CONFORMATIONAL STUDIES OF MONOCYCLIC AND BICYCLIC PARATHYROID HORMONE-RELATED PROTEIN-DERIVED AGONISTS

Parathyroid hormone-related protein (PTHrP) is expressed in a wide var iety of cells where it acts as an autocrine and/or paracrine factor in volved in regulation of cellular growth, differentiation, and embryoni c development. It may also play a physiological endocrine role in calc ium transport across the placenta or during lactation. The N-terminal portion, PTHrP-(1-34), retains all the calciotropic parathyroid hormon e-like activity and is a lead structure for the design of novel, bone anabolic agents for the treatment of bone disorders such as osteoporos is. To characterize the putative bioactive conformation, we have carri ed out a detailed structural analysis of a series of three conformatio nally constrained PTHrP-(1-34)-based mono- and bicyclic lactam-contain ing biologically active analogs: (I) [Lys(13),Asp(17)]PTHrP-(1-34)NH2; (II) [Lys(26),Asp(30)]PTHrP-(1-34)NH2, and (III) [Lys(13), Asp(17), L ys(26), Asp(30)]PTHrP-(1-34)NH2. The conformational properties were st udied 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 similar to 22%; that of the bicyc lic analog In is similar to 40%. In 30% TFE, all analogs reached a max imal helical content of 80%, corresponding to 26 or 27 residues out of 34 in a helical conformation. High-resolution structures obtained wit h 50:50 TFE/water revealed that all three analogs display two helical domains and a hinge region around Gly(12)-Lys(13). The highly potent m ono-and bicyclic agonists I and III display a second hinge around Arg( 19)-Arg(20) which is shifted to Ser(14)-Asp(17) in the weakly potent m onocyclic agonist H. 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 nuc leation at the N-terminal sequence an essential for generating a PTH/P THrP receptor-compatible bioactive conformation.