Investigating hydrophobic ligand–receptor interactions in parathyroid hormone receptor using peptide probes M. E. Cupp, B. Song, P. Kibler, U. S. Raghavender, S. K. Nayak, W. Thomsen and A. K. Galande* With an increasing number of new chemical entities entering clinical studies, and an increasing share of the market, peptides and peptidomimetics constitute one of the most promising classes of therapeutics. The success of synthetic peptides as therapeutics relies on the lead optimization step in which the lead candidates are modified to improve drug-like properties of peptides related to potency, pharmacokinetics, solubility, and stability, among others. Peptidomimetics based on the N-terminal stretch of the first 11 amino acids of the PTH have been investigated as potential lead compounds for the treatment of osteoporosis. On the basis of a peptide reported in the literature, referred to here as the Parent Peptide (H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH 2 ), we conducted systematic SAR analyses to investigate the effects of altering peptide hydrophobicity on PTH receptor functional potency as measured by the cAMP (cyclic adenosine monophosphate) accumulation and b-arrestin recruitment assays. Among hydrophobic residues, we found that the Val2 position shows the least flexibility in terms of the SAR studies, whereas the Leu7 position appeared to be most flexible. Through circular dichroism and nuclear magnetic resonance spectroscopy studies, we were able to establish that changes in hydrophobic residues significantly change the extent of peptide helicity and that the helical character correlates well with receptor agonist activity. Here, we report several novel PTH 1–11 peptidomimetics that show comparable or enhanced potency to stimulate G s -signaling over b-arrestin recruitment as compared with such properties of PTH 1–34 and the Parent Peptide. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: parathyroid hormone; structure–activity relationship; peptidomimetics; hydrophobicity Introduction Synthetic peptides and peptidomimetics constitute one of the most important classes of therapeutics in today’s pharmaceutical industry. For example, lisinopril, a peptidomimetic inhibitor of angiotensin-converting enzyme, is one of the most prescribed drugs, with the number of annual prescriptions approaching 90 mil- lion in the USA alone [1]. Several major therapeutic areas, including diabetes, cancer, and multiple sclerosis, have benefitted enormously due to the emergence of novel peptide-based therapeutics such as exenatide, bortezomib, and glatiramer acetate, respectively. PTH represents another important opportunity in peptide-based thera- peutics, as evidenced by the success of Lilly’s branded teriparatide (PTH 1–34) drug Forteo W (Indianapolis, IN, USA.). On the basis of Lilly’s 2011 annual report, the sales for this drug are approaching close to $1bn worldwide, fueled by the demands from countries such as Japan with a large percentage of aging population. Parathyroid glands secrete PTH as an 84-amino acid peptide in response to low calcium or high phosphate levels in the circulation, and this hormone regulates calcium homeostasis in blood and kidney as well as bone remodeling. In 2002, the FDA approved Forteo – recombinant PTH 1–34 – which retains full calciotropic activity of PTH 1–84 and is currently approved in the USA for the treatment of postmenopausal osteoporosis, idiopathic or hypogonadal osteoporosis in men, and glucocorticoid-induced osteoporosis. Teriparatide’s therapeutic scope is expected to widen even further because of its unique anabolic properties on bone [2]. Researchers are devising strategies to further optimize teriparatide by focusing on ways to enhance therapeutic efficacy, mitigate short-term and long-term adverse effects, and eliminate the need for subcutaneous injection by designing an orally bioavailable form. Shortened peptidomimetic analogs of PTH 1–34 are currently being investigated as one such strategy that can potentially lead to an orally bioavailable molecule with enhanced anabolic efficacy and minimal adverse effects [3]. * Correspondence to: Galande, Amit, Biosciences Division, SRI International, Harrisonburg, VA 22801, USA. E-mail: amit.galande@sri.com Biosciences Division, SRI International, Harrisonburg, VA, 22801, USA Abbreviations: 1-Napa, 1-napthylalanine; 2-Napa, 2-Napthylalanine; 2D NMR, two-dimensional nuclear magnetic resonance; 2D TOCSY, two-dimensional Total Correlation Spectroscopy; Abu, 2-aminobutyric acid; bVal, beta-valine; cAMP, cyclic adenosine monophosphate; CD, circular dichroism; Cha, cyclohexyl alanine; Chg, cyclohexylglycine; CHO, Chinese hamster ovary; CSD, chemical shift deviations; D 2 O, deuterated water; DIC, diisopropylcarbodiimide; DSS, sodium 4,4-dimethyl-4-silapentane-1-sulfonate; ECD, extracellular N-terminal domain; ERK, extracellular signal-related kinase; Fmoc, fluorenylmethyloxycarbonyl chloride; GPCR, G-protein coupled receptor; HLeu, homoleucine; HOBt, 1-hydroxybenzotriazole; HPhe, homophenylalanine; MALDI, matrix-assisted laser desorption ionization; Npg, neopentylglycine; Pfp, pentafluorophenylalanine; Phg, phenylglycine; PTH, parathyroid hormone; PTH1R, parathyroid hormone receptor type 1; PTHrP, parathyroid hormone-related protein; ROESY, rotating- frame nuclear Overhauser effect spectroscopy; FID, free induction decay; RP-HPLC, reversed-phase high-performance liquid chromatography; TFE-d 3 , deuterated TFE; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Tle, tertiary leucine; TOF, time-of-flight. J. Pept. Sci. 2013; 19: 337–344 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 7 December 2012 Revised: 29 January 2013 Accepted: 31 January 2013 Published online in Wiley Online Library: 8 April 2013 (wileyonlinelibrary.com) DOI 10.1002/psc.2502 337