High-potency ligands for DREADD imaging and activation in rodents and monkeys

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated ¹⁸F positron emission tomography (PET) DREADD radiotracer, [¹⁸F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [¹⁸F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.

Fig. 1

New DREADD ligands displaying high in vitro DREADD affinity and potency. a Compound 13 (C13) and Compound 22 (C22) structures. b Binding competition curves of [³H]CLZ versus increasing concentrations of C13 and C22 in HEK-293 cells expressing DREADDs. C13 and C22 exhibit comparable DREADD affinity to clozapine (CLZ) with C13 showing ~twofold greater affinity than C22. CLZ and C21 competition curves from Supplementary Fig. 1 are overlaid for comparison. c, d C13 selectively blocks [³H]CLZ binding to DREADDs in mouse slices at 10 nM. Representative images of sections collected from 3 different mice are displayed and quantified in (d) as mean ± SEM. Two-way ANOVA followed by Dunnett’s test, ^*p < 0.05 and ^**p < 0.01 compared with the respective vehicle. e–h [³H]C13 binds with greater selectivity than [³H]CLZ to DREADDs in mouse and monkey brain tissue expressing AAV-hM3Dq and AAV-hM4Di, respectively. i Intraperitoneal (IP) injection of [³H]C13 readily enters the brain and accumulates in DREADDs expression areas in D1-DREADD mice. Representative images from 3 mice per condition. j–l JHU37107 (J07), JHU37152 (J52), and JHU37160 (J60) are high-affinity DREADD ligands. m Docking and molecular dynamics simulation of J60 in the ligand binding pocket of a hM4Di model. n, o J60 and J52 selectively displace [³H]CLZ at a concentration of 1 and 10 nM from hM3Dq and hM4Di expressed in mouse brain sections (n = 3 mice per condition). p, q J60 and J52 activate hM3Dq and hM4Di expressed in HEK293 cells with high potency (experiments performed 3–5 times). In all cases, data are represented as mean ± SEM. Scale bars are 1 mm. Source data are provided as a Source Data file

Fig. 2

JHU37152 and JHU37160 exhibit high in vivo DREADD occupancy. a Structures of JHU37152 (J52) and JHU37160 (J60). b Brain and serum concentrations and ratios of J52 and J60 in mice (n = 4 mice per condition) at different time points after a 0.1 mg kg⁻¹ (IP) injection. C21 (1 mg kg⁻¹, IP) data are same as shown in Supplementary Figures for comparison purposes. c, d J52 and J60 (0.1 mg kg⁻¹, IP) displace in vivo [¹¹C]clozapine binding to DREADDs in AAV-DREADD-expressing mice (n = 5 mice). e, f J60 (0.1 mg kg⁻¹, IP) selectively blocks in vivo [¹¹C]clozapine binding to DREADDs in rats (n = 3 rats). g, h J60 (0.1 mg kg⁻¹) blocks in vivo [¹¹C]clozapine binding to hM4Di in the monkey. All data represented are mean ± SEM except in (h) where individual values are displayed. Source data are provided as a Source Data file

Fig. 3

JHU37152 and JHU37160 exhibit high in vivo DREADD potency. a–c J60 and J52 produce potent inhibition of locomotor activity in transgenic D1-DREADD mice but not in wild-type (WT) mice (n = 7 to 19 mice per condition). Two-way repeated measures ANOVA followed by Dunnett’s multiple comparison tests were performed, ^*p < 0.05 and ^**p < 0.01 compared with the respective vehicle. d, e DREADD-assisted metabolic mapping (DREAMM) using [¹⁸F]FDG in D1-hM3Dq and D1-hM4Di mice (n = 4 mice per condition) reveals opposing and differential recruitment of whole-brain functional networks. f, g J52 and J60 produce potent activation of locomotor activity in rats (n = 7 rats per condition) expressing hM3Dq in tyrosine hydroxylase (TH)-expressing neurons in the ventral tegmental area. One-way repeated measures ANOVA followed by Dunnett’s multiple comparison tests were performed, ^*p < 0.05 and ^**p < 0.01 compared with the respective vehicle. h–j Design of in vivo electrophysiological experiment and IHC showing hM4Di (green) and ChrimsonR (red) expression in the medial division of the medial geniculate nucleus (MGM) and lateral amygdala (LA). k, l J60 (0.1 mg kg⁻¹) produces rapid and potent hM4Di-driven inhibition of light-evoked neuronal activation. Data are represented as mean ± SEM, ^*p < 0.05, ^***p < 0.001. Source data are provided as a Source Data file

Fig. 4

[¹⁸F]JHU37107 enables noninvasive detection of DREADD in locally-targeted cells and at their long-range projections. a Structure of [¹⁸F]JHU37107. b–d [¹⁸F]JHU37107 selectively binds to DREADDs in the brain of transgenic D1-DREADD mice (n = 3 mice per condition) and is blocked by 0.1 mg kg⁻¹ of JHU37160. e–j [¹⁸F]JHU37107 selectively binds to AAV-DREADDs expressed in the rat cortex and enables noninvasive and longitudinal mapping of both local (injection site) and long-range projections of motor cortex circuitry (ventrolateral thalamus shown as a main hub). Representative immunohistochemical images showing GFP (green) or HA-tagged DREADDs (red) from representative rats are shown side by side with their corresponding [¹⁸F]JHU37107 PET images. The white arrows point at corresponding anatomical regions. k–m [¹⁸F]JHU37107 binds to hM4Di expressed in the monkey amygdala and at putative projection sites. All data are represented as mean ± SEM except in (l) and (m) where individual values are displayed. Scale bars are 1 mm. Source data are provided as a Source Data file and the raw PET data are available upon request