Conformational studies of a potent Leu11, D‐Trp12‐containing lactam‐bridged parathyroid hormone‐related protein‐derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu11,d-Trp12,Lys26,Asp30]PTHrP(7-34)NH2 (hybrid-lactam) in a 1:1 mixture of H2O/TFE-d3 at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (Kb= 650 nm) combines two modifications: Leu11,d-Trp12 (Kb= 5.1 nm), reported to eliminate partial agonism and enhance potency, and Lys26-Asp30 lactamization (Kb= 31 nm), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d-Trp12-Thr33 segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly12-Lys13 and Arg19. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.