160P London, UK
Pharmacology 2017

 

 

Functional and kinetic binding analysis of clinical antipsychotics at the 5HT1A receptor

H. M. Comfort, S. J. Charlton, D. A. Sykes. University of Nottingham, Nottingham, United Kingdom.

Introduction: Serotonin (5HT) receptors are implicated in the action of antipsychotic drugs (APDs). It is thought that agonism at the 5HT1A receptors (5HT1ARs) may reduce extrapyramidal symptoms (EPS) caused by dopamine D2receptor blockade, and improve cognitive impairment in schizophrenia (1). To investigate this we formulated a [35S]-GTPƴS binding assay to calculate relative values of efficacy for sixteen clinically used APDs at 5HT1ARs. Findings indicate that kinetic parameters play an important role in the action of APDs. Therefore, we have measured the association and dissociation rates of these APDs at the 5HT1AR using a novel TR-FRET based competition association binding assay.

Method: [35S]GTPƴS binding was performed using CHO-5HT1A membranes (10μg/well) incubated with compounds at room temperature for 1hr in assay buffer containing 20mM HEPES, 100mM NaCl, 10mM MgCl2 and 10μM GDP. [35S]-GTPƴS (100pM) was added for 40min before the reaction was terminated by filtration through GF/B plates. Competition binding was carried out as previously described (2), using fluorescently labelled NAN190 (F-NAN190, 40nM) to assess the kinetics of unlabelled compounds. The assay was performed at 37°C in HBSS containing 5 mM HEPES, 1% DMSO, and 0.02% pluronic acid. Binding was initiated by the addition of membranes (0.5μg/well) in the presence of 100μM GppNHp. Non-specific binding was determined by the addition of 120μM spiperone.

Results: F-NAN190 bound with an affinity of 4.37 nM, with association and dissociation rates of 5.73x107 M-1 min-1 and 0.17 min-1 respectively. The intrinsic activity (IA) of APDs determined in the [35S]GTPƴS assay is expressed as a percentage of the maximum response (% Emax) produced by 5HT (1μM).

Table 1. Functional and kinetic parameters. Data are mean ± SEM (n≥3)

Competition Association Kinetics [35S]-GTPƴS Assay Clinical data (3,4)
Koff (min-1) Kon (M-1min-1) pKd pEC50 IA (% Emax) EPS (odds ratio)
SGA/Atypical
Clozapine 0.59 ± 0.22 4.31 ± 2.83 x104 4.67 ± 0.12 5.87 54.08 0.3
Quetiapine 0.08 ± 0.01 2.85 ± 0.64 x102 4.53 ± 0.11 5.86 76.33 1.01
Amisulpride 0.69 ± 0.22 1.84 ± 0.32 x104 4.48 ± 0.10 4.89 -29.18 1.60
Sertindole 0.39 ± 0.09 1.60 ± 0.47 x105 5.56 ± 0.12 5.64 -25.4 0.81
Aripiprazole 0.64 ± 0.18 3.94 ± 1.18 x106 6.79 ± 0.16 6.82 48.44 1.20
FGA/Typical
Haloperidol 0.36 ± 0.16 9.94 ± 3.36 x104 5.47 ± 0.04 5.35 -27.47 4.76
Nemonapride 0.41 ± 0.09 1.39 ± 0.87 x106 6.30 ± 0.26 7.29 92.10 High
Chlorpromazine 0.47 ± 0.13 2.75 ± 0.79 x105 5.75 ± 0.06 6.00 -27.04 2.65

Conclusions: All APDs tested showed some affinity for human 5HT1ARs. A number of APDs with low clinically observed EPS (3) show agonism at 5HT1ARs (e.g. clozapine, quetiapine, and aripiprazole). However, there are notable exceptions such as sertidole which exhibits low EPS but is an inverse agonist at 5HT1ARs, and nemonapride which has high EPS (4) but is in fact a highly efficacious agonist. This suggests that 5HT1AR agonism may not be the most important factor in the reduced incidence of EPS displayed by certain APDs.

References:

(1) HY Meltzer and BW Massey (2011). Current Opinion in Pharmacology 11:59-67.

(2) Klein-Herenbrink C, et al. (2016). Nature communications 6:10842.

(3) Stefan Leucht et al. (2013). Lancet 382:951-62

(4) Tsuyoshi Kondo, et al. (2002). Progress in Neuro-Psychopharmacology & Biological Psychiatry 26:287-91.