Fig. 3
From: Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells
CETSA-guided screening distinguishes TH5427 as a lead NUDT5 inhibitor. a Biochemical characterization of compounds shown in Table 2 by enzyme-coupled malachite green assay (MG assay). Compounds of particular interest are labeled with their code names and MG assay IC50 values (nM). b Compounds with MG assay IC50 < 100 nM and reasonable solubility were tested by high-throughput CETSA (HT-CETSA) in HL-60 cell lysates. Values shown are relative to NUDT5 band intensity at 37 °C, normalized to SOD1 and a representative of two experiments. Cropped, representative blots are included. c Compounds with > 50 % stabilization in cell lysate CETSA were tested for target engagement at 20 µM with intact HL-60 cells by CETSA. Values are plotted relative to NUDT5 band intensity at 37 °C and normalized to SOD1. Data are means ± SEM and individual points from n = 2 experiments. Cropped, representative blots are shown. NS not significant, *p < 0.05, **p < 0.01; one-way ANOVA analysis. d HL-60 cells were treated with the same compounds from c but titrated by serial dilution from 20 µM to 0.06 µM for isothermal dose-response fingerprint CETSA (ITDRFCETSA). Data for TH5427 comprise data points from three independent experiments and representative experiments are shown for the remaining compounds. Representative, cropped blots are also shown. In all cases above, compounds colored in red progressed to the next stage of evaluation. e Structure of NUDT5 with TH5427 bound. Coordination of TH5427 (cyan) by NUDT5 dimer (chain A – limon green and chain B – green) via stacking interactions between Trp28 of chain A and Trp46 of chain B and hydrogen bonds to the amide nitrogen of Glu47 and side chain of Arg51
