Vol. 305, Issue 1, 57-69, April 2003

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

Ge Zhang, Ning Huang, Yu-Wen Li, Xiaoping Qi, Anne P. Marshall, Xiao-Xin Yan, Geraldine Hill, Cynthia Rominger, Shimoga R. Prakash, Rajagopal Bakthavatchalam, David H. Rominger, Paul J. Gilligan and Robert Zaczek

CNS 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.), Drug Metabolism (S.R.P.), and Chemical and Physical Sciences (R.B., P.J.G.), the Bristol-Myers Squibb Pharmaceuticals Research Institute, Wilmington, Delaware

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) binding K_i 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 the B_max of [¹²⁵I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF₁ receptors, [³H]SN003 binding to rat cortex and human CRF₁HEK293e cell membranes was characterized and shown to be reversible and saturable, with K_D values of 4.8 and 4.6 nM, and B_max values of 0.142 and 7.42 pmol/mg protein, respectively. The association and dissociation rate constants of [³H]SN003 (k₊₁ 0.292 nM¹ min¹ and k₁ 0.992 × 10² min¹) 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.