2.3. In Vitro GEF Assays

Screenings were performed using a fluorescence-based assay (adapted from [23]) to quantitate the exchange activity of the SOS-CAT on RAS. Briefly, 200 pmoles purified HRAS protein were incubated with 50 pmoles BODIPY^®-FL-GDP (ThermoFisher scientific) 1 h in the darkness. To a flat-bottom 96-wells black plate we added reaction buffer (20 mM Tris-ClH pH 7.5 + 150 mM NaCl + 1 mM MgCl₂ + 0.01% NP40 + 1 mM DTT) to a final volume of 100 μL (volume variable depending on protein concentration), 10 μL 1 mM GTP, either 5 μL of 200 μM (NACTS library) or 1 μL of 1 mM (Biomar MT collection) compound solutions (not to positive controls) were added for the screening reactions, adding 50 pmoles SOS1-CAT (not to negative controls) before measurement. The RAS-BODIPY-FL-GDP mixture was added, and the reaction was immediately measured in a fluorescent plate reader (TECAN, Männedorf, Switzerland) for 15 min (Exc.485 nm-Em.535 nm). Negative controls with reaction mix minus SOS and/or RAS proteins were performed for the compounds selected in the screening. In addition, 1 μL of 1 mM BI-2852 (Boehringer Ingelheim, Ingelheim, Germany) was used as a positive control of the inhibition on the exchange reaction. The assays for IC50 determinations maintained similar concentrations of RAS, SOS1, BODIPY^®-FL-GDP and GTP and final volume (100μL) while adjusting the volume of added compound to obtain final concentrations ranging from 0.1 μM to 100 μM.

For determination of IC50 values, we quantitated the percentage reduction of the initial rate of the GEF reaction caused by the different compound concentrations tested (from 0.1 μM to 100 μM) during the initial 200 s of reaction (ΔF/t; ΔF = total reduction of fluorescence units; t = 200 s). Normalized values of the percentage (%) inhibition produced by each compound concentration (X) tested were calculated according to the formula [100 – [(ΔF/t)_X – (ΔF/t)_C−/(ΔF/t)_C+ – (ΔF/t)_C−] × 100 = % inhibition] where the X, C+ and C− underscripts identify parameter values obtained with each compound concentration and their corresponding positive and negative controls. The IC50 assigned to each compound corresponded to the concentration yielding 50% inhibition of the initial reaction rate in each case.