CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens.

The proof of target engagement (TE) is a key element for evaluating potential investment in drug development. The cellular thermal shift assay (CETSA) is expected to facilitate direct measurement of intracellular TE at all stages of drug development. However, there have been no reports of applying this technology to comprehensive animal and clinical studies. This report demonstrates that CETSA can not only quantitatively evaluate the drug-TE in mouse peripheral blood, but also confirm TE in animal tissues exemplified by using the receptor interacting protein 1 kinase (RIPK1) lead compound we have developed. Our established semi-automated system allows evaluation of the structure-activity relationship using native RIPK1 in culture cell lines, and also enables estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells. Moreover, optimized tissue homogenisation enables monitoring of the in vivo drug-TE in spleen and brain. Our results indicate that CETSA methodology will provide an efficient tool for preclinical and clinical drug development.