Probing the Bimolecular Interactions of Parathyroid Hormone with the Human Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor. 1. Design, Synthesis and Characterization of Photoreactive Benzophenone-Containing Analogs of Parathyroid Hormone

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-proteincoupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in “photoaffinity scanning” of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physicochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle^8,18,Lys¹³(ε-pBz₂),L-2-Nal²³,Tyr³⁴]bPTH(1-34)NH₂ (K13) (K_b = 13 nM; K_m = 2.7 nM) and [Nle^8,18,L-Bpa²³,Tyr³⁴[bPTH(1-34)NH₂ (L-Bpa²³) (K_b = 42 nM; K_m = 8.5 nM). Another BP-containing analog, [Nle_8,18,D-2-Nal¹²,Lys¹³(ε-pBz₂),L-2-Nal²³, Tyr³⁴]bPTH(7-34)NH₂, was a potent antagonist (k_b = 95 nM; K_i = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe⁷, Gly¹², Lys¹³, Trp²³, and Lys²⁶). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the “contact points” between ligand and receptor.