title: 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.
abstract: The in vitro pharmacological profile of a novel small molecule corticotropin-releasing factor 1 (CRF(1)) 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 ([(3)H]SN003) are described. SN003 has high affinity and selectivity for CRF(1) receptors expressed in rat cortex, pituitary, and recombinant HEK293EBNA (HEK293e) cells with respective radiolabeled ovine CRF ([(125)I]oCRF) binding K(i) values of 2.5, 7.9, and 6.8 nM. SN003 was shown to be a CRF(1) receptor antagonist inasmuch as it inhibited CRF-induced cAMP accumulation in human CRF(1)HEK293e cells and CRF-stimulated adrenocorticotropin hormone release from rat pituitary cells without agonist activities. Significant decreases in the B(max) of [(125)I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF(1) receptors, [(3)H]SN003 binding to rat cortex and human CRF(1)HEK293e 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 [(3)H]SN003 (k(+1) 0.292 nM(-1) min(-1) and k(-1) 0.992 x 10(-2) min(-1)) were also assessed using human CRF(1)HEK293e cell membranes, giving an equilibrium dissociation constant of 3.4 nM. Moreover, [(3)H]SN003 binding displayed a single affinity state and insensitivity to 5'-guanylylimidodiphosphate, consistent with characteristics of antagonist binding. Incomplete inhibition of [(3)H]SN003 binding by CRF peptides also suggests that SN003 is not simply competitive with CRF at CRF(1) receptors. The distribution of [(3)H]SN003 binding sites was consistent with the expression pattern of CRF(1) receptors in rat brain regions. Small molecule CRF(1) antagonist radioligands like [(3)H]SN003 should enable a better understanding of small molecule interactions with the CRF(1) receptor.
authors.affiliation: CNS Diseases Research, the Bristol-Myers Squibb Pharmaceuticals Research Institute, Wilmington, Delaware, USA. ge.zhang@bms.com
authors.forename: Ge
authors.initials: G
authors.lastname: Zhang
authors.forename: Ning
authors.initials: N
authors.lastname: Huang
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Li
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qi
authors.forename: Anne P
authors.initials: AP
authors.lastname: Marshall
authors.forename: Xiao-Xin
authors.initials: XX
authors.lastname: Yan
authors.forename: Geraldine
authors.initials: G
authors.lastname: Hill
authors.forename: Cynthia
authors.initials: C
authors.lastname: Rominger
authors.forename: Shimoga R
authors.initials: SR
authors.lastname: Prakash
authors.forename: Rajagopal
authors.initials: R
authors.lastname: Bakthavatchalam
authors.forename: David H
authors.initials: DH
authors.lastname: Rominger
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Gilligan
authors.forename: Robert
authors.initials: R
authors.lastname: Zaczek
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008297:Male; D011725:Pyridines / Q000494:pharmacology*; D011869:Radioligand Assay; D019275:Radiopharmaceuticals / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology*; D014316:Tritium; D000099052:CRF Receptor, Type 1
chemical_list: C101531:CRF receptor type 2; D011725:Pyridines; D019275:Radiopharmaceuticals; D018019:Receptors, Corticotropin-Releasing Hormone; C473245:SN003; D014230:Triazoles; D014316:Tritium; D000099052:CRF Receptor, Type 1; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP