PCO371 intracellular agonism at the parathyroid hormone 1 receptor produces pan-activation of signalling partners

Small-molecule agonists of class B1 G-protein-coupled receptors (GPCRs) remain rare because these receptors typically require large extracellular peptide ligands for activation. PCO371 is a notable exception: an intracellular agonist, originally developed for osteoporosis treatment, that activates parathyroid hormone 1 receptor (PTH1R) from the cytoplasmic face of the receptor. In this study, we compared the functional, pharmacological and structural properties of PCO371 with the canonical extracellular peptide PTH1-34 at the PTH1R to define the mechanism underlying PCO371’s unusual signalling profile. Functionally, PCO371 exhibited markedly lower functional affinity and a strong dependence on receptor reserve, achieving full agonism only at high receptor expression, whereas PTH1-34 maintained robust signalling under receptor depletion. Across Gαs, Gαi3, Gαq(R183Q), cAMP, and β-arrestin-2 pathways, operational model analysis showed that PCO371 is non-biased, engaging the same transducers as PTH1-34 but with ∼1000-fold lower potency. Our findings establish PCO371 as a non-biased but globally less potent agonist, compared to PTH1-34, whose signalling efficacy depends on receptor reserve and G-protein engagement. PCO371 binding is β-arrestin-compatible but only drives measurable β-arrestin-2 recruitment when PTH1R is highly expressed. Overall, these insights define the mechanistic basis of intracellular agonism at a class B1 GPCR and provide a framework for designing next-generation small-molecule modulators that exploit this emerging pharmacological space.