Fig. 6: Potential targets for therapeutics development in B. duncani.

a, Drugs are categorized as follows: Red, effective against Babesia parasites and used clinically; Yellow, effective against Babesia parasites but have not been evaluated clinically; Pink, clinically approved drugs for other diseases but have not been tested against Babesia parasites; and Blue, drugs under clinical evaluation for the treatment of other diseases but have not yet been tested against Babesia parasite inhibitors. Drug and protein abbreviations are as follows: Lys-TRNAS, Lysyl tRNA Synthase; Pro-TRNAS, Prolyl tRNA Synthase; Thre-TRNAS, Theronyl/alanyl tRNA Synthase; Tryp-TRNAS, Tryptophanyl tRNA Synthase; DHFR, Dihydrofolate reductase; P-ATPase, P-type ATPase; CytB, Cytochrome bc1 complex subunit 7 superfamily; Translation EFG/EF2, Elongation factor EFG (EFG); PI4PK𝛄, Phosphatidylinositol 4-kinase gamma; FNTB, Farnesyltransferase subunit beta; CPSF2, Polyadenylation specificity factor subunit 2; DXR, MEP Synthase; FBR, Febrifugine; HAL, Halofuginone; CLD, Cladosporin; MUP, Mupirocin; BRD, Borrelidin; DDD1, DDD107498; KDU6, KDU691; MV48, MMV048; KAI4, KAI4KAI407; BQR6, BQR695; BRD, BRD73842; ATV, Atovaquone; TBZ, Tetracyclic Benzothiazepine; GW8, GW844520; GSK9, GSK932121; ELQ, Endochin-like quinolones; KAE6, KAE609; GNF4, GNF-Pf-4492; PA21, PA21A092; SJ73, SJ733; AN36, AN3661; FOSM, Fosmidomycin; MV81, MMV008138; BMS3, BMS-388891; MV19, MMV019066; PYR, Pyrimethamine; PGL, Proguanil; MV27, MMV027634. b, Overview of folate metabolism and inhibitors. Synthesis of DHF precursor by DHP(F)S and reduction of DHF to tetrahydrofolate (THF) catalysed by the bifunctional enzyme DHFR by using NADPH as electron donor. The thymidylate synthase (TS) catalyses the reductive methylation of deoxyuridine monophosphate (dUMP) in deoxythymidine monophosphate (dTMP) using the tetrahydrofolate (5,10-Methylene THF) as cofactor via a hydroxymethyl transfer, mediated by serine hydroxy methyltransferase (SHMT). Common DHFR inhibitors are marked in grey box. c, Sequence comparison of DHFR-TS sequences between B. duncani, B. microti, B. bovis and P. falciparum pyrimethamine-sensitive (3D7) and resistant (HB3) parasites with highlighted resistance-associated residues in their primary sequences (black arrows). d, Dose-response curves with IC₅₀ values of antifolates pyrimethamine and WR-99210 and their inhibitory effect on B. duncani intraerythrocytic parasite growth. Data presented as mean ± s.d. from three independent experiments performed in triplicates. e, Enzymatic activity inhibition of BdDHFR-TS and BmDHFR-TS by WR-99210. Data presented as mean ± s.d. from three independent experiments performed in triplicates.