Abstract: Obesity is a serious, chronic disease currently affecting about 38% of the global population and expected to rise to over 50% by 2035 according to a recent report from the World Obesity Atlas (WOA)1. Single GLP-1R agonist, Semaglutide (Novo-Nordisk), recently spiked market interest as an approved drug for obesity management. Now, many pharmaceutical companies are pursuing a strategy of targeting multiple receptors simultaneously for additive benefits, the most attractive of these being dual GLP-1R/GIPR agonist, Tirzepatide (approved drug developed by Eli Lilly), and triple GLP-1R/GIPR/GCGR agonist, Retatrutide (in clinical development by Eli Lilly). With anti-obesity target combination therapies quickly gaining market interest, the need for a focused set of cell-based assays ideal for testing functional activity of desired therapies against multiple disease-relevant targets in one convenient screen became apparent. The obesityLITE panel of cell-based assays includes 25 assays targeting leptin receptor and G-protein-coupled receptors (GPCRs) implicated in metabolic regulation including GLP-1R, GIPR, GCGR, MC4R, CCKAR, amylin, neuropeptide Y, ghrelin, and orexin receptors. Relevant obesity pharmacophores targeting single (semaglutide), dual (tirzepatide), or triple (retatrutide) receptors were assessed in the obesityLITE panel of assays for selectivity. Single point testing at 1 µM demonstrated the highly specific nature of these compounds against their respective targets. Activity was confirmed by dose-response screening for potency determination with sub-nanomolar EC50s measured in cyclic adenosine monophosphate (cAMP) signaling assays and nanomolar EC50s measured in β-arrestin signaling assays. To determine if these potency differences reflected GPCR signaling bias, we employed a method that utilizes the Black-Leff operation model2,3,4 to derive ligand bias values for GLP-1R for all three agonists. Minimal bias was observed using semaglutide for GLP-1R signaling, while tirzepatide and retatrutide were slightly, but significantly, biased toward cAMP signaling over β-arrestin. Tirzepatide additionally was imbalanced and displayed a significant shift in relative intrinsic efficacy toward cAMP signaling. Semaglutide was further evaluated for selectivity and specificity against 168 GPCR targets in the gpcrMAX panel and was highly specific, with no off-target response measured at 10 µM in agonist or antagonist mode. Taken together, we have demonstrated the use of a single platform of cell-based assays to characterize anti-obesity compounds against relevant metabolic targets for specificity, potency and biased signaling.
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