title: Amino-terminal parathyroid hormone fragment analogs containing alpha,alpha-di-alkyl amino acids at positions 1 and 3.
abstract: To define and minimize the N-terminal PTH pharmacophore, the effects of introducing different conformationally constraining di-alkyl amino acids at positions 1 and 3 of PTH(1-14) analogs were studied. Improvements in PTH receptor-binding affinity and signaling potency were found, although some substitutions resulted in partial agonism.
  
  
  BACKGROUND
  The N-terminal portion of parathyroid hormone (PTH) plays a critical role in PTH-1 receptor (P1R) activation. To investigate the mechanisms underlying this action and to minimize the N-terminal PTH pharmacophore, we employed the PTH(1-14) fragment as a scaffold for structure-activity relationship studies, and thus previously found that substitutions of the conformationally constraining, di-alkyl amino acid, alpha-amino-isobutyric acid (Aib), at positions 1 and 3 increase the P1R-binding affinity and signaling potency of the analog approximately 100-fold. Here we extend these findings by investigating the effects of other constrained di-alkyl amino acids at positions 1 and/or 3 on PTH analog activity.
  
  
  METHODS
  The di-alkyl amino acids, 1-aminocycloalkane-carboxylic acid (Ac(x)c, x = 3, 5, or 6) or diethylglycine (Deg), representing alkyl configurations of varying volumes and shape (cyclic and linear), were introduced into the parent peptide, [M]PTH(1-14) (M = Ala(1,3,12),Gln(10),Har(11),Trp(14)), and the analogs were tested for activity in P1R-expressing cells.
  
  
  RESULTS
  Relative to the binding affinity and cAMP-stimulating potency of the parent peptide (IC(50) = 27 mM; EC(50) = 220 nM), PTH(1-14) analogs substituted at position 1 exhibited 2- (Ac(3)c) to 60-fold (Ac(5)c) increases in affinity and potency, as measured in LLC-PK1 cells stably expressing the cloned P1R. Combining the substitutions of Ac(5)c(1) and Aib(3) yielded the highest affinity and most potent PTH(1-14) and shorter-length analogs to date: [Ac(5)c(1), Aib(3),M]PTH(1-X) (X = 14, 11, and 10; IC(50)s = 80 nM, 260 nM, and 850 microM; EC(50)s = 1.7 nM, 3.1 nM, and 1.9 microM, respectively). The effects of Ac(6)c(1) were similar to those of Ac(5)c(1). A dissociation of binding affinity and signaling activity occurred with Deg, as [Deg1,3,M]PTH(1-14) was a partial agonist.
  
  
  CONCLUSIONS
  Constraining the N-terminal PTH backbone conformation with di-alkyl amino acids at positions 1 and 3 may be a general strategy for optimizing and minimizing the PTH pharmacophore; however, inhibitory side-chain effects may be encountered. The new analogs presented should be useful as minimum-length functional probes of the PTH-PTH receptor interaction mechanism.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Naoto
authors.initials: N
authors.lastname: Shimizu
authors.forename: Thomas
authors.initials: T
authors.lastname: Dean
authors.forename: Ashok
authors.initials: A
authors.lastname: Khatri
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gardella
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D007295:Inositol Phosphates / Q000737:chemistry; D008956:Models, Chemical; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013552:Swine; D010738:Type C Phospholipases / Q000737:chemistry
chemical_list: D000596:Amino Acids; D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010455:Peptides; C040288:amino-terminal parathyroid hormone; D000242:Cyclic AMP; D010738:Type C Phospholipases