Cellular thermal shift assay of subcellular isolates to evaluate drug-membrane target interactions

The cellular thermal shift assay (CETSA) is a tool for target identification and validation in drug discovery. It relies on thermal melting curves to indicate drug binding and is performed in cells, cell lysates, or purified protein. These approaches can disrupt the structural integrity of membrane proteins, hindering drug-target engagement. We describe the first application of CETSA in isolated mitochondria and show the effects of this approach on the analysis of the compound UK5099 and its known binding target, the mitochondrial pyruvate carrier (MPC), a mitochondrial inner membrane-localized protein complex. Our analysis supports a model in which the MPC must remain structurally intact for UK5099 binding. We demonstrate that the binding of UK5099 to the MPC is disrupted in cells and cell lysates, whereas isolating mitochondria maintains the binding interaction between drug and target observable using CETSA. These data suggest that isolating membrane-bound organelles through subcellular CETSA stabilizes membrane-bound proteins in their native conformation, allowing the identification of membrane-localized drug binding targets that might otherwise be missed.•CETSA on subcellular isolates preserves membrane target in native conformation.•UK5099-MPC binding is preserved in mitochondrial isolates but not cells or lysates.•This approach validates direct-binding interaction of membrane proteins.