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 PCO371s 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 Gs, Gi3, Gq(R183Q), 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. 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 {beta}-arrestin-compatible but only drives measurable {beta}-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. HIGHLIGHTSO_LIPCO371 requires high PTH1R expression to achieve full agonism. C_LIO_LIPCO371 activates Gas, Gai and Gaq families but is significantly less potent than PTH1-34. Operational modelling shows no detectable signalling bias for PCO371; reduced signalling is global rather than pathway selective. C_LIO_LIPCO371-bound PTH1R structure is compatible with {beta}-arrestin engagement, where PCO371 elicits a measurable {beta}-arrestin-2 response only under high receptor expression. C_LIO_LIMolecular dynamic simulations reveal that PCO371 becomes stably bound only within a preassembled PTH1R-G-protein complex. C_LIO_LIPCO371 stabilises a distinct rearrangement in PTH1R:Gs/Gq/Gi3 TM6 and the TM1/TM7 bundle and forms G-protein subtype specific 5 helix interactions. C_LIO_LIEstablish a mechanistic basis for intracellular agonism, informing future design of therapeutically relevant modulators. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/728112v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@13cb194org.highwire.dtl.DTLVardef@1ae82bforg.highwire.dtl.DTLVardef@a00d4org.highwire.dtl.DTLVardef@bc657f_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG