A further analogue 3 was identified from the 11 confirmed hits. Unlike the pteridine compounds, 3 exhibited no activity in the ADP-Glo TM assay (0 -32% maximal inhibition, Table ); in this case however, an observed response in the counter-screen assay suggested interference with the ADP-Glo TM readout. SPA assay activity revealed a more potent compound of 19 µM with good maximal inhibition (77%) and no response in the SPA counter-screen assay. Further investigation of this compound revealed excellent selectivity for the MSH3 subunit (Table and Figure ). 3 (WZ3146) has previously been described as an EGFR inhibitor and we undertook a limited hit expansion exercise through purchasing and evaluating other structurally related kinase inhibitors. From these analogues 4 (Avitinib), 19 another EGFR inhibitor, emerged as a leading compound in this series with SPA potency of 3.5 µM, 97% maximal inhibition. Profiling of this analogue (Table ) revealed a competitive mode of action for inhibition, although a mixed mode component appeared to be present as indicated by the decrease in Vmax (Figure ). When measuring ATP binding in the UV-crosslinking experiment, the compound showed selectivity for the MSH3 subunit. Covalent binding this compound, which was a concern with the presence of the potentially reactive acrylamide motif, was ruled out through a jump dilution experiment: no shift in the measured IC50 suggested reversible binding. This observation was confirmed through preparation of the non-reactive saturated analogue 5 which displayed similar levels of inhibition (SPA IC50: 3.7 µM, 98% maximal inhibition) to the acrylamide analogue 4. Through this modification an entirely competitive binding mode was obtained as shown by an increase in Km and stable Vmax (Figure ).