We applied a multitarget-based approach to the development of novel therapies for HAT and leishmaniasis, in which we focused on pteridine-based inhibitors of L. major and T. brucei PTR1 and DHFR, and successfully designed the first known apparent picomolar inhibitors of TbPTR1. While LmPTR1/LmDHFR inhibition was previously explored for this compound class , we here demonstrated the potential of pteridine-based inhibitors against TbPTR1 and TbDHFR. We solved a crystal structure of the reference compound 1b bound to TbPTR1 to confirm the overlap in observed binding modes between LmPTR1 and TbPTR1 and the preference of the methotrexate inhibitor-like bound orientation in TbPTR1. Guided by our detailed comparative study of on-and off-targets in the parasitic and human folate pathway , crystal structures of reference compounds, and enzymatic evaluation of published reference pteridines , we designed 26 new pteridine derivatives that mostly have improved activity and selectivity. For their synthesis, we made use of an advanced MW-assisted protocol to improve the reaction yield of the pteroid step with reduced reaction time compared to previous synthetic procedures. Further determination of the crystal structures of complexes and computational docking enabled us to obtain a complete characterization of the binding modes of the pteridines to their molecular targets and supported the derivation of a SAR. The compounds were also tested against the human off-targets hDHFR and hTS. While they were and.