Figure 2: Primary screen using CETSA to measure target engagement of human thymidylate synthase.
(a) Scatter plot illustrating normalized screen data, where 0% corresponds to the TS signal observed in the presence of DMSO only (magenta square) and 100% corresponds to the TS signal observed in the presence of 100 nM raltitrexed (green triangle). Data for library compounds at a concentration of 50 μM are shown in blue (blue circle). The hit limit was calculated based on the average plus three standard deviations for the library compounds and is illustrated as a black solid line at 11.7%. The locations of the Prestwick drug set (yellow) and a nucleoside subset (purple) are highlighted. (b) ITDRFCETSA data illustrating the ranking of floxuridine (blue upwards triangle), 5-fluorouridine (FUR) (green downwards triangle), and 5-FU (lavender blue square) after 2 h of preincubation time. Data are also included for CBK115334 (magenta circle).The solid lines represent best fits to a saturation binding curve resulting in an apparent EC50 of TS at a concentration of 65±9 pM, 47±15 nM, 19±4 μM and 0.46±0.08 mM, respectively. Data are provided as the average and s.e.m. from one independent hit confirmation experiment done in quadruplicate. (c) Structures of known drugs and hit compounds discussed in the main text. (d) Structure of CBK115334 (magenta) and dUMP bound to TS, shown overlayed on the structure of the complex of raltitrexed (white) and dUMP (PDB 1HVY).
