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.

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.

Figure 1

Structures of the arylpiperazine compounds employed in this study.

Figure 2

Affinity of compounds for human 5-HT₇ receptors expressed in HEK293 cells from competitive binding experiments in membrane homogenates. Competition by MEL-9 and LP-211 for binding of [³H]-SB-269970 (2 nmol/L) to membrane homogenates. Total radioactivity bound was less than 20% of total radioactivity added in all the experiments. Data are the mean values ± SEM from three independent experiments performed in duplicate. The data were fitted to a sigmoidal dose–response curve (n_H = 1) or to a sigmoidal dose–response curve (variable slope), according to the best fit model (P < 0.05, extra sum-of-squares F-test).

Figure 3

Concentration–response curves for MEL-9 and LP-211 on 5 nmol/L or 1 μmol/L 5-CT-stimulated cAMP production in HEK-hu5-HT₇ cells. Cells were preincubated in the absence (control) or presence of the compounds at different concentrations (100 pmol/L–10 μmol/L) for 15 min prior to the addition of 5 nmol/L or 1 μmol/L 5-CT and incubation for 15 min. Basal values were subtracted from 5-CT-stimulated values and expressed as percent of the control 5-CT response. 5 nmol/L 5-CT elicited a cAMP response over basal equal to 44.3 ± 1.6% of the response elicited by 1 μmol/L 5-CT. Data are the mean values ± SEM from three independent experiments performed in triplicate.

Figure 4

Schild analysis for the antagonism of 5-CT-stimulated cAMP signaling by MEL-9 and LP-211 in HEK-hu5-HT₇ cells. Concentration–response curves for cAMP production stimulated by 5-CT in the absence (control) or presence of 10 nmol/L, 100 nmol/L or 1 μmol/L of MEL-9 (A) or LP-211 (B). Cells were preincubated with the compounds for 15 min prior to the addition of 5-CT and incubation for 15 min. Basal values were subtracted from 5-CT-stimulated values and expressed as percent of the control maximal 5-CT response. Data are the mean values ± SEM from three independent experiments performed in duplicate.

Figure 5

Concentration–response curves for the inhibition of 10 μmol/L 5-CT-stimulated cAMP production by MEL-9 and LP-211 in HEK-hu5-HT₇ cells in preincubation/washout experiments. Cells were preincubated in the absence (control) or presence of the compounds at increasing concentrations (ranging from 100 pmol/L to 10 μmol/L) for 30 min. After a compound washout procedure (see Materials and Methods), the cells were stimulated with 10 μmol/L 5-CT for 30 min. Basal values were subtracted from 5-CT-stimulated values and expressed as percent of the control 5-CT response. Data are the mean values ± SEM from three independent experiments performed in triplicate.

Figure 6

Concentration–response curves for the inhibition of 5-HT₇ binding sites by MEL-9 and LP-211 in preincubation/washout experiments in radioligand binding studies in whole HEK-hu5-HT₇ cells. Cells were preincubated in the absence (control) or presence of either MEL-9 or LP-211 at increasing concentrations (ranging from 100 pmol/L to 10 μmol/L), 10 μmol/L clozapine (Δ) as reference competitive antagonist, or 1 μmol/L methiothepin (▲) as reference irreversible antagonist, for 30 min. After a compound washout procedure (see Materials and Methods), the cells were subjected to whole-cell radioligand binding assays to determine specific [³H]-SB-269970 binding. [³H]-SB-269970 specific binding is expressed in each case as percent of [³H]-SB-269970 specific binding in cells preincubated in the absence of compound (control). The total radioactivity bound was less than 20% of total radioactivity added in all the experiments. Data are the mean values ± SEM from three to five independent experiments performed in duplicate. Concentration–response data were fitted to a sigmoidal dose–response curve (n_H = 1).

Figure 7

Effect of compounds MEL-9 and LP-211 on cAMP signaling stimulated by forskolin in HEK-hu5-HT₇ cells. (A, B) Concentration–response curves for MEL-9 (A) and LP-211 (B) on 10 μmol/L forskolin-stimulated cAMP production. Cells were preincubated in the absence (control) or presence of the compounds at different concentrations (100 pmol/L – 10 μmol/L) for 15 min prior to the addition of 10 μmol/L forskolin and incubation for 15 min. Basal values were subtracted from forskolin-stimulated values and expressed as percent of the control forskolin response. Data are the mean values ± SEM from three independent experiments performed in triplicate. (A, B Inset) Lack of effect of 1 μmol/L or 10 μmol/L MEL-9 (A, Inset) and 1 μmol/L or 10 μmol/L LP-211 (B, Inset) on forskolin-stimulated cAMP production in parental HEK293 cells. (C, D) Concentration–response curves for the inhibition of 10 μmol/L forskolin-stimulated cAMP production by MEL-9 (C) and LP-211 (D) in HEK-hu5-HT₇ cells in preincubation/washout experiments. Cells were preincubated in the absence (control) or presence of the compounds at increasing concentrations (ranging from 100 pmol/L to 10 μmol/L) for 30 min. After a compound washout procedure (see Materials and Methods), the cells were stimulated with 10 μmol/L forskolin for 15 min. Basal values were subtracted from forskolin-stimulated values and expressed as percent of the control forskolin response. Data are the mean values ± SEM from three independent experiments performed in triplicate.

Figure 8

Long-lasting inhibition of [³H]-SB-269970-binding sites by MEL-9 and LP-211 in HEK-hu5-HT₇ cells in preincubation/washout experiments. Cells were preincubated in the absence (control) or presence of 10 μmol/L MEL-9, 10 μmol/L LP-211, or 10 μmol/L RA-7 for 30 min. After a compound washout procedure (see Materials and Methods), cells were further incubated at 37°C for the indicated times before being subjected to whole-cell radioligand binding assays for determination of specific [³H]-SB-269970 binding. Total radioactivity bound was less than 20% of total radioactivity added in all the experiments. Specific binding is expressed as percent of the specific binding of control at each time point. Data are the mean values ± SEM from three independent experiments performed in duplicate.