Understanding drug target engagement and the relationship to downstream pharmacology is critical for drug discovery. Here we have evaluated target engagement of Chk1 by the small-molecule inhibitor V158411 using two different target engagement methods (autophosphorylation and cellular thermal shift assay [CETSA]). Target engagement measured by these methods was subsequently related to Chk1 inhibitor-dependent pharmacology. Inhibition of autophosphorylation was a robust method for measuring V158411 Chk1 target engagement. In comparison, while target engagement determined using CETSA appeared robust, the V158411 CETSA target engagement EC50 values were 43- and 19-fold greater than the autophosphorylation IC50 values. This difference was attributed to the higher cell density in the CETSA assay configuration. pChk1 (S296) IC50 values determined using the CETSA assay conditions were 54- and 33-fold greater than those determined under standard conditions and were equivalent to the CETSA EC50 values. Cellular conditions, especially cell density, influenced the target engagement of V158411 for Chk1. The effects of high cell density on apparent compound target engagement potency should be evaluated when using target engagement assays that necessitate high cell densities (such as the CETSA conditions used in this study). In such cases, the subsequent relation of these data to downstream pharmacological changes should therefore be interpreted with care.
Keywords: CETSA; Chk1; autophosphorylation; kinase inhibitor; target engagement.