Eneboparatide induces prolonged 24-hour PTH 1 receptor activation

Chronic hypoparathyroidism (HypoPT) leads to hypocalcemia due to insufficient parathyroid hormone (PTH), with conventional oral calcium and vitamin D therapy often failing to provide stable regulation. Eneboparatide is a peptide designed to activate the PTH 1 receptor (PTH1R) with prolonged effects.

We investigated the mechanism by which eneboparatide prolongs PTH1R activation.

This study explored eneboparatide receptor engagement, cellular signaling, tissue distribution, and structural determinants using in vitro and in vivo models.

Activation of PTH1R and PTH 2 receptor (PTH2R) was assessed using a cell-based cyclic adenosine monophosphate (cAMP) assay. Receptor binding affinity was determined through a radioligand competition assay. To examine the kinetics of PTH1R activation, cAMP production was measured for 24 hours following ligand washout in PTH1R-expressing cells. Tissue distribution and retention were evaluated in rats using quantitative whole-body autoradiography. Structure-function analyses investigated how amino acid alterations influenced the kinetics of PTH1R activation.

Eneboparatide activated PTH1R and PTH2R, establishing it as an agonist at both receptors. Eneboparatide demonstrated high affinity for the R0 conformation of PTH1R, supporting persistent receptor activation and extended cAMP signaling up to 24 hours in contrast to the transient effects of native PTH(1-34). When compared with PTH(1-34), autoradiography in rats revealed prolonged retention of eneboparatide in the kidney, bone, and liver, all of which express PTH1R, consistent with enhanced receptor occupancy in target tissues. Structure-function studies established that sustained PTH1R activation depends on targeted amino acid substitutions in the PTH segment and its direct connection with the PTH-related peptide region, extending at least through residue 29. These features enable eneboparatide to confer robust and extended control of serum calcium despite rapid systemic clearance.

These results mechanistically delineate the long-acting profile of eneboparatide and support its ongoing development as a therapeutic option for patients with HypoPT.