Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

Uncoupling protein 1 (UCP1) catalyzes mitochondrial proton leak in brown adipose tissue for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we have utilized membrane protein thermostability shift analysis to characterize the interaction of activating molecules with purified UCP1. We reveal that activators influence the protein through a specific destabilizing interaction, behaving as transport substrates that shift UCP1 to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the drug ibuprofen, where ligand analysis indicates a relatively wide structural specificity for interacting molecules. Ibuprofen induces UCP1 activity in liposomes and isolated brown fat mitochondria, but not in cultured brown adipocytes. Though the drug does induce activity in UCP1-expressing HEK293 cells, demonstrating that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.