title: The arylpiperazine derivatives N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide and N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide exert a long-lasting inhibition of human serotonin 5-HT7 receptor binding and cAMP signaling.
abstract: We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT7) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [(3)H]-(2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([(3)H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT7-expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT7-binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT7 receptors unresponsive to 5-CT and also rendered 5-HT7-expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT7 receptors may benefit the study of 5-HT7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT7 receptors.
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: Patricio
authors.initials: P
authors.lastname: Atanes
authors.affiliation: Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari "A. Moro" Bari, Italy.
authors.forename: Enza
authors.initials: E
authors.lastname: Lacivita
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Rodríguez
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Brea
authors.affiliation: Esteve; Drug Discovery and Preclinical Development Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Burgueño
authors.affiliation: Esteve; Drug Discovery and Preclinical Development Barcelona, Spain.
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Vela
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Cadavid
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Loza
authors.affiliation: Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari "A. Moro" Bari, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Leopoldo
authors.affiliation: Biofarma Research Group, Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela Santiago de Compostela, Spain.
authors.forename: Marián
authors.initials: M
authors.lastname: Castro
keywords: HEK293 cells; LP-211; Schild analysis; [3H]-SB-269970 binding; arylpiperazine derivative; cAMP signaling; insurmountable antagonism; irreversible inhibition; long-lasting inhibition; preincubation/washout experiments; serotonin 5-HT7 receptors