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 approximately equal to 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.

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: (III) 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 approximately 22%; that of the bicyclic analog III is approximately 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 II. 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.

Mono- and bicyclic analogs of parathyroid hormone-related protein. 2. Conformational analysis of antagonists by CD, NMR, and distance geometry calculations.

The conformation of the three cyclic antagonist analogs of parathyroid hormone-related protein (PTHrP)-(7-34) [[Lys13,Asp17]PTHrP-(7-34)NH2,[Lys26,Asp30 ]PTHrP-(7-34)NH2,[Lys13,Asp17,Lys26, Asp30]PTHrP-(7-34)NH2] is investigated by CD, NMR, and extensive computer simulations in aqueous solution and a TFE:water mixture. The structural analysis of these peptides, designed to stabilize different regions of the sequence in alpha-helical conformations, is an important step in addressing the correlation between helical content and binding affinity and bioactivity in this hormone-receptor system. Results from CD and NMR spectroscopy of all three analogues in aqueous solution indicate the presence of alpha-helix only in regions containing a 20-membered lactam ring. Upon addition of TFE, the three analogues display differences in the anticipated increase in helical content. The high-resolution structures produced at 50:50 TFE:water indicate specific differences in the extent and location of the helical regions. These conformations provide insight into the biological profiles of these analogues, reported in the previous manuscript [Bisello et al. (1997) Biochemistry 36, 3293-3299]. Since all three analogues are alpha-helical in the C-terminal region (residues 25-34 have been previously identified as containing the binding domain) and display similar binding affinities, we conclude that this conformational feature is important for the interaction between the peptide and the receptor. The extent of the helix (toward the N-terminus) and the presence of a hinge in the central region of the peptide play roles in the observed efficacy as measured by antagonism of PTH-stimulated adenylyl cyclase activity. The most active analogue consists of helical segments from residues 13-18 and 20-34, separated by a kink centered at Arg19.

Conformation of parathyroid hormone antagonists by CD, NMR, and molecular dynamics simulations.

The conformation of two highly potent parathyroid hormone (PTH) antagonists was investigated in water/2,2,2-trifluoroethanol mixtures. The two peptides are derived from the sequence (7-34) of PTH and of PTH-related protein (PTHrP) and have a D-Trp replacing Gly in position 12. In the analogue derived from PTHrP, Lys11 was replaced by Leu to remove the residual agonist activity. The study was conducted by CD and two-dimensional proton magnetic resonance spectroscopy, and the nuclear Overhauser effects found were utilized in restrained distance geometry and molecular dynamics simulations. Both peptides adopt a helical C-terminal conformation, which seems more stable in the case of the PTHrP analogue. A type II' beta-turn centered around D-Trp12 and Lys13 is present in both structures.