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 non-peptide agonist, originally developed for osteoporosis treatment, that activates parathyroid hormone 1 receptor (PTH1R) from the cytoplasmic face of the receptor and is considered Gs-protein biased. In this study, we compared the functional, pharmacological and structural properties of PCO371 with the canonical extracellular peptide agonist PTH 1-34 at the PTH1R to define the molecular mechanisms underlying PCO371's unusual signalling profile. By manipulating receptor reserve, we demonstrate that PCO371 is a partial agonist relative to PTH 1-34, contrary to previous observations. Across Gs, Gi3, Gq, cAMP, and {beta}-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. Mechanistic modelling using the Cubic Ternary Complex Activation model revealed that reduced intrinsic efficacy is sufficient to reproduce PCO371's distinct monotonic signalling kinetics, providing a quantitative explanation for its partial agonism. PCO371 binding is {beta}-arrestin-compatible but only drives measurable {beta}-arrestin-2 recruitment when PTH1R is highly expressed. Molecular modelling suggests that PC0371 stable binding needs an intact receptor-G protein complex. 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.