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 K_d 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.

References:

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.