CONFORMATIONAL STUDIES OF A POTENT LEU(11),D-TRP(12)-CONTAINING LACTAM-BRIDGED PARATHYROID HORMONE-RELATED PROTEIN-DERIVED ANTAGONIST

Human parathyroid hormone-related protein (PTHrP) is expressed in vari ous tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. A s for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 f ragment can reproduce the full spectrum of calciotropic activities inh erent in full-length PTH. Truncation of six amino acid residues from t he N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTH rP(7-34) acts as partial agonist against the receptor shared by both h ormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu(11),D-Trp(12),Lys(26),ASp(30)]PTHrP(7-34)NH2 (hy brid-lactam) in a 1:1 mixture of H2O/TFE-d(3) at pH similar to 4 by ci rcular dichroism, nuclear magnetic resonance and distance geometry cal culations. This weak antagonist (K-b = 650 nM) combines two modificati ons: Leu(11),D-Trp(12) (K-b = 5.1 nM), reported to eliminate partial a gonism and enhance potency, and Lys(26)-Asp(30) lactamization (K-b = 3 1 nM), aimed to stabilize the helical structure of the principal bindi ng domain attributed to residues 25-34. The helical content in 30% tri fluoroethanol is 88%, i.e., higher than the corresponding linear analo g, and comprises the D-Trp(12)-Thr(33) segment. This hybrid lactam con tains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg(19-20), but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and an tagonists studied so far, the dominant structural motif consists of tw o helical domains at the two ends of the sequence and of two hinge reg ions centered around Gly(12)-Lys(13) and Arg(19). The weakly active ag onists and antagonists do not exhibit the ''hinge'' around position 19 . These findings suggest that the presence and location of discrete hi nge.