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. 2018 Sep 14;1(1):61-72.
doi: 10.1021/acsptsci.8b00012. Epub 2018 Jul 27.

DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo

Affiliations

DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo

Karen J Thompson et al. ACS Pharmacol Transl Sci. .

Abstract

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Binding of muscarinic DREADD ligands to DREADDs and wildtype receptors. Displacement of [3H]-NMS by increasing concentrations of ACh, CNO, C21, or perlapine at (A) hM1, (B) hM4, (C) hM1Dq, and (D) hM4Di. All experiments were performed using a Kd concentration of [3H]-NMS. Data represents the mean ± SEM of at least three experiments performed in duplicate.
Figure 2
Figure 2
Signaling at hM1 and hM1Dq mediated by muscarinic DREADD ligands. Concentration response curves for ACh, CNO, C21, and perlapine in (A) IP1 accumulation mediated by hM1, (B) ERK 1/2 activation mediated by hM1, (C) IP1 accumulation mediated by hM1Dq, and (D) ERK 1/2 activation mediated by hM1Dq. Data represents the mean ± SEM of at least three experiments performed in duplicate.
Figure 3
Figure 3
Signaling at hM4 and hM4Di mediated by muscarinic DREADD ligands. Concentration response curves for ACh, CNO, C21, and perlapine in (A) inhibition of isoproterenol elevated cAMP by hM4Di, (B) ERK 1/2 activation mediated by hM4Di, (C) inhibition of isoproterenol elevated cAMP by hM4, and (D) ERK 1/2 activation mediated by hM4. Data represents the mean ± SEM of at least three experiments performed in duplicate.
Figure 4
Figure 4
Determination of C21 and perlapine binding affinities at a panel of GPCR drug targets. Binding affinity (pKi) was determined at indicated receptors and targets using radioligand binding assays with membrane preparations and provided by NIMH PDSP. Results were presented as mean ± SEM from a minimum of 3 independent assays, each in triplicate. Indicated are those receptors where the pKi values are less than 5. The raw data is presented in Table S1.
Figure 5
Figure 5
Assessment of off-target activity of C21 against 318 nonolfactory GPCR targets. Agonist activity of C21 at 318 nonolfactory human GPCRs at a final of 5 μM. Results were represented as fold of basal in quadruplicate. Dopamine receptor DRD2 with 100 nM Quinpirole served as an assay control (Control). The GPCRome screening assay was carried out as outlined in the Methods section and plotted using Prism. The raw data is presented in Table S2.
Figure 6
Figure 6
Assessment of activity of C21 at dopamine D1–D3 and histamine H4 receptors. Activation of cAMP signaling at (A) dopamine D1 receptors stimulated with C21 and SKF81297, (B) dopamine D2 receptors stimulated with C21 and quinpirole, (C) dopamine D3 receptors stimulated with C21 and quinpirole, and (D) histamine H4 receptors stimulated with C21 and histamine. Data shown represents the mean ± SEM of at least three independent experiments performed in duplicate.
Figure 7
Figure 7
Analysis of brain and plasma exposure for CNO, C21, and perlapine. (A) Unbound fraction of plasma and brain CNO and clozapine levels following i.p. administration of various concentrations of CNO. (B) Unbound fraction of plasma and brain levels of perlapine following i.p. administration of various concentrations of perlapine. (C) Unbound fraction of plasma and brain levels of C21 following i.p. administration of various concentrations of C21. (D) Time course of C21 brain and plasma exposure following i.p. administration of C21 (5 mg/kg). (E) Percentage protein binding of C21 in human and mouse plasma and mouse brain homogenate. Data shown represents the mean ± SEM of at least three independent experiments.
Figure 8
Figure 8
C21 and CNO activates hM3Dq in neuronal cultures and in vivo. Recordings from lateral hypothalamic vGAT neurons infected with AAV encoding either hM3Dq-mCherry or mCherry. Neurons were exposed to C21 (1 μM) in the presence of tetrodotoxin (TTX, 500 nM). (A) Representative trace and (B) histogram on the right is the mean ± SEM with n = 6 cells for the hM3Dq and n = 3 cells for the mCherry (* indicates p < 0.05, Student’s t test). (C–F) Food intake in animals in which hM3Dq or mCherry was virally induced to be expressed in LH-vGAT neurons followed by administration of (C) C21 (0.3 mg/kg) (D) C21 (1.0 mg/kg), (E) C21 (3.0 mg/kg), and (F) CNO (3.0 mg/kg). Data represents the mean ± SEM of 11 mCherry mice and 10 hM3Dq mice.
Figure 9
Figure 9
In vivo activation of hM3Dq expressed in ARCAgRP-neurons by C21 and CNO increases feeding behavior in sated mice. Light cycle food intake was monitored following CNO or C21 administration at various concentrations to calorically replete AgRP-ires-CRE-expressing animals infected with (A–C (n = 6)) AAV-DIO-hM3Dq-mCherry or (D–F (n = 6)) control non-CRE-expressing animals infected with AAV-DIO-hM3Dq-mCherry. Data represents the mean ± SEM.
Figure 10
Figure 10
In vivo activation of hM4Di expressed in PVHSIM1-neurons by C21 and CNO increases feeding behavior in sated mice. Light cycle food intake was monitored following CNO or C21 administration at various concentrations to calorically replete SIM1-CRE-expressing animals infected with (A, B (n = 6)) AAV-DIO-hM4Di-mCherry or (C, D (n = 6)) control non-CRE-expressing animals infected with AAV-DIO-hM4-mCherry. Data represents the mean ± SEM.

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