title: A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.
abstract: The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1β that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598.
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States. Electronic address: blord@its.jnj.com.
authors.forename: Brian
authors.initials: B
authors.lastname: Lord
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Ameriks
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Fourgeaud
authors.affiliation: Janssen Research & Development, LLC, Drug Safety Sciences, Turnhoutseweg 30, Beerse 2340, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Vliegen
authors.affiliation: Janssen Research & Development, LLC, Drug Safety Sciences, Turnhoutseweg 30, Beerse 2340, Belgium.
authors.forename: Willy
authors.initials: W
authors.lastname: Verluyten
authors.affiliation: Janssen Research & Development, LLC, Drug Safety Sciences, Turnhoutseweg 30, Beerse 2340, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Haspeslagh
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Carruthers
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Lovenberg
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Pascal
authors.initials: P
authors.lastname: Bonaventure
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Letavic
authors.affiliation: Janssen Research & Development, LLC, Neuroscience Drug Discovery, 3210 Merryfield Row, San Diego, CA 92121-1126, United States.
authors.forename: Anindya
authors.initials: A
authors.lastname: Bhattacharya
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D006146:Guanidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding / Q000502:physiology; D058920:Purinergic P2X Receptor Antagonists / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011719:Pyrazines / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D014230:Triazoles / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C539459:2-cyano-1-((1S)-1-phenylethyl)-3-quinolin-5-ylguanidine; C000609564:7-(2-chloro-3-(trifluoromethyl)benzyl)-6-methyl-3-pyrimidin-2-yl-6,7-dihydro(1,2,4)triazolo(4,3-a)pyrazin-8(5H)-one; D006146:Guanidines; D007473:Ion Channels; D058920:Purinergic P2X Receptor Antagonists; D011719:Pyrazines; D011804:Quinolines; D058486:Receptors, Purinergic P2X7; D014230:Triazoles; D014316:Tritium; D000255:Adenosine Triphosphate
keywords: Autoradiography; Depression; JNJ-54140515; JNJ-54232334; P2X7; Radioligand binding; Receptor occupancy