029P Nottingham, UK
7th Focused Meeting on Cell Signalling



Agonist activity of clozapine at muscarinic DREADD receptors

K. J. Thompson1, E. Khajehali2, C. Molloy1, S. J. Bradley1, A. Christopoulos2, A. B. Tobin1. 1Institute of Molecular, Cell and Systems Biology, Glasgow, United Kingdom, 2Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Parkville, Australia.

Introduction: Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a chemogenetic tool widely used to dissect signalling in vitro and in vivo1, of which muscarinic DREADDs - hM1Dq, hM3Dq, hM4Di - are most widely used2. Transmembrane mutations render these receptors largely unresponsive to endogenous acetylcholine, but sensitive to the otherwise inert clozapine-N-oxide (CNO). Recent reports suggest back-metabolised clozapine, rather than CNO, is the muscarinic DREADD activator in vivo1. We therefore investigated clozapine agonism at hM1Dq and hM4Di in vitro, and whether back-metabolised clozapine could be detected in CNO-injected mice.

Methods: [3H]-NMS Displacement: Confluent monolayers of FLP-in CHO cells expressing hM1WT, hM4WT, hM1Dq, or hM4Di were incubated in Kreb’s buffer with [3H]-NMS at Kd concentration and increasing ligand concentrations for 2 hr at 37°C. Cells were washed then lysed. Liquid scintillation counting determined bound radioactivity. Functional assays: Assays were carried out according to IP-One-Gq and phosphoERK1/2 (Thr202/Tyr204) Kits (CisBio, France). For IP1, washed and detached cells were resuspended in Stimulation Buffer and stimulated for 1 hr at 37°C. For Thr202/Tyr204 phosphorylation, serum-starved cells were stimulated for 5 min at 37°C then lysed. Resulting IP1 accumulation or Thr202/Tyr204 phosphorylation were determined with cryptate/D2 antibodies and HTRF. Pharmacokinetics: C57bl/6J mice were injected with varying CNO concentrations. After 30 min mice were exsanguinated and brains removed to assess plasma and brain drug exposure (in accordance with ASPA 2012). Data Analysis: All data analysis used GraphPad Prism 7.

Results: Clozapine and CNO displaced [3H]-NMS in hM1WT, hM1Dq, hM4WT, and hM4Di receptor cell lines (Table 1; n=3) but demonstrated no wild type receptor agonism in functional assays (n=3). In contrast, clozapine stimulated Thr202/Tyr204 phosphorylation at hM4Di receptors (Table 1; n=3) and IP1 accumulation in hM1Dq receptors (Table 1; n=3) more potently than CNO. C57bl/6J mice exhibited a dose-dependent increase in plasma CNO following 0.3, 1, or 1.5 mg/kg CNO injection (50.1 nM ± 0.16; 575.44 nM ± 0.03; 467.7 nM ± 0.09; n=3), yet CNO brain exposure was not detected. However, clozapine was detected in plasma and brains of these mice, indicating CNO back-metabolism.

Conclusions: Clozapine has greater affinity and potency than CNO at muscarinic DREADDs in vitro. Furthermore, CNO was back-metabolism to brain-penetrating clozapine in vivo. Collectively, this suggests clozapine may be a muscarinic DREADD agonist in vivo.


1. Gomez, J.L. et al. (2017). Science, 357, 503-507.

2. Armbruster, B. N. et al. (2007). Proc Natl Acad Sci USA, 104 (12), 5163-8. 4.

Log (M) pKi and EC50 values at hM1WT, hM1Dq, hM4WT and hM4Di receptors
[3H]-NMS Displacement LogpKi (M) (S.E.M.) IP1 Accumulation or Thr202/Tyr204 phosphorylation LogEC50 (M) (S.E.M.)
ACh CNO Clozapine ACh CNO Clozapine
hM1WT -5.02
(± 0.04)
(± 0.09)
(± 0.04)
(± 0.14)
hM1Dq -2.95
(± 0.08)
(± 0.06)
(± 0.07)
(± 0.13)
(± 0.08)
(+/- 0.2)
hM4WT -4.55
(± 0.33)
(± 0.05)
(± 0.28)
(± 0.13)
hM4Di -2.66
(± 0.11)
(± 0.03)
(± 0.432)
(± 0.09)
(± 0.14)
(+/- 0.26)