Molecular Targeted Engagement of DPP9 in Rat Tissue\nUsing CETSA, SP3 Processing, and Absolute Quantitation Mass Spectrometry

The\ncellular thermal shift assay (CETSA) provides a means of understanding\nthe extent to which a small molecule ligand associates with a protein\ntarget of therapeutic interest, thereby inferring target engagement.\nBetter analytical detection methods, including mass spectrometry,\nare being implemented to improve quantitation within these assays,\nproviding both absolute quantitation and a very high analyte specificity.\nTo understand the target engagement, and hence inhibition, of the\nprotein dipeptidyl peptidase 9 (DPP9) in rat tissue, CETSA experiments,\ncoupled with single-pot, solid-phase-enhanced sample preparation (“SP3”)\nand absolute quantitation by high-resolution mass spectrometry, demonstrated\na temperature-dependent “melting curve” by ex vivo incubation\nof compound with rat tissue and further demonstrated in vivo engagement\nby a dose-dependent response to treatment. These experiments illustrate\nthe ability to extend the CETSA to in vivo dosed-animal samples using\nabsolute quantitation of DPP9 by mass spectrometry and demonstrate\na viable path for interrogating therapeutic molecules for drug discovery.