Development of a Parathyroid Hormone Receptor Antagonist with High Proteolytic Stability

Presentation Number: SAT 354
Date of Presentation: April 2nd, 2016

Ross W. Cheloha*1, Tomoyuki Watanabe2, Thomas Dean2, Samuel H Gellman1 and Thomas J Gardella2
1University of Wisconsin, Madison, WI, 2Massachusetts General Hospital, Boston, MA


Cell signaling processes mediated by parathyroid hormone receptor-1 (PTHR1) enable regulation of several important physiological processes including calcium homeostasis and bone turnover. While PTHR1-medaited signaling is activated by parathyroid hormone (PTH), past work has shown that some derivatives of PTH lacking important N-terminal residues, such as PTH(7-34) bind to PTHR1 and do not activate intracellular signaling. These derivatives act as competitive antagonists of PTH-induced intracellular signaling. Further modification of the of the PTH scaffold via incorporation of a Gly-to-dTrp12 substitution provides a compound (dTrp12-PTH(7-34)) which, in addition to acting as a PTHR1 antagonist, also inhibits ligand-independent signaling at disease-associated PTHR1 variants with elevated levels of constitutive activity (inverse agonism). PTHR1 antagonists and inverse agonists may be useful therapeutically for preventing or reversing PTH receptor over activation and associated symptoms, such as humoral hypercalcemia of malignancy and Jansen’s chondroplasia; however, conventional PTH peptides exhibit low stability in the presence of proteases, which likely diminishes their therapeutic utility. We sought to address this liability via incorporation of amino acid derivatives containing backbone structural modifications, which prior work suggests may enhance resistance to proteolytic degradation, into the dTrp12‑PTH(7‑34) scaffold. One derivative containing six backbone modified residues (antag-D6) was compared to prototype dTrp12‑PTH(7‑34). We assessed how backbone modification affected PTHR1 binding, antagonist activity, inverse agonist activity, and stability in the presence of proteases. Binding assays showed that antag-D6 and dTrp12-PTH(7-34) bound PTHR1 with indistinguishable affinity and in assays using SGS-72 cells, derived from the human osteosarcoma cell line Saos-2 stably transfected with Glosensor cAMP reporter protein, antag-D6 inhibited the cAMP signal induced by PTH(1-34) as effectively as dTrp12-PTH(7-34). Similarly, antag-D6 and dTrp12-PTH(7-34) exhibited indistinguishable inverse agonist activities in HEK293 cells stably expressing constitutively active PTHR1 variants (H223R or T410P) associated with Jansen’s chondrodysplasia. Protease stability experiments revealed that antag-D6 was over 10-fold more stable than dTrp12-PTH(7-34). Thus, antag-D6 was as effective as a conventional PTHR1 antagonist ligand in cell and receptor-based evaluations, but showed higher stability with respect to degradation by proteases. Peptide backbone modification thus offers a straightforward approach for enhancing the stabilities of peptidic antagonists and inverse agonists for the PTHR1, which could facilitate the development of more effective therapeutics.


Nothing to Disclose: RWC, TW, TD, SHG, TJG