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.

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 α7^{L131G,Q139L,Y217F}-GlyR. D) Varenicline whole cell voltage clamp response at α7^{L131G,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 P_A-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.