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

and 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 culture condition suitable for the continuous propagation of and in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy 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 pyrimethamine-resistant strain HB3, with IC values below 10 nM, and display excellent therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs.