Psychedelic-inspired drug discovery using an engineered biosensor

This study describes the development and use of an engineered biosensor (PsychLight) that detects relevant serotonin release to predict the hallucinogenic behavioral effects of psychedelics. This tool is used to identify non-hallucinogenic psychedelic compounds that still elicit antidepressant-like effects.

Abstract

“Ligands can induce G protein-coupled receptors (GPCRs) to adopt a myriad of conformations, many of which play critical roles in determining the activation of specific signaling cascades associated with distinct functional and behavioral consequences. For example, the 5-hydroxytryptamine 2A receptor (5-HT2AR) is the target of classic hallucinogens, atypical antipsychotics, and psychoplastogens. However, currently available methods are inadequate for directly assessing 5-HT2AR conformation both in vitro and in vivo. Here, we developed psychLight, a genetically encoded fluorescent sensor based on the 5-HT2AR structure. PsychLight detects behaviorally relevant serotonin release and correctly predicts the hallucinogenic behavioral effects of structurally similar 5-HT2AR ligands. We further used psychLight to identify a non-hallucinogenic psychedelic analog, which produced rapid-onset and long-lasting antidepressant-like effects after a single administration. The advent of psychLight will enable in vivo detection of serotonin dynamics, early identification of designer drugs of abuse, and the development of 5-HT2AR-dependent non-hallucinogenic therapeutics.”

Authors: Chunyang Dong, Calvin Ly, Lee E. Dunlap, Maxemiliano V. Vargas, Junqing Sun, In-Wook Hwang, Arya Azinfar, Won Chan Oh, William C. Wetsel, David E. Olson & Lin Tian

Notes

This research was highlighted in Nature News. A researcher, not affiliated with the current group, said the method is an “innovative approach” but that the translation from research in mice to men is always a difficult bridge to gap.

You can also find a breakdown on The Tab.

Highlights from the authors/journal:

• “Engineered psychLight—a genetically encoded 5-HT sensor based on the 5-HT2AR
• PsychLight can measure 5-HT dynamics in behaving mice
• A psychLight-based cellular imaging platform predicts hallucinogenic potential
• Identified a non-hallucinogenic psychedelic analog with antidepressant properties“

PsychLight is a sensor that can highlight activity at our most psychedelic of receptors. This paper describes how the sensor works and how it has already been used to identify promising molecules.

The sensor works by lighting up when psychedelic molecules bind to it. And lighting up is meant literally, the sensor is fluorescent. The structure of the receptor is near-identical to the serotonin (5HT) 2a receptor. This makes it so that psychedelics which usually bind to this receptor, also bind to the sensor. The big difference is that PsychLight, well, lights up if this happens.

Highlights from the paper

• The researchers inserted the sensor in the brains of mice and then gave them a variety of psychedelics, which lit up the sensor
• But when given ketanserin, something that blocks psychedelics, the sensor didn’t light up, even if psychedelics were given afterwards
• The research group also tested novel compounds and found that ‘AAZ-A-154’ didn’t light up the sensor, but did show promising neurogenesis effects

With the caveat of this study being in mice, this sensor could become a vital tool in the identification of new psychedelics. These can be molecules that can possibly help heal mental health disorders, possibly without the acute trip.

Summary

INTRODUCTION

G protein-coupled receptors (GPCRs) represent attractive therapeutic targets with nearly 35% of all FDA-approved medications affecting this class of proteins. Biased ligands have the potential to reduce undesirable side effects while maintaining efficacy.

Ligands for the 5-HT2AR are some of the first small molecules to demonstrate biased agonism, and are also some of the most important drugs in neuropsychiatry.

Psychedelics may prove useful for treating a variety of neuropsychiatric diseases, including depression, post-traumatic stress disorder, and substance use disorder. However, it is currently unclear if the subjective effects of these drugs are necessary to ameliorate disease symptoms.

Recent breakthroughs have increased our understanding of how receptor activation leads to coupling with various signal transducers, but there are still no assays capable of directly measuring hallucinogenic potential across a wide range of structurally diverse 5-HT2AR ligands.

We developed a modular strategy for creating genetically encoded fluorescent sensors that are activated by ligand-induced conformational changes in GPCRs. We used this strategy to identify previously unknown hallucinogenic drugs.

Development of psychLight

To develop a sensor for the human 5-HT2AR, we replaced the third intracellular loop (IL3) with a circularly permuted green fluorescent protein (cpGFP) and optimized the insertion site of cpGFP. We named the top-performing variant psychLight1.

We expressed psychLight1 in HEK293T cells and found that 5-HT activated the sensor, and other agonists were able to increase the sensor’s fluorescence intensity to varying degrees. The traditional 5-HT2AR antagonists had minimal effect on psychLight1 fluorescence.

Two-photon imaging of endogenous serotonin dynamics ex vivo and in vivo

We performed two-photon imaging in frame-scan mode (33 Hz) and triggered endogenous 5-HT release by electrical stimulation. PsychLight2 was able to detect electrically evoked 5-HT release in single trials, and its response could be enhanced by incubation with 50 mM escitalopram.

We injected psychLight2 into the basolateral amygdala, dorsal raphe nucleus, or orbitofrontal cortex of freely behaving mice, and measured 5-HT transients during an auditory fear conditioning experiment. We observed a robust increase in fluorescence intensity immediately after the onset of foot-shock, followed by a sharp decline during the shock. PsychLight2 detects endogenous 5-HT2AR ligands in freely behaving animals, but psychLight0 has a key point mutation that completely prevents agonist binding.

PsychLight activity differentiates hallucinogenic and non-hallucinogenic drugs

We administered 5-MeO-DMT to mice to determine if the sensor could faithfully report 5-HT2AR activation in vivo. The sensor responded to both agonist- and antagonist-induced conformational changes in vivo.

We tested the sensor’s ability to differentiate between known hallucinogenic agonists and structurally similar non-hallucinogenic analogs. The sensor performed exceptionally well in rat drug discrimination (DD) and mouse HTR assays.

All four hallucinogenic compounds activated psychLight1 when expressed in HEK293T cells, with half maximal effective concentrations ranging from 18.8 – 627 nM. None of the non-hallucinogenic congeners were able to increase the sensor’s response.

PsychLight1 potencies correlate exceptionally well with hallucinogenic potencies in humans, but not with efficacies. Furthermore, ligand activation of psychLight1 appears to be distinct from other measures of 5-HT2AR activation.

Development of a psychLight-based medium-throughput pharmacological assay

To enable medium-throughput identification of hallucinogenic designer drugs of abuse and non-hallucinogenic therapeutics targeting 5-HT2ARs, a screening platform based on wide-field high content imaging was developed.

We first tested a panel of ligands with similar molecular structures to 5-HT and found that subtle differences in ligand structure can significantly modulate the fluorescence signal generated by PSYLI2 cells.

We screened a library of eighty-three compounds for 5-HT2AR agonist or antagonist activity. Serotonergic hallucinogens reliably activated the sensor, while non-hallucinogenic 5-HT2AR ligands did not.

We suspected that the inherent fluorescence of 2-bromolysergic acid diethylamide and bromocriptine was resulting in false-positive signals, so we performed concentration-response experiments in PSYLI2 cells and under cell-free conditions.

potentials of designer drugs

We screened a library of thirty-four compounds with unknown hallucinogenic potentials and found that 5-F-DMT and 5-Cl-DMT were hallucinogenic, while 5-Br-DMT failed to induce HTRs at any dose.

We used psychLight to identify non-hallucinogenic 5-HT2AR ligands occupying previously unknown chemical space. AAZ-A-154 failed to produce any head-twitches in mice, but decreased locomotion, indicating that this compound can still impact behavior without producing hallucinogenic effects.

Characterizing the antidepressant-like effects of AAZ-A-154

AAZ-A-154, a new compound, increased dendritic arbor complexity in cultured rat embryonic cortical neurons to a comparable extent as the fast-acting antidepressant ketamine, and its antidepressant potential was assessed using behavioral assays relevant to active stress-coping strategies and anhedonia.

AAZ-A-154 decreased immobility in the forced swim test (FST) in C57BL/6J mice, and produced both rapid (30 min) and long-lasting (1 week) antidepressant-like effects after a single administration.

To determine if AAZ-A-154 could ameliorate anhedonia, we used VMAT2 heterozygous (VMAT2-HET) mice. AAZ-A-154 improved anhedonia in VMAT2-HET mice immediately following a single administration, and persisted for at least 12 days before the treated VMAT2-HET animals began to display reduced sucrose preference.

DISCUSSION

We developed psychLight, a 5-HT2AR-based fluorescent sensor, which can detect endogenous 5-HT dynamics and detect hallucinogenic conformations of the receptor. PsychLight exhibits millisecond off kinetics and can be used to detect both fast and slow decaying 5-HT signals in acute BNST slices following electrical stimulation.

PsychLight is based on the 5-HT2AR, which plays an essential role in the hallucinogenic effects of psychedelics. It provides direct measurement of 5-HT2AR conformational change, which overcomes the limitations of existing methods that either provide a snap-shot view of the interaction or depend on slow, indirect secondary signaling.

PsychLight fills the gap between in vitro testing of novel compounds and in vivo behavioral studies. It can be used to rapidly assess the hallucinogenic potential of novel compounds across a wide range of chemical structures.

PsychLight can be used to identify non-hallucinogenic 5-HT2AR antagonists or non-hallucinogenic biased agonists, including AAZ-A-154, a non-hallucinogenic analog of a psychedelic compound that promotes neuronal growth and produces long-lasting beneficial behavioral effects in rodents following a single administration.

The precise mechanisms of action of hallucinogens at molecular and circuit levels remain largely unknown. Genetic tools, such as reporters, sensors, and effectors, will be useful for dissecting the circuits involved in hallucinogenic versus antidepressant effects.

Limitations of study

This study has a number of limitations that should be addressed by follow-up experiments. These limitations include the need for a full pharmacological profile of AAZ-A-154, which should include information on mechanism of action, off-target effects, pharmacokinetics, full dose-response studies, potential toxicity, and efficacy validated using other established assays.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animals d METHOD DETAILS B

Lead contact

The chemical names of the drugs methamphetamine, 5-Br-DMT, 5-Cl-DMT, 5-F-DMT, 6-MeO, 8-OH-DPAT, and 5-HT2AR are given. Several drugs are used to treat depression, including escitalopram oxalate, ketamine, lisuride, NMT, norepinephrine, and S-amphetamine. The effects of these drugs vary depending on the drug and the individual. SEM, STD, VEH, VMAT2-HET, WT, TBG, DF/F, and WT = wild-type, vesicular monoamine transporter 2 heterozygous, and water.

Compounds

The NIH Drug Supply Program provided lysergic acid diethylamide hemitartrate, psilocin, psilocybin, 2-(4-Iodo-2,5-dimethoxyphenyl)ethan-1-amine hydrochloride (2C-I), 2-bromo-lysergic acid diethylamide tartrate (BOL-148), i All compounds used in these studies were synthesized in house and judged to be pure based on NMR and UHPLC-MS. The key step involved the regioselective ring opening of enantiopure Boc-protected aziridines derived from R- and S-alaninol, respectively.

PsychLight Development and Characterization

PsychLight1 and PsychLight2 were developed using circular polymerase extension cloning (CPEC), restriction cloning, and gBlock gene fragments. FLAG epitopes were placed at the 50 end of the constructs and cpGFP modules were inserted into the human 5-HT2AR using CPEC.

Tissue Culture

HEK293T cells were grown in DMEM, transfected with Effectene, washed with HBSS, and imaged in HBSS containing Mg2+ and Ca2+.

Confocal Microscopy Experiments

HEK293T cells were transfected with psychLight1, grown on 12 mm coverslips, perfused with 0.1% DMSO, and then exposed to ascending concentrations of drugs from 1 pM to 10 mM in a solution of 100 nM 5-HT. Images were recorded using a 465 nm laser and a 40x oil objection (0.55 N.A.).

Two-Photon Uncaging Experiments

Organotypic slice cultures from the frontal cortex were prepared from postnatal day 2 – 3 C57BL/6J mice, and infected with psychLight2 virus 19 – 20 days prior to imaging. Two-photon imaging and uncaging were performed on transfected layer 2/3 pyramidal neurons after 21 – 23 days in vitro at 30 C in recirculating artificial cerebrospinal fluid with 2 mM CaCl2, 1 mM MgCl2, 0.1 mM RuBi-5-HT, and 0.001 mM tetrodotoxin. Two-photon uncaging was achieved using RuBi-5-HT and a pulsed Ti::sapphire laser. Line-scan recording of fluorescence transients was performed simultaneous with 5-HT uncaging on layer 2/3 pyramidal neurons using two pulsed Ti::sapphire lasers for imaging and uncaging at wavelengths of 920 nm and 810 nm, respectively. To measure changes in psychLight fluorescence intensities following 5-HT bath application, bleed-through-corrected and background subtracted green (psychLight) and red (tdTomato) fluorescence intensities were calculated.

Brain Slices for Two-Photon Imaging

A 9-channel programmable pulse stimulator was used to image the area within approximately 20 mm of the electrode. Drugs were dissolved in imaging HBSS buffer and diluted at 1:1000 prior to application in the perfusion system.

General

Mice were anesthetized with isoflurane, Carprofen and Buprenorphine were subcutaneously injected, and body temperature was maintained with a heating pad. A sterile scalpel was used to make an incision, and optical fiber was implanted and secured with metabond and dental cement.

Optical Fiber Implantation

Optical fibers were inserted into the brain of mice after viral injection and secured with Metabond and dental cement.

Auditory Fear Conditioning

ROC analysis was done by a custom MATLAB script that calculated the true detection rate and false positive rate for the sensor response to the shock. The discriminability index was approximated by the area under the ROC curve.

Head-twitch Response with Fiber Photometry

Three animals were used for experiments measuring sensor activity in the prelimbic cortex. The DF/F values were calculated by fitting the 405 nm signals to the 465 nm channels and dividing by the fitted 405 nm signal.

Agonist Mode

For agonist mode experiments, 180 mL of imaging media were added to each well of the assay plate, and then 5 regions of interest (ROIs) were taken per well using the default 5 ROI pattern with no bias to location or overlap of the ROIs.

Once imaging was complete, the images were exported and analyzed using a self-written MATLAB script. The DF/F values were calculated for each well and the triplicate mean was obtained.

Antagonist Mode

For antagonist mode experiments, 160 mL of imaging media was added to each well of the assay plate, and 5 regions of interest (ROIs) were taken per well using a Lecia DMi8 at 40x (N.A. = 0.6) with no bias to location and no overlap of the ROIs.

Imaging was performed on HEK293T cells, and DF/F values were calculated using a self-written MATLAB script. Compounds unlikely to bind to the sensor should produce minimal to no response in either agonist or antagonist mode.

Schild Regression Analysis

A treatment plate was prepared by pre-mixing various concentrations of a non-hallucinogenic compound with increasing concentrations of 5-HT. Images were taken using a Lecia DMi8 at 40x with 5 regions of interest taken per well using the default 5 ROI pattern.

Forced Swim Test (FST)

Male and female C57BL/6J mice were housed 4 – 5 mice of the same sex/cage in a UCD vivarium following an IACUC approved protocol. One week after the initial handling, the mice underwent a 6 min swim session in a clear Plexiglas cylinder filled with 30 cm of 24 1 C water. All FSTs were performed between the hours of 0800 and 1300 h, and immobility time was scored for the last 4 min of the 6 min trial.

Head-Twitch Response (HTR) and Locomotion Assays

The HTR assay was performed using both male and female C57BL/6J mice. Compounds were administered 5 mL/kg, i.p., using 0.9% saline as the vehicle, and HTRs were videotaped and scored later by two blinded observers.

Sucrose Preference

Adult male and female mice were used for these experiments. They were housed individually 48 h prior to the experiment and were given ad libitum access to chow and water.

AAZ-A-154 (15 mg/kg, i.p.) was administered to mice on day 1 and the next day, the mouse was presented with a water-sucrose pairing. The mouse’s preference for the sucrose bottle was calculated as the volume of sucrose consumed minus the volume of water consumed.

(R)-2-(dimethylamino)propan-1-ol

A reaction of R-alaninol with glacial acetic acid and sodium cyanoborohydride was carried out, followed by the addition of 37% formaldehyde(aq). The product was concentrated under reduced pressure to yield a colorless oil.

(R)-1-chloro-N,N-dimethylpropan-2-amine hydrochloride

(R)-2-(dimethylamino)propan-1-ol was added to a solution of SOCl2 and heated to reflux for 4 h to yield the desired product.

AAZ-A-154

To a solution of 5-methoxyindole in DMSO, 1-chloro-N,N-dimethylpropan-2-amine hydrochloride, potassium iodide, and potassium tert-butoxide were added, and the mixture was stirred for 24 h. The organic extracts were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to yield the desired product.

LED-C-233

5-fluoroindole was dissolved in DMSO, and the reaction mixture was stirred for 24 h. The organic extracts were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to yield a colorless oil, which was purified by flash chromatography.

QUANTIFICATION AND STATISTICAL ANALYSIS

Treatments were randomized, and statistical analyses were performed using GraphPad Prism. The sucrose preference and the volume of liquid consumed in the anhedonia test were analyzed separately by repeated-measures ANOVA.