Ultrapotent chemogenetics for research and potential clinical applications

Abstract

Chemogenetics enables noninvasive chemical control over cell populations in behaving animals. However, existing small-molecule agonists show insufficient potency or selectivity. There is also a need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the smoking cessation drug varenicline. We then synthesized subnanomolar-potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.

Conflict of interest statement

Competing interests: S.M.S., C.J.M., and P.H.L. have pending patents on this technology and own stock in Redpin Therapeutics, LLC, which is a biotech company focusing on therapeutic applications of chemogenetics. S.M.S. is a cofounder and consultant for Redpin Therapeutics. M.M. is a cofounder and owns stock in Metis Laboratories, Inc.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1.

Screen of mutant α7 nAChR LBD ion channel activity against clinically used drugs. A) Schematic of modular PSAM-IPD chimeric channels for cell activation and inhibition. B) Screen of 41 α7–5HT3 channels with mutant LBDs against 44 clinically used drugs. C) Key molecules. D) Granisetron sensitivity conferred by Trp77→Phe. E) Tropisetron potency improved by Gln79 mutations.

Figure 2.

Development of an ultrapotent PSAM for varenicline. A) Varenicline (left) and tropisetron (right) bind AChBPs in distinct orientations. Dashed line: H-bond with Trp147. Parentheses: homologous α7 nAChR pre-protein numbering. B) Leu131→Gly oppositely affects potency for varenicline, ACh, and other α7 nAChR agonists. C) Potency enhancement of varenicline for α7L131G,Q139L,Y217F-GlyR. D) Varenicline whole cell voltage clamp response at α7L131G,Q139L,Y217F-GlyR. Baseline shifts reflect slow washout (dashed lines). E) Cortical layer 2/3 neurons expressing EGFP and PSAM4-GlyR with non-permeabilized cell-surface labeling by α-Bungarotoxin-Alexa594 (αBgt-594). F-H) Action potential firing strongly suppressed by varenicline strongly in neurons expressing PSAM4-GlyR (F) due to reduced input resistance (G) and elevated rheobase (H). I) Varenicline binding to PSAM4-5HT3 elicits a weakly desensitizing current with high potency. Baseline shifts reflect slow washout. J) αBgt-594 labeling of PSAM4-5HT3 HC (red) on cell surface of a cortical layer 2/3 neuron co-expressing EGFP. K,L) Varenicline (15 nM) depolarizes (K) and elicits action potentials (L) in neurons expressing PSAM4-5HT3 HC. Error bars are SEM. Mann Whitney U-test, n.s. P>0.05, ***P<0.001.

Figure 3.

PSAM4-GlyR neuron silencing in mice and a monkey. A) PSAM4-GlyR—IRES—EGFP targeted unilaterally to the SNr. Inset: schematic of unilateral SNr transduction and contralateral rotation. Asterisk: non-specific immunofluorescence. B) Low doses of intraperitoneal varenicline elicit contraversive rotation for mice expressing PSAM4-GlyR (n=9 mice) but not sham operated or EGFP-alone expressing mice (n=6 mice). C) Two doses of varenicline separated by 5 h give similar proportion of total rotation (n=4 mice). D) Timecourse of rotation response normalized to maximum rotation for each mouse (n=4 mice). Pink arrows: amphetamine injections, cyan arrow: varenicline injection. E) Coronal diagram of rhesus macaque brain showing location of GPi targeted for AAV injection and in vivo electrophysiological recordings along trajectory shown by dotted line. Box: area of interest for (F). OT:optic tract, GPi: internal globus pallidus, GPe: external globus pallidus. F-H) EGFP marker gene expression near injection site. Boxes denote area of interest for subsequent panel, arrowheads (H) indicate EGFP-positive neuronal profiles visualized by 3–3’-diaminobenzidine polymerization. (I-L) In one monkey, electrophysiological reduction of overall neuronal firing rate (I) and burst firing rate (J) in GPi after peripheral varenicline injection. Coefficient of variation of ISI (CV of ISI) (K) and percent time in burst firing (L) were not affected (pre-AAV: n=8 neurons, post-PSAM4-GlyR AAV: n=10 neurons). Error bars are SEM. Mann-Whitney U-test, n.s. P>0.05, **P<0.01.

Figure 4.

Highly selective chemogenetic agonists. A) Synthetic pathways (letters) for uPSEM agonists (see Methods). B) Comparison of uPSEM agonist EC50s at PSAM4 channels and endogenous varenicline targets, as well as IC50 for α4β2 nAChR with 1 μM ACh. LED: lowest effective dose for mice in SNr rotation assay. Units: nM; parentheses: SEM. Selectivity relative to PSAM4-GlyR in bold. C,D) Displacement of [3H]ASEM at PSAM4-GlyR by uPSEMs (C) and Ki values (D). Units: nM. Parentheses: SEM. E,F) Dose response curves for PSAM4-GlyR, α4β2 nAChR, 5HT3-R. uPSEM792 (E) is a 10% partial agonist of α4β2 nAChR and uPSEM817 (F) inhibits α4β2 nAChR. G,H) uPSEM 792 (G) and uPSEM817 (H) strongly suppress firing in neurons expressing PSAM4-GlyR. I) Efflux ratio<2, indicating uPSEM792 and uPSEM817 are not PgP substrates. PB-A and PA-B are basal and apical permeability across Caco-2 cell line monolayer (n = 2 replicates). J,K) In vivo PET imaging after [18F]-ASEM injection showing cortical localization of PSAM4-GlyR in horizontal view through head and upper torso (J) and coronal view of the head (K). Arrow shows site of PSAM4-GlyR expression. Asterisks show accumulation of [18F] outside the brain. (L) [18F]-ASEM binding to PSAM4-GlyR under baseline conditions and with competition by uPSEMs and varenicline. uPSEM792 (1 mg/kg), uPSEM793 (0.3 mg/kg), uPSEM815 (0.3 mg/kg), uPSEM817 (0.3 mg/kg), varenicline (0.3 mg/kg). M) Tomographic plane showing [18F]-ASEM binding to PSAM4-GlyR in vivo. N) Competition of [18F]-ASEM binding by intraperitoneally administered uPSEM792. O,P) Ex vivo fluorescence image (O) and autoradiographic image of [3H]-ASEM binding (P) of corresponding brain slice expressing PSAM4-GlyR—IRES—EGFP in left dorsal striatum. Right striatum was injected with EGFP-alone expressing virus. Error bars are SEM.

Figure 5.

Neuron silencing with uPSEMs and PSAM4-GlyR in vivo. (A) Experimental design for monitoring hippocampal CA1 neuron silencing with PSAM4-GlyR and uPSEM792 using in vivo two photon Ca2+ imaging in head-fixed mice running on a treadmill with textural landmarks. PSAM4-GlyR—IRES—EGFP was virally expressed in hippocampal CA1 pyramidal neurons in Thy1-GCaMP6f mice. Neurons co-expressing GCaMP6f and viral PSAM4-GlyR—IRES—EGFP are solid green, while neurons expressing only GCaMP6f are green outline. (B) Z-stack projections (green) overlaid with maximum intensity projections of GCaMP6f fluorescence in time (magenta) overlaid with an example from a saline treated (top) and uPSEM792 treated (bottom) mouse. Red outline encloses densely transduced region, which also shows strong uPSEM792-mediated reduction of neuron activity (reduced magenta signals). Outside the red boundary is the sparsely transduced region. Neurons depicted in (C) are circled. (C) Representative ROIs (left) and somatic Ca2+ traces (right). Transduced PSAM4-GlyR(+) pyramidal neurons are identified by EGFP-filled somata, whereas PSAM4-GlyR(−) cells have only cytoplasmic GCaMP6f fluorescence. (D) Activity rate (area under ΔF/F trace of Ca2+ transients divided by epoch duration (AUC/min)) for episodes in which mice were running and not running on the treadmill. Densely transduced: run, n = 68 neurons, no-run, n = 58 neurons from 2 mice; sparsely transduced: run, n= 103 neurons, no-run, n= 96 neurons from 2 mice. Mann-Whitney U-test and signed rank test with Holm-Sidak correction. (E) Place field activity following uPSEM792 administration. Somatic ROI outlines before and after treatment (left), ΔF/F activity (center top) with associated position of the mouse on the belt (center bottom), and raster plots with mean ΔF/F activity in each 2 cm spatial bin across laps (right). Asterisks denote the location of significant running calcium transients along the belt. (F-I) In vivo uPSEM dose responses for mice (n = 4) expressing PSAM4-GlyR unilaterally in SNr. Behavioral response and timecourse for uPSEM793 (F,G) and uPSEM792 (H,I). Time course of rotation response normalized to maximum rotation for each mouse. Pink arrows: amphetamine injections, blue arrow: uPSEM injection. Error bars are SEM. n.s. P>0.05, **P<0.01, ***P<0.001.