The measured inhibitory activities of compounds 2a-e, 3a-e and 4a-j against the targets TbPTR1, TbDHFR, LmPTR1, LmDHFR, and the off-targets hDHFR and hTS, are given in . Greener boxes show higher inhibition and selectivity. $ indicates that a precise activity value could not be determined as the tight binding limit was approached. used to characterize and rank compounds when screening for enzyme inhibition in drug discovery projects. Overall, the inhibitory activities against the PTR1 targets for the designed compounds are improved, as are PTR1 vs. off-target selectivities. Indeed, for a small number of compounds (2c, 2d, 4c, 4d, 4e, 5c and 5d), the IC50 values for inhibition were determined to be either 1 nM or less than 0.1 nM against TbPTR1. As the TbPTR1 assay makes use of low nanomolar concentrations of enzyme, for these very potent compounds, the tight binding limit was approached and therefore, accurate values of the IC50 values could not be determined. 10 nM. This notable improvement of LmPTR1 activity is in line with its predicted good steric fit to the LmPTR1 binding pocket shape (compare Figures and). Full enclosure and stabilizing interactions with hydrophobic residues lining the pocket entrance likewise probably contribute to an around 10-fold higher potency of 3d and 3e against TbPTR1 than the most similar reference compound 1c (lacking an N10 substitution) (IC50 3d, 3e: 10 nM; 1c: 110 nM).