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

Area of Science:

• Pharmacology
• Biotechnology
• Drug Development

Background:

• Proof of target engagement (TE) is critical for drug development investment.
• Cellular thermal shift assay (CETSA) measures intracellular TE but lacks comprehensive animal and clinical application data.

Purpose of the Study:

• To demonstrate CETSA's utility in quantitative evaluation of drug-TE in animal models and human clinical studies.
• To establish a semi-automated system for evaluating structure-activity relationships and drug occupancy.
• To optimize tissue homogenization for in vivo TE monitoring.

Main Methods:

• Quantitative evaluation of drug-TE in mouse peripheral blood.
• Confirmation of TE in animal tissues using a RIPK1 kinase inhibitor.
• Semi-automated system for structure-activity relationship analysis in cell lines.
• Estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells.
• Optimized tissue homogenization for spleen and brain TE monitoring.

Main Results:

• CETSA successfully quantified drug-TE in mouse peripheral blood.
• CETSA confirmed TE in animal tissues, including spleen and brain, using a RIPK1 inhibitor.
• The semi-automated system enabled structure-activity relationship evaluation and drug occupancy estimation.
• Optimized tissue homogenization facilitated in vivo TE monitoring.

Conclusions:

• CETSA is a viable method for quantitative drug-TE assessment in preclinical animal studies.
• CETSA can be applied to monitor in vivo drug-TE in various animal tissues.
• The developed methodology offers an efficient tool for both preclinical and clinical drug development stages.