Summary The objective of this project is to identify small non-peptidic molecule modulators of the parathyroid hormone type 1 receptor (PTHR), a medically important G protein-coupled receptor (GPCR) regulating blood calcium and phosphate homeostasis, and bone remodeling in response to PTH. Detrimental hypercalcemia caused by excess of blood PTH level, or polymorphisms of PTHR resulting in receptor hyperactivity, osteoporosis, and hypocalce- mia caused by defective PTHR signaling, underlie numerous bone and mineral-ion pathologies affecting public health. Previous support via R01DK-116780 has solved the near-atomic structure of PTHR in complex with Gs and made discoveries that led us to formulate the hypothesis that integrated computational, and pharmacological studies, using high-resolution structures of the PTHR and medicinal chemistry methods, may address the chal- lenge of discovering and developing small molecules capable of allosterically modulating PTHR signaling. Aim 1 will use a combination of computational approaches, including molecular dynamics (MD) simula- tions coupled to elastic network model (ENM)-based methods, to predict PTHR sites that are simultaneously druggable and allosteric, and identify small molecules that can potentially serve as allosteric modulators. These compounds will be iteratively refined and validated with feedback from experiments conducted in Aims 2 and 3. Aim 2 will characterize and optimize (via synthesis and/or modifications using advanced medicinal chem- istry methods) the pharmacological profile and cellular impact of these hit compounds by determining the binding, signaling, and trafficking properties of PTHR in the presence of these compounds. Cell studies will test the func- tional actions of compounds with most interesting pharmacological profiles on osteoblast mineralization and expression of 25-hydroxyvitamin D 1-a-hydroxylase, rate limiting enzyme for the biosynthesis of vitamin D). Aim 3 will carry out single particle cryo-EM studies to solve the structure of PTHR in complex with Gq, the G protein that mediates PTH signaling through Ca2+ and PKC signaling pathways. The new structure(s) will permit us to determine the mechanistic basis by which different G proteins –Gq and Gs– reciprocally couple to the PTHR. Structural data will be further used for simulating the collective dynamics of the complex and the mechanism of allosteric communication between the Gq protein and the orthosteric and allosteric ligand-binding pockets. The significance of this research program lies in its first of its kind computational pipeline integrated with multidisciplinary experimental characterization and validation studies, permitting a precise identification of drug- gable allosteric sites in the PTHR to identify novel small molecules with potential therapeutic utility for the treat- ment of bone and mineral ion diseases. Our research may ultimately prove useful to other peptide hormone GPCRs to identify small molecul...