Screening of purine nucleoside analogues against intracellular Toxoplasma gondii

Toxoplasmosis remains a world-wide public health concern, especially for the immunocompromised. Although this population segment is increasing due to therapeutic interventions, organ transplants and infections including HIV, treatment relies almost exclusively on sulfadoxine and pyrimethamine, antifolates developed against malaria but with only moderate efficacy against acute toxoplasmosis and no effect on the chronic stage. Here we explore whether 7-substituted analogues of 7-deazaadenosine (tubercidin) that have shown remarkable efficacy against other protozoan pathogens, might also show anti-toxoplasmic activity. Tubercidin and a series of eleven 7-substituted analogues including 2'-deoxy and 3'-deoxyribofuranoses was tested against intracellular Toxoplasma gondii tachyzoites. The test compounds yielded EC₅₀ values between 0.012 and 1.72 μM, well below those of the control drug sulfadiazine (11.9 μM) and the previously identified purine analogue adenosine arabinoside (Ara-A; 11.4 μM). The tubercidin analogues displayed at most moderate toxicity to HFF cells, with the most efficacious compound, 7-(3,4-di-Cl-phenyl)-3'-deoxytubercidin (FH8513) reaching a selectivity index of >2500. These nucleosides are most likely taken up by T. gondii through one of the four Equilibrative Nucleoside Transporters (ENTs) encoded by the parasites. However, deletion of TgENT2 and/or TgENT3 had no effect on the EC₅₀ values, and deletion of TgAT1 actually sensitised the tachyzoites to most of the tubercidin analogues. We propose that these nucleosides are internalised through the TgENT1 uridine transporter and that the sensitisation in ΔTgAT1 cells is the result of reduced uptake of adenosine that competes with the tubercidin analogues for metabolic enzymes such as adenosine kinase.