Abstract
Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors are a powerful tool to test causality in basic neuroscience, but are also potentially amenable to clinical translation. A major obstacle is however that the widely-used agonist clozapine-N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations, but is not approved for use in humans by the Food and Drug Administration or European Medicines Agency, limiting its translational potential. Here we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a full and potent agonist of the human muscarinic-receptor M4-based DREADD, facilitating clinical translation of chemogenetics to treat CNS diseases.
Introduction
CNS diseases caused by abnormal circuit function represent a major burden to society. Although many respond to conventional small molecule treatment, some diseases such as intractable pain and refractory epilepsy account for a substantial unmet need. Drug-resistant focal epilepsy alone affects approximately 0.2% of the entire population (1, 2). Although surgical resection of the epileptogenic zone is effective, it is contraindicated in the overwhelming majority of patients because of high risks of permanent disability associated with brain tissue removal (3). Several gene therapies for refractory epilepsy, based on altering the balance of excitation and inhibition, have been validated in preclinical models (4-8). Chemogenetics using viral vector-mediated expression of inhibitory DREADDs is especially promising because the therapeutic effect can be titrated by adjusting the dose of the activating ligand (9).
A potential limitation to clinical translation of DREADD technology is that most studies to date have used clozapine-N-Oxide (CNO), the inactive metabolite of the atypical antipsychotic drug clozapine (CZP) (10), as the ligand. CNO is not approved for clinical use, and recent evidence shows that CNO is actively exported from the CNS and back-converted to CZP, which crosses the blood brain barrier and subsequently acts as the ligand activating the DREADD (11, 12). CZP has a relatively high EC50 (~57nM) at the muscarinic receptor M4-based Gi-coupled DREADD (hM4D(Gi)) (10). This is up to 5-fold higher than the EC50 or IC50 at receptors relevant to its action as an antipsychotic drug (13). In addition, patients on CZP can develop agranulocytosis, myoclonus and generalized seizures (14–16). Although recent studies highlight the ability of hM4D(Gi) expressed in epileptogenic zones to suppress focal seizures when activated (8, 17, 18), the pharmacological profile of CZP is far from optimal for clinical translation. Related antipsychotic drugs have been proposed as potential agonists (10, 11), and two other drugs activating DREADDs have recently been described: “Compound 21” (C21) and perlapine (PLP) (19, 20). Although perlapine has previously been used as a mild sedative anti-histamine drug in Japan, neither it nor C21 is approved for clinical use by the FDA or EMA. Identification of an FDA/EMA-approved drug for repurposing as a DREADD activator would facilitate clinical translation of DREADD technology to treat CNS diseases.
Results
hM4D(Gi)-dependent Kir3.1 and Kir3.2 activation
In order to measure Gi-coupled hM4D(Gi) activation, we established an electrophysiological screen based on measuring the potentiation of the inward-rectifying potassium current in a HEK cell line stably expressing Kir3.1 and Kir3.2 (21) (Fig. 1a). We verified the sensitivity of the system by estimating the EC50 of CZP as 61±19nM (mean ± SEM, n=6), close to the reported EC50 of 57nM (10)). We used CNO (1µM) as a positive control to define maximal activation of hM4D(Gi) (Fig 1b), and confirmed that CZP, PLP and C21 are full agonists (efficacy in comparison to 1µM CNO: CZP/CNO=1.14±0.06, n=6; PLP/CNO=1.17±0.16, n=9; C21/CNO=1.11±0.07). C21 showed a significant lower EC50 than CZP (CZP EC50=61±19nM; PLP EC50=40±10nM; C21 EC50=20±4nM; p<0.05 one-way ANOVA with Bonferroni post-hoc test). We performed a shape (3D) (22, 23) and 2-D similarity (2D) screen (24) in order to identify FDA/EMA-approved drugs with structural and electrochemical properties similar to those of C21 (Fig. 2a). Prioritized drugs with similarity indicated by the TanimotoCombo score for the 3D screen, and by similarity for the 2D based screen, are listed in Fig. 2b and Supplementary Table 1.
hM4D(Gi) activation with drugs identified by similarity screens
We tested olanzapine (OZP, 3D rank 1), promazine (PZN, 3D rank 2), triptilennamine (TNA, 3D rank 5), diphenhydramine (DPH, 3D rank 6), chlorprothixen (CZX, 3D rank 9), and amoxapine (AXN, 2D rank 2). We also tested the first, putative active metabolite of quetiapine (2D rank 6), norquetiapine (NQN) (25) (for chemical structures of all tested molecules – Supplementary Table 2). Of all drugs tested, only OZP was able to fully activate hM4D(Gi) at a concentration between 100-300nM, using 1µM CNO as control as above (OZP/CNO=1.18±0.13; n=3) (Fig. 2b). A full dose-response curve for OZP revealed an EC50 of 5±2nM (n=6), significantly lower than CZP (EC50=61±19nM, n=6; p=0.0128, Student’s t-test) (Fig. 3a).
In vivo verification that olanzapine activates hM4D(Gi)
To test whether OZP is effective in vivo we redesigned a codon-optimized version of hM4D(Gi) linked via a viral self-cleaving 2A-peptide to GFP (hM4D(Gi)opt), and put it under control of a human CamKIIα promoter for selective expression in excitatory neurons (26). We verified that the EC50 of OZP at hM4D(Gi)opt was similar to that at the original hM4D(Gi): OZP hM4D(Gi)opt: EC50=7±2nM, n=6; OZP hM4D(Gi): EC50=5±2nM, n=6). P0 mice were randomized for injection of either 2.5µl AAV2/8-hM4D(Gi)opt or 2.5µL AAV2/8-CamKII-copGFP (control) into both lateral ventricles (Fig. 3b right). A third group of mice received no injection. After a period of training, their performance on the rotarod was then tested at ~P42, while blinded to the viral injection. All mice received an acclimatization session on the rotarod on the day of testing, followed by two test sessions, one before and one after ligand injection. We injected OZP at a dose of 0.1mg/kg intraperitoneally, approximately 10-fold lower than the dose reported to achieve a peak plasma concentration equivalent to that obtained in patients treated with a typical anti-psychotic dose (27). OZP significantly reduced the latency to fall from 149±14sec to 111±11 sec (n=15; p=0.002; paired Student’s t-test) in hM4D(Gi) injected animals. In contrast GFP-injected animals showed no significant difference pre- and post-OZP (control: 180±21, OZP 0.1mg/kg: 169±25; n=15; p=0.568; paired Student’s t-test) (Fig. 3b). OZP also had no effect in mice that had received no viral injection (control: 212±21, OZP 0.1mg/kg: 189±20; n=6; p=0.109; paired Student’s t-test).
Discussion
Although recent papers highlight the potential of PLP or C21 as full and potent activators of the hM4D(Gi), they would require extensive screening to be approved for clinical use (28).PLP has been used clinically in Japan but was subsequently withdrawn from the market, calling for an alternative licensed drug that can be repurposed as an activator of hM4D(Gi) for clinical translation of DREADD technology. The present study shows that OZP (ranked first in the 3D-based in silico screen) is a full and potent activator of hM4D(Gi). Its EC50 in comparison to EC50 and IC50 at targets related to its clinical use as an antipsychotic, is lower than for CZP (13). OZP is a second-generation atypical antipsychotic which is approved by the FDA and EMA for treatment of schizophrenia and manic episodes in bipolar disorder. A common side effect of OZP at doses used in schizophrenia and bipolar disorder is a small degree of weight gain (29). OZP is a D2-receptor antagonist and its side effect profile therefore also includes akathisia, extrapyramidal symptoms, tardive dyskinesia, and neuroleptic malignant syndrome, although these are much less common than for first-generation antipsychotic drugs such as haloperidol and chlorpromazine. The in vitro EC50 of OZP at hM4D(Gi) is in the range of affinities reported for its native drug targets (30). The ability to affect performance on the rotarod with 0.1mg/kg OZP reported here is consistent with the principle of receptor reserve, whereby GPCR-mediated effects can be achieved with low doses of agonist (31). Given that CZP is typically only prescribed for treatment-resistant patients, due to its unfavorable side effect profile (13), CZP is much less suitable for repurposing as a DREADD activator. We therefore propose that OZP, which is widely available in oral, intramuscular and intravenous formulations, is suited for clinical translation of hM4D(Gi)-based chemogenetics to treat CNS diseases, including refractory epilepsy.
Materials and Methods
Voltage clamp recordings
A cell line stable expressing Kir3.1/3.2 (32) was cultured in DMEM GlutaMax® (Gibco), supplemented with 10% fetal bovine serum (Gibco), Penicillin/Streptomycin (50 I.U./m, Gibco), and contained 500µg/ml Geneticin® (Gibco) as a selection marker. Cells were transiently transfected with TurboFect® transfection reagent (Thermo Fisher Scientific) with 3 µg of hM4D(Gi)-plasmid (Addgene, 45548) and 1 µg of CMV-GFP for cell identification. Standard whole-cell patch-clamp experiments were performed after 2-3 days as previously described (26). Briefly, borosilicate-glass electrodes were pulled (Sutter Instruments) and fire polished (Narishige), with a final resistance of 2-4.5 MΩ. The extracellular recording solution contained (in mM): KCl 140, CaCl2 2.6, and MgCl2 1.2, HEPES 10, adjusted to pH 7.4 with KOH. The intracellular recording solution contained (in mM): KCl 107, MgCl2 1.2, CaCl2 1, EGTA 10, HEPES 5, Mg-ATP 2, and NA2-GTP 0.3, adjusted with KOH to pH 7.2. Cells were voltage-clamped at a holding potential of 0 mV, and a 100 ms step depolarization from −100 mV to 50 mV was applied in 10 mV increments and a 30 sec inter-pulse interval. Whole cell currents were low-pass filtered at 2 kHz (Axopatch 1-D, Axon Instruments) and digitized at 10 kHz. The membrane leak conductance in each cell was estimated from a linear fit to currents measured between 0 and +50 mV. The inward rectifying conductance mediated by Kir3.1/3.2 was estimated from a linear fit to currents between −100 and 0 mV after subtracting the leak conductance (Fig. 1a). All recordings were performed at room temperature, and the different drugs were applied by a custom-built perfusion system. Clozapine-N-oxide (CNO, Generon, #HY17366), perlapine (PLP, Tocris, #5549), compound 21 (C21, HelloBio, #HB6124), clozapine (CZP, Cayman Chemicals, #12059), olanzapine (OZP, Santa Cruz Biotechnology, #sc-212469), promazine (PZN, Sigma Aldrich, #46674), triptilennamine (TNA, Santa Cruz Biotechnology, #sc-229608), diphenhydramine (DPH, Cerilliant, #D-015), chlorprothixen (CZX, Santa Cruz Biotechnology, #sc-211077), norquetiapine (NQN, BioVision, #2362), and amoxapine (AXN, LKT labs, #A5059) were dissolved in either DMSO or extracellular recording solution at a stock concentration of 1mM, and subsequently diluted to specified concentrations. CNO (1 µM) was routinely tested to estimate maximal activation of hM4D(Gi) in each cell, and the Kir3.1/3.2-mediated conductance activated by each agonist application was therefore related to that evoked by 1 µM CNO.
Molecular biology
The hM4D(Gi)-plasmid was purchased from Addgene (#45548). Standard molecular biology techniques were used to clone dscGFP-T2A into an AAV2 transfer plasmid. The codon-optimized version of the HA-hM4D(Gi) (GeneOptimizer®, GeneArt®, Thermo Fisher Scientific) was linked to dscGFP via a viral 2A peptide. For in the in vivo experiments the CMV promoter was replaced with a CamKIIα-promoter to allow expression in excitatory neurons (26), the antibiotic resistance was changed from ampicillin to kanamycin, and a restriction site after the 2A peptide was removed.
In silico screening
One low energy conformation of C21 calculated with Omega 2.3.2 (33, 34) was used as the query for the generation of the shape-based model. The default model was modified and the final model only contained the color features shown in Fig. 2a. A maximum number of 200 conformers were generated for Drugbank version 5.0.7 (32) with Omega 2.3.2 (33, 34). The default settings of vROCS 3.0.0 (22, 23) were used for screening and hits were ranked according to the TanimotoCombo score.
The ChEMBL (24) web service (https://www.ebi.ac.uk/chembl/; access date 2017/06/21) was employed to find FDA/EMA-approved drugs with similar 2D structure as C21.
Viral injections
All animal procedures were performed in accordance with the [Author University] animal care committee’s regulations. Viral aliquots were prepared of AAV2/8-CamKIIα-GFP-T2A-hM4D(Gi)opt or AAV2/8-CamKIIα-GFP (both titres >1011 GC/ml; Vectorbuilder) and coded by a researcher conducting neither surgical procedures nor behavioral analyses. P0 neonatal CL57BL/6 mice were anaesthetized with intraperitoneal ketamine 6mg/kg and midazolam 0.2mg/kg. A 10μl microinjection syringe fitted with a 32G angled needle (Hamilton) was filled with one or other virus. Mouse pups (n=30) were divided equally between viral types and manually injected with 2.5μl into each lateral ventricle, approximately 1mm lateral from the sagittal suture and halfway between lambda and bregma, to optimize widespread cerebral transduction. 6 pups received no injection. Pups’ paws were marked with green tattoo ink to allow differentiation between viral types, and after recovery they were returned to their home cage.
Behavioral analysis
At P35 mice were trained on a mouse Rotarod (Ugo Basile). Mice were initially acclimatized for 10 minutes on the Rotarod turning at 5 revolutions per minute (RPM), replacing them each time they fell off, followed by acceleration over a 5-minute period from 5 to 40 RPM. The sequence was repeated 4 times. The latency to fall or to three consecutive cartwheels was recorded. Training was repeated daily until every mouse’s performance reached a plateau, taking approximately 2 weeks.
On the day of DREADD agonist testing the mice had a further acclimatization session (5 minutes at 5RPM followed by 4 accelerations) followed by a break of at least 30 minutes. They were then tested twice, with the same protocol as the acclimatization session, before and 20 minutes after intraperitoneal injection of OZP 0.1mg/kg. The latency to falling off or cartwheeling was recorded. Trial times were recorded using Excel (Microsoft), and statistical analyses performed with Graph Pad Prism 5.01, by a researcher blinded to viral treatment.
Confocal Fluorescence
To establish the extent of viral transduction mice were anaesthetized with 150mg/kg pentobarbital (Boehringer Ingelheim) and transcardially perfused with 20ml heparinized (80mg/L) phosphate-buffered saline (PBS, Sigma-Aldrich) until the perfusate was clear, then switched to 4% paraformaldehyde (Tocris) (20ml). Brains were extracted and immersed in 4% PFA for a further 24 hours, before vibratome slicing (VT1000S Leica) at 50μm, mounting on slides with Vectashield mounting medium with DAPI (Vector Labs) and confocal fluorescence imaging (Zeiss LSM 710) to visualize GFP expression.
Statistical Analysis
Statistical analysis was performed with Graph Pad Prism 5.01. Unpaired/paired Student’s t-test, or one-way ANOVA with Bonferroni post-hoc test was used as indicated. Data are shown as mean±sem, and the significant level was set to an α-error of p<0.05.
Funding
This work was supported by the Medical Research Council and Wellcome Trust.
Author contributions
A.L. and D.M.K designed all experiments and wrote the manuscript. A.L and S.K. performed and analyzed all in vitro experiments. T.K designed, performed, and analyzed all in silico experiments. M.W. and G.L. performed the vector injections, and M.W. performed and analyzed all in vivo experiments. J.C. and A.S. cloned the constructs. A.L., D.M.K., S.S., G.L., S.K., A.S., J.C., T.K., and M.W. revised the manuscript.
Competing interests
S.S. and D.M.K hold a patent on the use of DREADD technology to treat epilepsy.
Data and materials availability
All data are available upon request.
Acknowledgments
We thank OpenEye for providing software free of charge. We are grateful to B. Roth for highlighting the potential of atypical antipsychotic drugs, and A. Tinker for the gift of the Kir3.1/3.2 expressing cell line.