Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice.

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca2+ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.