Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([3H]SN003) for Corticotropin-Releasing Factor1 Receptors

  1. Ge Zhang1,
  2. Ning Huang1,
  3. Yu-Wen Li1,
  4. Xiaoping Qi1,
  5. Anne P. Marshall1,
  6. Xiao-Xin Yan1,
  7. Geraldine Hill1,
  8. Cynthia Rominger1,
  9. Shimoga R. Prakash2,
  10. Rajagopal Bakthavatchalam3,
  11. David H. Rominger1,
  12. Paul J. Gilligan3 and
  13. Robert Zaczek1
  1. 1CNS Diseases Research (G.Z., N.H., Y.-W.L., X.Q., A.P.M., X.-X.Y., G.H., C.R., D.H.R., R.Z.), 2Drug Metabolism (S.R.P.), and 3Chemical and Physical Sciences (R.B., P.J.G.), the Bristol-Myers Squibb Pharmaceuticals Research Institute, Wilmington, Delaware
  1. Dr. Ge Zhang, The Bristol-Myers Squibb Company, Building 21/Room 2344A, 311 Pennington-Rocky Hill Rd., Hopewell, NJ 08534. E-mail: ge.zhang{at}bms.com

Abstract

The in vitro pharmacological profile of a novel small molecule corticotropin-releasing factor 1 (CRF1) receptor antagonist, (±)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine (SN003), and the characteristics of its radioligand ([3H]SN003) are described. SN003 has high affinity and selectivity for CRF1 receptors expressed in rat cortex, pituitary, and recombinant HEK293EBNA (HEK293e) cells with respective radiolabeled ovine CRF ([125I]oCRF) bindingKi values of 2.5, 7.9, and 6.8 nM. SN003 was shown to be a CRF1 receptor antagonist inasmuch as it inhibited CRF-induced cAMP accumulation in human CRF1HEK293e cells and CRF-stimulated adrenocorticotropin hormone release from rat pituitary cells without agonist activities. Significant decreases in theBmax of [125I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF1receptors, [3H]SN003 binding to rat cortex and human CRF1HEK293e cell membranes was characterized and shown to be reversible and saturable, with KD values of 4.8 and 4.6 nM, and Bmax values of 0.142 and 7.42 pmol/mg protein, respectively. The association and dissociation rate constants of [3H]SN003 (k+1 0.292 nM−1min−1 and k−1 0.992 × 10−2 min−1) were also assessed using human CRF1HEK293e cell membranes, giving an equilibrium dissociation constant of 3.4 nM. Moreover, [3H]SN003 binding displayed a single affinity state and insensitivity to 5′-guanylylimidodiphosphate, consistent with characteristics of antagonist binding. Incomplete inhibition of [3H]SN003 binding by CRF peptides also suggests that SN003 is not simply competitive with CRF at CRF1 receptors. The distribution of [3H]SN003 binding sites was consistent with the expression pattern of CRF1 receptors in rat brain regions. Small molecule CRF1 antagonist radioligands like [3H]SN003 should enable a better understanding of small molecule interactions with the CRF1 receptor.

Footnotes

  • DOI: 10.1124/jpet.102.046128

  • Abbreviations:
    CRF
    corticotropin-releasing factor
    ACTH
    adrenocorticotropin hormone
    CP-154,526
    butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]ethylamine
    SN003
    (±)-N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c] pyridin-4-amine
    NBI 27914
    5-chloro-N-(cyclopropylmethyl)-2-methyl-N-propyl-N′-(2,4,6-trichlorophenyl)-4,6-pyrimidinediamine hydrochloride
    SSR125543A
    4-(2-chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride
    hrCRF
    human/rat corticotropin-releasing factor
    oCRF
    ovine corticotropin-releasing factor
    α-helical CRF
    α-helical corticotropin-releasing factor9–41
    d-PheCRF
    [d-Phe12,Nle21,38,Ca-MeLeu37]-CRF12–41
    Gpp(NH)p
    5′-guanylylimidodiphosphate
    HEK
    human embryonic kidney
    PBS
    phosphate-buffered saline
    DMP696
    4-(1,3-dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine
    DMP904
    4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine
    R-121919
    3-[6-dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine
    • Received October 29, 2002.
    • Accepted January 3, 2003.
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  1. Published online before print January 21, 2003, doi: 10.1124/jpet.102.046128 JPET April 1, 2003 vol. 305 no. 1 57-69
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