Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys

Abstract

The chemogenetic technology, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), affords reversible and remote control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, the sluggish kinetics, metabolic liabilities, and potential for off-target effects of CNO represent areas for improvement. Here we provide a new agonist deschloroclozapine (DCZ), which displays high affinity and selectivity for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μ g/kg) enhanced neuronal activity via hM 3 Dq within minutes in mice and monkeys. Intramuscular injection of DCZ (100 μ g/kg) reversibly induced behavioral deficits in hM 4 Di-expressing monkeys.DCZ represents the most potent, selective, metabolically stable and fast-acting DREADD agonist reported with utility in both mice and non-human primates for a variety of applications. 7 at dopaminergic (D 1 and D 2 ) and serotonergic (5-HT 2A and 5-HT 2C ) receptors compared with clozapine 14 . We determined that DCZ has 100-fold improved affinity and stronger agonist potency for hM 3 Dq and hM 4 Di relative to CNO or C21, while reducing off-target binding compared with clozapine in vitro. Using positron emission tomography (PET), we demonstrated that DCZ rapidly penetrates into the brain and selectively binds to DREADDs, and that DCZ doses for DREADD occupancy are 20-and 60-fold smaller than CNO and C21,respectively. Finally, we demonstrated that DCZ is capable of rapidly (<10 min post-injection) activating hM 3 Dq and hM 4 Di in both mice and monkeys without discernible off-target action. Thus, DCZ represents a potent and selective chemogenetic actuator for muscarinic-based DREADDs in mice and primates that is useful for a variety of in vitro and in vivo contexts with high translational potential. 8 RESULTS DCZ selectively binds to DREADDs in vitro and in vivoWe first assessed the binding affinities of DCZ to hM 3 Dq and hM 4 Di, and compared them to those of clozapine, CNO and C21 using radioligand competition binding assays. DCZ inhibited [ 3 H]quinuclidinyl benzilate (QNB) binding to hM 3 Dq and hM 4 Di with nanomolar affinity (inhibition constants; hM3Dq K i = 6.3 nM; hM4Di K i = 4.2 nM), which was comparable to clozapine ( hM3Dq K i = 5.9 nM; hM4Di Ki = 0.89), while CNO and C21 were about 100-and 50-fold weaker, respectively (CNO, hM3Dq K i = 680 nM; hM4Di K i = 360 nM; C21, hM3Dq K i = 850 nM; hM4Di K i = 180 nM)( Table 1). Unlike clozapine ( Fig. 1c and Supplementary Table 1), DCZ had negligible affinities for a large number of tested G protein-coupled receptors (GPCRs), ion channels or transporters (K i 's > 100 nM) and relatively low affinities for a few endogenous receptors such as muscarinic acetylcholine ( hM1 K i = 83 nM; hM5 K i = 55 nM) and serotonin receptors ( 5-HT2A K i = 87 nM)( Fig. 1b and Supplement...

Single caudate neurons encode temporally discounted value for formulating motivation for action

“…A monkey (#214) was injected with AAV2-CMV-mKO (1.7 x 10e 13 particles/mL) into the left DLPFC (S2A and S2B Figs). Details of the surgical procedures for the viral vector injection were described elsewhere [23]. The vector was injected at 1 to 2 μl per site.…”

“…Five to ten min following administration, the animal was allowed to start performing the tasks, which continued for 100 min. Based on a previous study, chemogenetic silencing would be effective 15-120 min after DCZ administration [23]. We performed at most one inactivation study per…”

“…However, the activity was not influenced by the level of satiation. To determine whether the value-related activity might be causally related to the behavior, pharmacological inactivation (local muscimol injection) and chemogenetic inactivation (designer receptor exclusively activated by designer drugs, DREADD) [23,24] of dCDh was carried out; both of these inactivations produced consistent impairments in motivational behaviors reflected as a distorted integration of reward size and delay, while behaviors based on the integration of reward size and physiological state remained intact.…”

Chemogenetic actuator drugs impair prefrontal cortex-dependent working memory in rhesus monkeys

“…However, drawbacks of CNO include poor brain penetrance, action as 74 a substrate for P-glycoprotein, and reverse metabolism to its parent compound (Gomez et deschloroclozapine -possess better brain penetrance and higher affinity and potency for 81 DREADD receptors than CNO. All of these actuators have been tested in rodent systems 82 (Gomez et al, 2017; Thompson et al, 2018;Weston et al, 2018) and low-dose clozapine 83 (Raper et al, 2019) and deschloroclozapine (Nagai et al, 2019) have been tested in nonhuman 84 primates. Clozapine and olanzapine are atypical antipsychotics designed for clinical use, and 85 deschloroclozapine is a metabolite of clozapine; each has high affinity for DREADD receptors 86 (Gomez et al, 2017;Weston et al, 2018;Nagai et al, 2019).…”

“…All of these actuators have been tested in rodent systems 82 (Gomez et al, 2017; Thompson et al, 2018;Weston et al, 2018) and low-dose clozapine 83 (Raper et al, 2019) and deschloroclozapine (Nagai et al, 2019) have been tested in nonhuman 84 primates. Clozapine and olanzapine are atypical antipsychotics designed for clinical use, and 85 deschloroclozapine is a metabolite of clozapine; each has high affinity for DREADD receptors 86 (Gomez et al, 2017;Weston et al, 2018;Nagai et al, 2019). Importantly, these drugs can 87 activate DREADD receptors at doses lower than those associated with their therapeutic effects 88 (Casey, 1993; Lidow and Goldman-Rakic, 1997; Murphy, 1997;Linn et al, 2003).…”