A Linear Hexapeptide Somatostatin Antagonist Blocks Somatostatin Activity In Vitro and Influences Growth Hormone Release in Rats

  1. William R. Baumbach1,
  2. Tikva A. Carrick1,1,
  3. Mark H. Pausch2,
  4. Brendan Bingham1,1,
  5. Danielle Carmignac3,
  6. Iain C. A. F. Robinson3,
  7. Richard Houghten4,
  8. C. Mark Eppler1,
  9. Laura A. Price2 and
  10. John R. Zysk2,1
  1. 1Molecular and Cellular Biology (W.R.B., T.A.C., B.B., C.M.E., J.R.Z.) and 2Molecular Genetic Screen Design (M.H.P., L.A.P.), American Cyanamid Company, Princeton, New Jersey 08543, 3Division of Neurophysiology and Neuropharmacology (D.C., I.C.A.F.R.), National Institute of Medical Research, Mill Hill, London, UK, and 4Torrey Pines Institute for Molecular Studies, San Diego, California 92121 (R.H.)

    Abstract

    Somatostatin (SRIF) is the main inhibitory peptide regulating growth hormone (GH) secretion. It has been difficult to establish the role of endogenous SRIF release in the absence of pure SRIF antagonists. Although several SRIF antagonists have recently been described, none have been shown to possess in vivo activity in the absence of added SRIF. Here, an SRIF antagonist with no detectable agonist activity has been identified from a synthetic combinatorial hexapeptide library containing 6.4 × 107 unique peptides. Each peptide in the library is amino-terminally acetylated and carboxyl-terminally amidated and consists entirely ofd-amino acids. A SRIF-responsive yeast growth assay was used as a primary screening tool, and cAMP accumulation, competitive binding, and microphysiometry also were used to confirm and further characterize SRIF antagonist activity. The hexapeptide library was screened in stepwise iterative fashion to identify AC-178,335, a pure SRIF antagonist of the sequence Ac-hfirwf-NH2. Thisd-hexapeptide bound SRIF receptor type 2 with an affinity constant (Ki) of 172 ± 12 nm, blocked SRIF inhibition of adenylate cyclase in vitro (IC50 = 5.1 ± 1.4 μm), and induced GH release when given alone (50 μg intravenously) to anesthetized rats with or without pretreatment with a long-acting SRIF agonist.

    Footnotes

    • Send reprint requests to: Dr. William R. Baumbach, American Cyanamid, P.O. Box 400, Princeton, NJ 08543. E-mail:baumbachb{at}pt.cyanamid.com

    • ↵1 Current affiliation: CNS Disorders, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543.

    • ↵2 Current affiliation: NovaScreen, 7170 Standard Dr., Hanover, MD 21076.

    • Abbreviations:
      SRIF
      somatostatin
      S-14
      somatostatin-14
      S-28
      somatostatin 28
      GH
      growth hormone
      SSTn
      somatostatin receptor subtype, wheren is the subtype
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      HEK
      human embryonic kidney
      TSH
      thyroid-stimulating hormone
      GRF
      growth hormone-releasing factor
      IGF-I
      insulin-like growth factor I
      • Received May 12, 1998.
      • Accepted July 28, 1998.
    « Previous | Next Article »Table of Contents

    This Article

    1. Molecular Pharmacology November 1, 1998 vol. 54 no. 5 864-873
    1. » Abstract
    2. Full Text
    3. Full Text (PDF)

    Classifications

    Responses

    1. Submit a response
    2. No responses published

    Current Issue

    1. November 2009, 76 (5)
    1. Current Issue
    1. Alert me to new issues of Molecular Pharmacology