotozoal agent
Diminazene Aceturate	632	A veterinary drug for blood protozoans

Parasites and animals

Babesia sp. Xinjiang (Bxj) was previously isolated from sheep bitten by Rhipicephalus sanguineus and Hyalomma anatolicum in Xinjiang Uygur Autonomous Region, China and preserved at Vector and Vector-Borne Diseases (VVBD) Laboratory, Lanzhou Veterinary Research Institute (LVRI) (CAAS Lanzhou, China) (Guan et al. 2016; Guan et al. 2012b; Guan et al. 2009). Babesia duncani (PRA-302) and Babesia microti (Franca) Reichenow (PRA-99) were purchased from American Type Culture Collection (ATCC) and cryopreserved in liquid nitrogen. LVG Golden Syrian Hamsters were obtained from Charles River Laboratories (Beijing, China) and bred at Laboratory Animal Center of Lanzhou Veterinary Research Institute in a clean environment. Female BALB/c mice were obtained from Laboratory Animal Center, LVRI.

In vitro culture of Babesia sp. Xinjiang

A monoclonal line (termed G5) of Bxj (BxjG5) was cultured according to a protocol previously described (Guan et al. 2012a). Briefly, the parasites were cultured in a complete medium (CM) consisting of 5% fresh sheep red blood cells (RBCs) (prepared as previously described (Guan et al. 2010)), 20% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, U.S.A), and Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza, Belgium) in a humidified 5% CO₂ atmosphere at 37 °C.

Anti-proliferation assay in Babesia sp. Xinjiang

The ten epigenetic inhibitors and imidocarb dipropionate were diluted in CM in a series of gradient concentrations from 200 μΜ to 0.005 μΜ. Initially, BxjG5-infected sheep erythrocytes were mixed with CM containing 5% fresh sheep red blood cells (RBCs) to reach a parasitemia of 1% and then cultured in 24-well plates (Corning, NY) supplemented with epigenetic inhibitors at different concentrations. Cultures were incubated for 48, 72 or 96 h. The medium was replaced with CM supplemented with inhibitors every 48 h. The same amount of DMSO as the added drug was used as a negative control and the maximum concentration of DMSO in culture medium was below 0.4%. The percentage of growth inhibition was calculated by measuring parasitemia on Giemsa-stained blood smears by manual microscopy. The growth inhibition rates were calculated as the following formula: (parasitemia in control wells—parasitemia in wells treated with inhibitors)/parasitemia in control wells × 100%. Three independent experiments with three replicates each were performed for these assays. IC₅₀ is the half maximal inhibitory concentration.

Drug effects on host cells

Sheep kidney epithelial cells (Madin-Darby ovine kidney, MDOK, KL-1633, ATCC) (stored and maintained daily at VVBD, LVRI) were used to evaluate the cytotoxicity of the ten epigenetic inhibitors. The cell line was cultured in a 96-well plate (Corning, NY) at 37℃ and 5% CO₂ with Dulbecco's modified Eagle's medium (DMEM) (BI, Israel) supplemented with 10% FBS. Approximately 2,500 cells were seeded in each well in a total volume of 100 μl and incubated for 24 h. After culturing for 24 h, the medium was replaced with CM supplemented with inhibitors, the concentrations of which ranged from 200 μM to 0.125 μM, with a twofold gradient dilution. The cells were cultured for 24 h, 48 h, or 72 h. Then, 10 μl of CCK-8 solution (Cell Counting Kit-8, Baisai, Shanghai, China) was added to each well of the culture plate, and incubated at 37℃ for 3 h. Cell viability was evaluated by measuring the absorbance at 450 nm using a microplate reader. DMSO served as a negative control. Independent experiments with technical replicates were performed.

In vivo anti-Babesia activity against B. duncani and B. microti

Twenty 12-week-old hamsters were infected with 2,500 B. duncani-infected RBCs by intraperitoneal injection. Then the inoculated hamsters were randomly and equally divided into four groups. When parasites were observed on Giemsa-stained blood smears by microscopy, the hamsters were treated via intraperitoneal injection of 200 μl/dose of vehicle control (DMSO/PBS (v/v) = 1:1) or furamidine (2 mg/kg bw) or BIX-01294 (7.35 mg/kg bw) or DA (20 mg/kg bw) daily for five consecutive days.

As we found some non-infectious events after inoculation with Babesia duncani-infected erythrocytes, we re-evaluated the efficacy of BIX-01294 and furamidine in the treatment of B. microti infection in BALB/c mice to avoid bias in the final results due to non-infectious events. Twenty female BALB/c mice (six weeks old) were intraperitoneally inoculated with 1 × 10⁷ B. microti-infected RBCs and then equally randomized into four groups. Parasitemia was monitored daily after inoculation as aforementioned. Treatment was started via intraperitoneal injection when parasites were detected on Giemsa-stained blood smears by microscopy. The four groups of mice received 100 μl/dose of vehicle control (DMSO/PBS (v/v) = 1:1) or furamidine (2 mg/kg bw) or BIX-01294 (10 mg/kg bw) or DA (30 mg/kg bw) daily for five consecutive days.

Infected animals that survived more than 50 days after inoculation and had no detectable parasite recurrence on blood smears were considered as cured. Animals were euthanized by inhalation of excessive amounts of isoflurane (Orbiepharm, Qingdao, China) in a Small Animal Anesthesia Machine (Yuyan Instruments, Shanghai) when the experiments ended.

In vivo safety evaluation of furamidine

To evaluate the adverse effects of furamidine at the therapeutic dosage (2 mg/kg bw), we conducted an in vivo safety experiment. Six to eight-week-old healthy BALB/c mice were randomly divided into two groups (n = 5 per group): a vehicle control group (DMSO/PBS) and a therapeutic dose group (2 mg/kg bw). According to the grouping, mice received a daily intraperitoneal injection of furamidine or an equivalent volume of the vehicle solvent for five consecutive days. Mice were observed twice daily (morning and afternoon) for clinical manifestations (including activity, responsiveness, fur condition, and respiration). Body weight was recorded every morning prior to dosing. 24 h after the last administration, mice were anesthetized by inhalation of excessive amounts of isoflurane (Orbiepharm, Qingdao, China), and blood samples were collected via the retro-orbital plexus. Each blood sample was divided into two equal portions. A portion of the blood was placed in EDTA-K₂-coated tubes for hematological analysis. And the remaining blood was centrifuged at 3,500 rpm at room temperature for 10 min to separate serum for biochemical analysis. After blood sampling, mice were euthanized by cervical dislocation. The liver was carefully excised, rinsed with cold PBS, and then, fixed in 4% paraformaldehyde for latter histopathological evaluations.

Statistical analysis

Parasitemia (PI) was determined by counting infected erythrocytes (iRBC) in 2,000 to 4,000 RBCs (total RBC, tRBC) on thin Giemsa-stained blood smears: PI = iRBCs/tRBCs × 100% (Guan et al. 2010). The curves between the inhibitor concentrations and inhibition rates were fitted using GraphPad Prism 7.0. Disparities among different groups were assessed by one-way analysis of variance (ANOVA), and multiple comparisons were performed by the post hoc Tukey–Kramer test. A p value below 0.05 was considered to indicate statistical significance.

Results

Antiproliferative effect of 10 epigenetic inhibitors against BxjG5

We had evaluated the effects of 10 epigenetic inhibitors on BxjG5 proliferation after incubation for 48, 72 and 96 h. As depicted in Fig. 1, furamidine and imidocarb dipropionate could effectively inhibit BxjG5 growth in vitro at nanomolar concentrations, outperforming the other nine inhibitors. Furthermore, the IC₅₀s of furamidine, which were 0.03 ± 0.55 μM at 48 h, 0.02 ± 0.50 μM at 72 h, and 0.02 ± 0.76 μM at 96 h, exhibited a slight decreasing trend with the extension of parasite incubation. Additionally, the in vitro growth of BxjG5 was also significantly inhibited by BIX-01294, for which the IC₅₀s were 0.47 ± 0.10 μΜ at 48 h, 0.35 ± 2.50 μΜ at 72 h, and 0.29 ± 1.67 μΜ at 96 h. Four of these compounds, including Sinefungin, UNC-1999, GSK343 and LLY-507, showed moderate effects on BxjG5 growth, with IC₅₀s ranging from ~ 2 to ~ 7 μM, whereas PH₂-HCl and amodiaquine exhibited relatively low activities inhibitory effects on proliferation, with an IC₅₀ of ~ 26 μM. Even when the concentrations of GSK3368715 and chaetocin increased to 200 μM, we still did not observe any obvious inhibitory effect on BxjG5 proliferation (data not shown).

Fig. 1.

Drug effects of the HMT inhibitors on BxjG5 proliferation in vitro

Cytotoxicity assays on host cells

To evaluate the cytotoxicity to host cells, the epigenetic compounds were diluted with CM at a series of concentrations and the inhibitory effects on MDOK cell viability were determined by a CCK8 assay. As shown in Fig. 2, the inhibition rates of chaetocin, LLY-507, BIX-01294 and UNC-1999 on MDOK cells were greater than 70% when the concentrations exceeded 10 μM, while the inhibitory effects of sinefungin on MDOK cells were the lowest, and the inhibition rates remained low (< 30%) even when the concentrations increased to 100 μM. The toxicity of furamidine, imidocarb dipropionate and amodiaquine were at intermediate levels; moreover, furamidine was less toxic than imidocarb dipropionate. Substantially, the IC₅₀s of these compounds in MDOK cells decreased with increasing incubation time.

Fig. 2.

Drug effects of the HMT inhibitors in host cell line (MDOK)

The therapeutic index (TI) is a safety indicator for drugs. In this study, the TI of furamidine (> 1,500) was even greater than that of imidocarb dipropionate (482 to 627) and was the highest among the tested compounds (Table 2). In general, furamidine appeared to be a novel potential drug for the treatment of babesiosis.

Table 2.

Activities of compounds in Babesia sp. Xinjiang and MODK cells

Compound	IC50 in Babesia sp. Xinjiang (μM)			IC50 in MDOK cells (μM)			Therapeutic index
	48 h	72 h	96 h	24 h	48 h	72 h
Furamidine	0.03 ± 0.55	0.02 ± 0.50	0.02 ± 0.76	98.81 ± 0.60	45.36 ± 0.43	43.01 ± 0.24	1,512 ~ 2,150
BIX-01294	0.47 ± 0.10	0.35 ± 2.50	0.29 ± 1.67	9.53 ± 0.70	6.98 ± 0.37	6.01 ± 0.37	14.9 ~ 17.2
Sinefungin	2.14 ± 0.07	2.25 ± 0.34	2.89 ± 1.10	> 100.00	> 100.00	> 100.00	> 41.1
PFI-2 hydrochloride	16.47 ± 0.86	10.68 ± 0.82	17.52 ± 1.09	> 100.00	> 100.00	> 100.00	> 6.1
UNC-1999	5.15 ± 1.31	3.88 ± 1.00	4.70 ± 1.03	7.92 ± 0.90	10.48 ± 0.51	9.23 ± 1.13	2.0 ~ 2.4
GSK3368715	> 200.00	> 200.00	> 200.00	> 100.00	> 100.00	> 100.00	-
Amodiaquine	33.16 ± 2.19	23.95 ± 1.47	26.55 ± 1.49	72.80 ± 1.35	20.52 ± 1.08	12.70 ± 2.02	0.5 ~ 0.6
LLY-507	4.02 ± 0.74	3.25 ± 0.46	2.58 ± 0.63	4.91 ± 0.69	5.16 ± 0.70	4.66 ± 1.17	1.3 ~ 1.4
GSK343	7.25 ± 0.75	6.88 ± 0.51	7.04 ± 0.56	17.68 ± 0.83	13.00 ± 0.47	11.33 ± 0.22	1.7 ~ 1.8
Chaetocin	> 200.00	> 200.00	> 200.00	< 0.13	< 0.13	< 0.13	< 0.00065
Imidocarb dipropionate	0.04 ± 0.82	0.03 ± 0.47	0.02 ± 0.24	24.08 ± 0.38	19.28 ± 0.39	18.80 ± 0.27	482 ~ 627

Drug effects on in vivo B. duncani-infected hamster models

The promising results of furamidine in the in vitro antiproliferation assay encouraged us to further estimate its’ in vivo efficacy as a single therapy against B. duncani-infected hamsters. Treatment was started when parasites were detectable (parasitemia ≈ 1%) on thin Giemsa-stained blood smears. As depicted in Fig. 3A, furamidine treatment effectively reduced the mean parasite burden to less than 10%, whereas the mean parasitemia in the BIX-01294 and vehicle control groups reached approximately 20%. Since B. duncani (WA-1 isolate) infection is lethal to LVG Syrian hamsters, in this study, all hamsters inoculated with 2,500 B. duncani-infected erythrocytes via intraperitoneal injection exhibited 100% mortality, with the exception of the DA-treated group. As shown in Fig. 3B, in the BIX-01294-, vehicle- and furamidine-treated groups, the mortality reached 100% on days 2 (5/5 deaths), 3 (3/3) and 6 (5/5) post-treatment, respectively, whereas in the DA-treated group, there were no deaths (0/4) until day 43 posttreatment. Furamidine treatment effectively prolonged the survival time of B. duncani-infected hamsters compared with those in the BIX-01294 and vehicle groups (p = 0.0013).

Fig. 3.

Drug effects of furamidine and BIX-01294 on Babesia duncani and Babesia microti proliferation in vivo. A Parasitemia of different groups of B. duncani-infected hamsters after treatment; B Kaplan–Meier curves of B. duncani-infected hamsters in each group; C Parasitemia of different groups of B. microti-infected BALB/c mice after treatment; D Heatmap of p values from Pairwise comparisons of parasitemia among different groups after treatment

Drug effects on in vivo B. microti BALB/c mice models

Based on the promising data obtained in the in vivo animal model of B. duncani infection, we further evaluated the drug effect of BIX-01294 and furamidine on a BALB/c mouse model of B. microti infection. On day 3 post inoculation (dpi 3), B. microti parasites were observed on Giemsa-stained thin blood smears (parasitemia ≈ 1%), and the mice were treated with a single dose of BIX-01294 (10 mg/kg bw), furamidine (2 mg/kg bw), DA (30 mg/kg bw) or vehicle control. DA and furamidine effectively decreased the parasite burden from 3 to 4 days posttreatment in comparison with BIX-01294 and the vehicle control. In fact, in the furamidine-treated group, parasitemia peaked on day 3 posttreatment and gradually declined to zero on day 9 posttreatment, while in the BIX-01294 and vehicle control groups, parasites were not detected for up to days 32 and 47 posttreatment, respectively (Fig. 3C). In addition, furamidine administration had effects on in vivo B. microti proliferation that were nearly comparable to those of DA, as no obvious difference in parasitemia was observed between the two groups during the treatment period (all p values < 0.01, Fig. 3D).

In vivo safety evaluation of furamidine

To evaluate the in vivo safety profile of furamidine at the therapeutic dosage, we conducted a systemic toxicity study in 6 to 8-week-old female BALB/c mice. The animals were randomly divided into two groups (n = 5 per group): an untreated control group receiving vehicle (DMSO) and a furamidine-treated group administered at a dosage of 2 mg/kg bw via intraperitoneal injection daily for 5 consecutive days.

Hematological and biochemical analyses revealed no significant differences between the furamidine-treated group and the vehicle control group. Complete blood count parameters, including red blood cells, white blood cells, and platelet counts, showed comparable values between both groups; similarly, serum biochemical parameters indicative of liver and kidney function demonstrated no statistically significant alterations following furamidine administration. Besides, no significant difference in body weight gain was observed between the two groups of mice during the treatment period (Fig. 4A).

Fig. 4.

In vivo safety evaluation of furamidine. A laboratory indices and body weight gain between furamidine treated and untreated groups; B The histological slides stained by hematoxylin–eosin (HE) of liver treated or untreated by furamidine (2 mg/kg bw) for five consecutive days. Scale bar = 100 μm. ALT, alanine aminotransferase; AST, aspartic aminotransferase; Crea, Creatinine; HCT, hematocrit; HGB, hemoglobin; Lymp, Lymphocyte; Neu, Neutrophile; PLT, Platelets; RBC, Red Blood Cell; UA, Uric Acid; WBC, White Blood Cell

Histopathological examination of liver tissues through Hematoxylin and Eosin (H&E) staining further supported the safety profile of furamidine. The hepatic architecture in furamidine-treated mice appeared normal, with preserved lobular structure, intact hepatocyte morphology, and absence of pathological features such as necrosis, inflammation, or steatosis (Fig. 4B). The histological findings were indistinguishable from those observed in the vehicle control group.

Discussion

At present, many investigations have revealed the clinical significance of chemicals targeting HMTs, and some of them have been approved by the Food and Drug Administration (FDA) for disease therapy (Sutopo et al. 2023; Xu et al. 2022). Interestingly, small molecules targeting HMTs have been shown to be promising for treating infectious diseases, such as malaria, schistosomiasis and trypanosomosis (Malmquist et al. 2012; Roquis et al. 2018; Verma 2012; Zuma et al. 2017). These encouraging findings prompted us to carry out the current study to investigate the effects of HMT inhibitors against Babesia parasites both in vitro and in vivo.

A total of ten HMT inhibitors were enrolled in this study. According to the results of in vitro drug experiments, furamidine and BIX-01294 exhibited relatively outstanding efficacies on BxjG5 growth (Figs. 1 and 2). In the study by Nicholas et al. (Malmquist et al. 2012), BIX-01294 showed good activity, with an IC₅₀ of 0.075 ± 0.036 μM against Plasmodium falciparum, whereas the IC₅₀ of BIX-01294 (ranging from 0.29 ± 1.67 to 0.47 ± 0.10 μM) in BxjG5 (Table 2), was significantly higher than that in P. falciparum (p < 0.05). The inhibition effect of BIX-01294 in BxjG5 was similar with that in Babesia divergens (IC50: 252 ± 0.82 nM) (Vanheer and Kafsack 2021). The IC₅₀s of BIX-01294 in human cell lines (JEG-3 placental choriocarcinoma cells; and human foreskin fibroblasts) and MDOK cells were at least 22-fold higher than those in parasites, indicating that BIX-01294 was, to some extent, selective for parasites over host cells. Interestingly, the IC50 of furamidine in BxjG5 was at least tenfold lower than that of BIX-01294, while, the IC50 of furamidine in MDOK cells was more than 30-fold higher than that of BIX-01294 (Table 2). The IC₅₀s of furamidine on BxjG5 (20 nM) were similar to that on B. divergens (IC₅₀ for strain 1903 was 36.8 nM, and 35.8 nM for strain 4201) (Nehrbass-Stuedli et al. 2011), implying that furamidine may be a broad-spectrum anti-Babesia epigenetic drug. What’s more, furamidine displayed the greatest inhibitory effect and TI among the ten HMT inhibitors in the treatment of babesiosis.

The encouraging results of in vitro experiments motivated us to evaluate the drug activities of BIX-01294 and furamidine in vivo. Unfortunately, the results from the in vivo drug experiments on B. duncani and B. microti indicated that BIX-01294 did not appear to inhibit Babesia growth in vivo (Fig. 3A and C). In contrast, BIX-01294 seemed to support the growth of babesia parasites in vivo at a therapeutic dose (7.35 mg/kg bw in hamsters or 10 mg/kg bw in BALB/c mice) because the parasitemia in the BIX-01294-treated groups infected with B. duncani or B. microti was higher than that in the vehicle control-treated groups (p < 0.05). This might be due to the high cytotoxicity (almost 100% inhibition) of BIX-01294 at concentrations greater than 10 μM (Fig. 2C). Consequently, BIX-01294 may not be suitable for the treatment of babesiosis despite its relatively good ability to inhibit babesia in vitro. Although furamidine has been proven to be effective in the treatment of trypanosomosis, its effectiveness against Babesia sp. Xinjiang and Babesia duncani has yet to be investigated. Interestingly, we first demonstrated that furamidine could effectively inhibit Babesia sp. Xinjiang and Babesia duncani growth both in vitro and in vivo (Figs. 1A and 3A). Although furamidine did not completely eliminate parasites from the blood of B. duncani-infected hamsters, it substantially extended the survival time of infected hamsters. To circumvent the impact of non-infectious events on the final results, we reassessed the efficacy of furamidine and BIX-01294 in B. microti infections. In this study, furamidine was also shown to have a comparable ability to DA to inhibit parasites in the blood of B. microti-infected BALB/c mice (Fig. 3C and D, all p < 0.05 during the treatment period). The anti-Babesia results of furamidine on B. microti in BALB/c mice in our study were similar with that in NMRI mice in the study by Nehrbass-Stuedli, et al. (Nehrbass-Stuedli et al. 2011). In Nehrbass-Stuedli’s study, furamidine showed excellent anti-parasite activity to totally cure B. microti-infected NMRI mouse model at 25 mg/kg, and could also completely suppress parasites through 13 days even at the lower dose of 3.125 mg/kg, while in our study furamidine could completely suppress parasitemia at 2 mg/kg even up to day 50 dpi. The disparities between Nehrbass-Stuedli’s study and our research may originate from the different routes of administration (Dirnena-Fusini et al. 2018). In addition, the antiparasitic effects of furamidine in B. microti-infected animal models were similar to that of FLLL-32 in a recent study by Shimaa, et al. (El-Sayed et al. 2023).

Furamidine has previously been employed in animal disease models at doses ranging from 1 to 25 mg/kg (Berger et al. 2011; Du et al. 2025; Wenzler et al. 2009). In our study, although the parasite load in B. duncani-infected hamsters was significantly reduced following furamidine treatment, all infected animals ultimately succumbed. To investigate the cause of mortality, we analyzed hematological and biochemical parameters, yet no marked differences were observed among the treatment groups (Supplementary Figure S1), suggesting that factors other than overt organ toxicity contributed to the deaths. We then explored whether a cytokine storm might be responsible. However, attempts to measure serum cytokines using clinical diagnostic kits yielded inconclusive results, likely due to poor cross-reactivity with hamster cytokines (data not shown).

Subsequently, we conducted a systematic safety evaluation of furamidine. Comprehensive analyses revealed a favorable in vivo safety profile for the compound. Hematological parameters remained unaltered, indicating an absence of bone marrow suppression or adverse effects on peripheral blood cells. No significant differences were observed in the biochemical indicators between the furamidine-treated and untreated groups, suggesting that furamidine has no obvious impact on hepatic and renal function (Fig. 4A). Histopathological examination of liver tissues via H&E staining further corroborated these findings, showing no evidence of hepatocyte damage, inflammatory infiltration, or necrosis after repeated administration of 2 mg/kg bw furamidine for five days (Fig. 4B). These results are particularly notable given the liver’s central role in drug metabolism and its susceptibility to drug-induced injury (Vaja and Rana 2020). The absence of hepatotoxicity at a therapeutically relevant dose supports the potential clinical translatability of furamidine. Nevertheless, longer-term studies and assessments at higher doses will be necessary to fully delineate its safety profile.

Additionally, some important issues should be addressed in this study. In contrast to previous in vivo drug experiments, in this study, treatment was not initiated until there was visible evidence of parasites in the blood of infected animal models. After 2,500 B. duncani-infected RBCs or 1 × 10⁷ B. microti-infected RBCs were used to inoculate LVG Syrian Golden hamsters or BALB/c mice, respectively, parasites were not detected on the Giemsa-stained blood smears of three of the twenty hamsters and two of the twenty mice even 50 days post infection (data not shown). This may falsely enhance the effectiveness of the drug against parasites if we start treatment soon after inoculation. Therefore, future studies should pay more attention to the effect of this variation on experimental results. In addition, since B. duncani is one of the most lethal Babesia species confirmed in animal models (e.g. hamsters and C3H mice), furamidine failed to completely eliminate B. duncani in this study, although it showed inhibitory effects on B. duncani proliferation to some extent. Therefore, there is still a need to find more potent derivatives of furamidine in the future to overcome this dilemma.

Conclusions

Taken together, our study revealed that furamidine may be a promising and safe anti-Babesia spp. drug for clinical babesiosis treatment. Future research could improve its efficacy in Babesia treatment by synthesizing more potent derivatives of furamidine or in combination with other anti-Babesia drugs.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Qindong Liang: Writing – original draft, Visualization, validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xiaoyun Li: Writing – original draft, Visualization, validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xinxin Zhang: Methodology, Investigation, Formal analysis, Data curation. Yuting Zhang: Investigation, Formal analysis, Data curation. Jinming Wang: Writing – original draft, Methodology. Zeen Liu: Methodology, Investigation, Formal analysis, Data curation. Yuxin Ye: Investigation, Formal analysis, Data curation. Yanan Bai: Investigation, Formal analysis, Data curation. Shuaiyang Zhao: Methodology. Jianxun Luo: Methodology. Hong Yin: Writing – review & editing, Supervision, Methodology, Conceptualization. Chongge You: Writing – review & editing, Supervision, Methodology, Conceptualization. Guiquan Guan: Writing – review & editing, Supervision, Methodology, Conceptualization.

Funding

This study was financially supported by grants from the National Key Research and Development Program of China (2022YFD1800200, 2024YFD1800100), the National Nature Science Foundation of China (№32573390), the Science Fund for Creative Research Groups of Gansu Province, China (24JRRA812, 22JR5RA024), the Agricultural Science and Technology Innovation Program, China (ASTIP) (CAAS-ASTIP-2016-LVRI), NBCIS (CARS-37), National Parasitic Resources Center, China (NPRC-2019–194-30), the Leading Fund of Lanzhou Veterinary Research Institute, China (LVRI-SZJJ-202105), and the hatching program of the State Key Laboratory for Animal Disease Control and Prevention, China (SKLVEB2021CGQD02 and SKLADCP2023HP04).

Data availability

The data used in this study are present in the paper.

Declarations

Ethical approval

This study was conducted with the approval of the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (permit No. LVRIAEC-2022–001). All animal experiments were performed in accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interest

The authors declare no competing interests.

Clinical trial number

Not applicable.