Human African trypanosomiasis (HAT) or sleeping sickness is a parasitic disease transmitted by tsetse flies with a relatively benign haemolymphatic followed by a lethal encephalitic stage. Treatment is increasingly compromised by emergence of drug resistance in addition to the known toxicity of current drugs. In response to this medical need, our previous hit-finding campaign identified nucleoside analogues that are highly potent and selective against trypanosomes in vitro and fully curative in vivo after oral administration (50 mg/kg for 5 days) in an acute mouse model. This project will make a structure-activity relationship by expanding two novel compound series to further optimize potency and to make drug uptake less dependent on a single transporter that is prone to resistance development. The chemical synthesis will be combined with detailed evaluation of compound efficacy using state-of-the art methodologies, including natural transmission models and in vivo bioluminescent imaging to assess overall impact of treatment. Aiming to comply with the desired target product profile for such drugs, potency will be evaluated in acute and chronic infections with cerebral involvement. Most promising compounds will be subjected to identification of the action mechanism using loss-of-function and protein biochemical approaches. Collectively, this project aims at providing a convincing proof-of-concept for the use of nucleoside analogues for the treatment of this neglected disease.