In vitro efficacy of next-generation dihydrotriazines and biguanides against babesiosis and malaria parasites

Abstract

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC₅₀ values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs.

Keywords: DHFR-TS; antifolates; antiparasitic drugs; apicomplexan; dihydrotriazine; nucleotide biosynthesis.

Fig 1

Conservation of the folate metabolic pathway among Babesia and Plasmodium parasites. (A) A schematic representation of folate metabolism in Babesia and Plasmodium parasites. The main enzymes (blue color) targeted by antifolates (violet color) are highlighted. Abbreviations (Abbs.): iRBC = infected red blood cell, DHP = dihydropteroate, DHPP = dihydropteroate pyrophosphate, DHPS = dihydropteroate synthase, DHF = dihydrofolate, DHFR = dihydrofolate reductase, PABA = p-aminobenzoic acid, SHMT = Serine hydroxy methyltransferase, THF = tetrahydrofolate, and TS = thymidylate synthase. (B) Alignment of a region of DHFR-TS enzymes from various Babesia species as well as pyrimethamine-sensitive (3D7) and -resistant (Dd2, HB3, and V1/S) P. falciparum strains. Residues known to be associated with susceptibility or tolerance to antifolates are marked in red. (C) Phylogenetic analysis of DHFR-TS enzymes from piroplasmids (tick-transmitted intraerythrocytic parasites) and various Plasmodium species. The tree was built using Phylogeny.fr pipeline combining Mafft, BMGE, and FastTree software applications. An advanced option was used to generate 1,000 bootstraps providing branch support. Clade supporting Piroplasmida sequences is shown in green; Plasmodium sequences are in blue, with branches of P. falciparum sequences represented in violet.

Fig 2

Inhibition of B. duncani DHFR-TS by JPC-2067 and JPC-2056. (A and B) Chemical structures and inhibition of B. duncani intraerythrocytic development by the prodrug, JPC-2056 (A), and its active drug JPC-2067 (B). The data represent the mean of two biological replicates with three technical replicates. Values represent mean ± SD. (C-D) A dose-dependent inhibition of purified the DHFR activity of the B. duncani (C) and B. microti (D) DHFR-TS enzymes by JPC-2067. EC₅₀ values were calculated from the inhibition curves and represent mean ± SD from three independent experiments, each performed in triplicate.

Fig 3

In vivo efficacy of JPC-2057 in mouse models of human babesiosis. (A) Parasitemia in female C3H/HeJ mice (n = 5) infected (intravenously) with 1 × 10⁴ B. duncani-iRBCs, which cause lethal infection in these mice, and treated with either vehicle (PEG-400) (red) or JPC-2056 (30 mg/kg, blue lines) administered via oral gavage once a day for 10 days (DPI-1 to 10). Treated mice were monitored daily and were euthanized when treatment was deemed not to be effective. Each data point depicts parasitemia measured via inspection of Giemsa-stained blood smears on the specified day. (B) Bar diagram depicting % parasitemia in the vehicle and JPC-2056 groups on the last day of treatment (DPI-10). Data are presented as mean ± SEM (n = 5 mice per group). (C) Kaplan-Meyer curve depicting survival of B. duncani-infected female C3H/HeJ mice followed by treatment with vehicle and JPC-2056. (D) Parasitemia in female CB17/SCID mice (n = 5) infected with 1 × 10⁴ B. microti infected RBCs (LabS1 strain) and treated with either vehicle (PEG-400, red lines) or JPC-2056 (blue lines) at 30 mg/kg once a day for 10 days. (E) Bar diagram showing the % parasitemia after 40 days post-infection in the vehicle and JPC-2056-treated groups. Data are presented as mean ± SEM (n = 5 mice per group).

Fig 4

Activity of DHTs and biguanides against Babesia and Plasmodium species and isolates in vitro. (A-F) In vitro efficacy of dihydrotriazines and biguanides against Babesia [B. duncani WA1 (A), B. divergens Rouen87 (B), and B.MO1 (C)] and Plasmodium species [3D7 (D), Dd2 (E), and HB3 (F)] at 5, 10, 50, and 100 nM drug concentrations. Color-coded heat maps represent mean (n = 3) percent inhibition of parasite growth, with dark blue representing 100% growth and dark red representing 100% inhibition.