title: Irreversible binding of N-methyl-N-[(1S)-1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl-3,4 -dichlorophenylacetamide to the cloned rat kappa opioid receptor.
abstract: N-Methyl-N-[(1S)-1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl-3,4- dichlorophenylacetamide (MITPD), is an isothiocyanate derivative of the kappa agonist ICI-199,441. In this study, interaction of MITPD with cloned mu, delta, and kappa opioid receptors was characterized. MITPD inhibited [3H]diprenorphine binding to kappa receptors with high affinity and with approximately 700- and approximately 870-fold selectivity over mu and delta receptors. Pretreatment with MITPD followed by extensive washing reduced kappa receptor binding with an IC50 value of 3.7 nM, but did not affect mu or delta binding at < or = 0.1 microM. Preincubation with 1 microM MITPD abolished [3H]diprenorphine binding, while pretreatment with 1 microM ICI-199,441 increased Kd of [3H]diprenorphine binding with no change in Bmax. Thus, MITPD is a selective kappa irreversible ligand. The region of the kappa receptor that conferred selectivity for MITPD was determined by examining its binding to four mu/kappa chimeras. IC50 values of MITPD for inhibition of [3H]diprenorphine binding were determined to be 430 nM for Chimera III (kappa1-141/mu151-398), 1.8 nM for Chimera IV (mu1-150/kappa142-380), 40 nM for Chimera XI (mu1-268/kappa263-380) and 14 nM for Chimera XII (kappa1-262/mu269-398). Pretreatment with MITPD followed by extensive washing reduced binding to chimera IV with an IC50 value of 75 nM, but did not affect III, XI or XII binding (IC50 >1 microM). Thus, the region from the third transmembrane helix to the C-terminus of the kappa receptor is important for the binding of MITPD.
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: C
authors.initials: C
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Yin
authors.forename: J G
authors.initials: JG
authors.lastname: Li
authors.forename: J C
authors.initials: JC
authors.lastname: Xue
authors.forename: S A
authors.initials: SA
authors.lastname: Weerawarna
authors.forename: W L
authors.initials: WL
authors.lastname: Nelson
authors.forename: L Y
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D000081:Acetamides / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D017879:Isothiocyanates / Q000378:metabolism*; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000081:Acetamides; D017879:Isothiocyanates; C107789:N-methyl-N-(1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl-3,4-dichlorophenyl)acetamide; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins