title: [¹²⁵I]YP20: a novel radioligand specific for the extracellular domain of the CRF₁ receptor.
abstract: The peptide corticotropin-releasing factor (CRF) binds to the CRF₁ receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or "J"-site. Known small molecule antagonists bind to the J-site while known CRF₁ receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF₁ receptor. This ligand, which we named [¹²⁵I]Yamada peptide 20 ([¹²⁵I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [¹²⁵I]CRF binding site on the hCRF₁ receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF₁-CHO cells. Under optimized conditions, 20 pM [¹²⁵I]YP20 reproducibly bound to hCRF₁-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF₁ receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²⁵I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²⁵I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t(1/2)=0.92 min and t(1/2)=11.7 min). While [¹²⁵I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF₁ receptor.
authors.affiliation: CNS Discovery, AstraZeneca, Wilmington, Delaware 19850-5437, USA. john.gordon@astrazeneca.com
authors.forename: John C
authors.initials: JC
authors.lastname: Gordon
authors.forename: Phil
authors.initials: P
authors.lastname: Edwards
authors.forename: Charles S
authors.initials: CS
authors.lastname: Elmore
authors.forename: Lois Ann
authors.initials: LA
authors.lastname: Lazor
authors.forename: Kathy
authors.initials: K
authors.lastname: Paschetto
authors.forename: Robert
authors.initials: R
authors.lastname: Bostwick
authors.forename: Mark
authors.initials: M
authors.lastname: Sylvester
authors.forename: Russ
authors.initials: R
authors.lastname: Mauger
authors.forename: Clay
authors.initials: C
authors.lastname: Scott
authors.forename: David
authors.initials: D
authors.lastname: Aharony
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D003346:Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology*; D010455:Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D054730:Protein Interaction Domains and Motifs; D011743:Pyrimidines / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D000099052:CRF Receptor, Type 1
chemical_list: D007457:Iodine Radioisotopes; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D011743:Pyrimidines; C412383:R 121919; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C556223:Yamada peptide 20; D006165:Guanylyl Imidodiphosphate; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases