3.4.1. Primary Screening

Kinetobox collection was tested on the recombinant Lm/TbPTR1 protein by a Cytochrome C (Cyt-C) coupled-spectrophotometric assay with a 96-well multiplate reader (Spectramax-190, Molecular Device) [40,41]. Each compound was properly diluted to have a final concentration of 10 µM and a DMSO percentage ≤ 1% in the enzyme mixture. Methotrexate (MTX) was included into the screening panel as C⁺ control at final concentration of 1 µM (IC50 equal to 1 µM and 0.5 µM for Tb- and Lm-PTR1, respectively) [42]. Then, 1 μL of each diluted compound stock (2 mM in DMSO) was manually added to the plate (in triplicate). The first and the last row of each plate were used for C⁺ (MTX) and C⁻ (no-inhibition) controls to reduce any positional and/or association bias. This step was followed by the addition of 100 μL of 20 mM sodium citrate pH 6.0, 80 μM Cyt-C, 3 μM and 0.3 μM BH2 (for Lm and Tb, respectively), 0.002 μM and 0.02 μM (for Lm and TbPTR1, respectively) and double-distilled water (0.2 µm filtered) to volume. After homogenization, 10 min of incubation at 30 °C and shaking for 1 min, 100 μL of activity buffer containing NADPH (500 μM) and sodium citrate 20 mM was added to each well. After brief shaking, the reading was performed for a total kinetic time of 10 min at 30 °C at 550 nm. Raw screening measurements were used to determine the slope of progression curves by linear regression for control and compound wells. The percent inhibition (%Inh) was calculated for each compound as follows: %Inh = 100 − [(dOD/dt)^well * 100]/µ^C−, where (dOD/dt)^well represents the slope of each compound well and µ^C− the average of no-inhibition controls [24]. The data results are the mean of two experiments performed in triplicate.