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Amino‐Terminal Parathyroid Hormone Fragment Analogs Containing α,α‐di‐alkyl Amino Acids at Positions 1 and 3
Author(s) -
Shimizu Naoto,
Dean Thomas,
Khatri Ashok,
Gardella Thomas J
Publication year - 2004
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.040914
Subject(s) - pharmacophore , chemistry , amino acid , parathyroid hormone , stereochemistry , peptide , alkyl , receptor , potency , biochemistry , calcium , in vitro , organic chemistry
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, α‐amino‐isobutyric acid (Aib), at positions 1 and 3 increase the P1R‐binding affinity and signaling potency of the analog ∼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 c1 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 μM; EC 50 s = 1.7 nM, 3.1 nM, and 1.9 μM, 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 [Deg 1,3 ,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.