Following the assessment of the improvement on the target inhibition level, we next determined the anti-parasitic effect on T. brucei brucei Lister 427 bloodstream forms and L. infantum intramacrophage amastigotes (Figure and Table ). The LmPTR1 and LmDHFR proteins are highly similar to the corresponding L. infantum proteins (91% and 96% sequence identity, respectively) but, in spite of the improved effect on both target proteins, the designed pteridines are mostly inactive against L. infantum. In contrast, the compounds show activity against T. brucei. Another reason for the low correlations between parasite and target protein inhibition could be transport issues. For example, the charged compound tail and possible polyglutamylation of the parent MTX (1a) have previously been suggested to influence compound transport. All the newly designed pteridines lack the glutamate tail, which may affect their in vivo activities, but there might be other structural or physicochemical features that render them more or less active against parasites despite similar target inhibition. For example, for T. brucei, we noticed that while 2c and 4e have similar effects on the targets TbPTR1 (0.1 nM) and TbDHFR (approx. 1 μM), they differ notably in their inhibitory effect on the parasite bloodstream forms (75 vs. 40%).