Conformational studies of mono- and bicyclic parathyroid hormone- related protein-derived agonists

Parathyroid hormone-related protein (PTHrP) is expressed in a wide variety of cells where it acts as an autocrine and/or paracrine factor involved in regulation of cellular growth, differentiation, and embryonic development. It may also play a physiological endocrine role in calcium transport across the placenta or during lactation. The N-terminal portion, PTHrP-(1-34), retains all the calciotropic parathyroid hormone-like activity and is a lead structure for the design of novel, bone anabolic agents for the treatment of bone disorders such as osteoporosis. To characterize the putative bioactive conformation, we have carried out a detailed structural analysis of a series of three conformationally constrained PTHrP-(1 34)- based mono- and bicyclic lactam-containing biologically active analogs: (I) [Lys¹³, Asp¹⁷]PTHrP-(1-34)NH₂; (II) [Lys²⁶, Asp³⁰]PTHrP-(1-34)NH₂, and (III) [Lys¹³, Asp¹⁷, Lys²⁶, Asp³⁰]PTHrP-(1-34)NH₂. The conformational properties were studied by circular dichroisim, nuclear magnetic resonance spectroscopy, distance geometry calculations, and molecular dynamic simulations in water/trifluoroethanol (TFE) mixtures. The helical content in water of both monocyclic analogs I and II is ~22%; that of the bicyclic analog III is ~40%. In 30% TFE, all analogs reached a maximal helical content of 80%, corresponding to 26 or 27 residues out of 34 in a helical conformation. High-resolution structures obtained with 50:50 TFE/water revealed that all three analogs display two helical domains and a hinge region around Gly¹²-Lys¹³. The highly potent mono- and bicyclic agonists I and III display a second hinge around Arg¹⁹-Arg²⁰ which is shifted to Ser¹⁴-Asp¹⁷ in the weakly potent monocyclic agonist I. We suggest that the presence and localization of discrete hinges in the sequence together with the high propensity for helicity of the C-terminal sequence and the enhancement of helical nucleation at the N-terminal sequence are essential for generating a PTH/PTHrP receptor-compatible bioactive conformation.