Amino-terminal parathyroid hormone fragment analogs containing alpha,alpha-di-alkyl amino acids at positions 1 and 3

J Bone Miner Res. 2004 Dec;19(12):2078-86. doi: 10.1359/JBMR.040914. Epub 2004 Sep 20.


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.

Introduction: 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.

Materials and 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.

Conclusion: 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Animals
  • COS Cells
  • Cell Line
  • Circular Dichroism
  • Cyclic AMP / metabolism
  • Dose-Response Relationship, Drug
  • Inhibitory Concentration 50
  • Inositol Phosphates / chemistry
  • Models, Chemical
  • Parathyroid Hormone / chemistry*
  • Parathyroid Hormone / pharmacology*
  • Peptide Fragments / chemistry*
  • Peptide Fragments / pharmacology*
  • Peptides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Signal Transduction
  • Structure-Activity Relationship
  • Swine
  • Type C Phospholipases / chemistry


  • Amino Acids
  • Inositol Phosphates
  • Parathyroid Hormone
  • Peptide Fragments
  • Peptides
  • amino-terminal parathyroid hormone
  • Cyclic AMP
  • Type C Phospholipases