brucei PTR1 (TbPTR1) and TbDHFR, in addition to the corresponding Leishmania targets, while ensuring selectivity against the off-target hDHFR. The enzymatic evaluation of reference pteridines reported earlier , our comparative study of trypanosomatid folate pathway proteins , and computational docking studies were first employed for the design of novel pteridine derivatives. Three new crystal structures of complexes of pteridines with TbPTR1 and a complex with LmPTR1 were determined and confirmed the predicted bound orientation of the novel pteridines. A systematic analysis of correlations between computed physicochemical molecular descriptors and observed anti-parasitic effects was then performed and allowed us to prioritize promising compounds for synthesis. In total, we identified 26 new pteridine-based multitarget inhibitors showing improved target inhibitory profiles for PTR1 and DHFR of both L. major and T. brucei. Among these inhibitors, we report the first, to the best of our knowledge, apparent picomolar inhibitors of TbPTR1 and several new low nanomolar inhibitors of LmPTR1, which mostly also show selective micromolar to submicromolar inhibition of the parasite DHFR variants. In vitro evaluations of the designed multi-target inhibitors against bloodstream forms of T. brucei brucei revealed low micromolar to submicromolar EC 50 values for three of these pteridines. Taken together, we here report a successful application of systematic multitarget design approach to yield selective pteridinebased anti-parasitic compounds affecting multiple trypanosomatid enzymes.