A non-hallucinogenic psychedelic analogue with therapeutic potential

Article

A non-hallucinogenic psychedelic analogue

with therapeutic potential

https://doi.org/10.1038/s41586-020-3008-z

Received: 17 January 2020

Accepted: 30 October 2020

Published online: xx xx xxxx

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Lindsay P. Cameron¹, Robert J. Tombari², Ju Lu³, Alexander J. Pell², Zefan Q. Hurley²,

Yann Ehinger⁴, Maxemiliano V. Vargas¹, Matthew N. McCarroll⁵, Jack C. Taylor⁵,

Douglas Myers-Turnbull^5,6, Taohui Liu³, Bianca Yaghoobi⁷, Lauren J. Laskowski⁸,

Emilie I. Anderson⁸, Guoliang Zhang², Jayashri Viswanathan², Brandon M. Brown⁹,

Michelle Tjia³, Lee E. Dunlap², Zachary T. Rabow¹⁰, Oliver Fiehn¹⁰, Heike Wulff⁹,

John D. McCorvy⁸, Pamela J. Lein⁷, David Kokel^5,11, Dorit Ron⁴, Jamie Peters^12,13, Yi Zuo³&

David E. Olson^2,14,15,16

ꢀ✉

The psychedelic alkaloid ibogaine has anti-addictive properties in both humans and

animals¹. Unlike most medications for the treatment of substance use disorders,

anecdotal reports suggest that ibogaine has the potential to treat addiction to various

substances, including opiates, alcohol and psychostimulants. The efects of ibogaine—

like those of other psychedelic compounds—are long-lasting², which has been

attributed to its ability to modify addiction-related neural circuitry through the

activation of neurotrophic factor signalling^3,4. However, several safety concerns have

hindered the clinical development of ibogaine, including its toxicity, hallucinogenic

potential and tendency to induce cardiac arrhythmias. Here we apply the principles of

function-oriented synthesis to identify the key structural elements of the potential

therapeutic pharmacophore of ibogaine, and we use this information to engineer

tabernanthalog—a water-soluble, non-hallucinogenic, non-toxic analogue of ibogaine

that can be prepared in a single step. In rodents, tabernanthalog was found to

promote structural neural plasticity, reduce alcohol- and heroin-seeking behaviour,

and produce antidepressant-like efects. This work demonstrates that, through

careful chemical design, it is possible to modify a psychedelic compound to produce a

safer, non-hallucinogenic variant that has therapeutic potential.

Ibogaine is the most abundant of the numerous alkaloids produced by that can last for more than 24 hours. Although ibogaine was once sold

Tabernanthe iboga⁵. Although it has not been tested in double-blind, in France as a medicine for the treatment of neuropsychiatric diseases,

placebo-controlled clinical trials, anecdotal reports and open-label it was removed from the market owing to its adverse effects¹.

studies suggest that it can reduce symptoms of drug withdrawal, reduce

Although the exact mechanism of action of ibogaine has not yet been

drug cravings and prevent relapse^1,6. However, the potential of ibogaine fully elucidated, evidence suggests that it might alter addiction-related

as a therapeutic is limited by several major issues. First, access to large circuitry by promoting neural plasticity. First, ibogaine has been shown

quantities is limited, both by overexploitation of the plant from which it to increase the expression of glial cell-line-derived neurotrophic factor

is derived and by the lack of a scalable, enantioselective, total synthesis⁶. (GDNF) in the ventral tegmental area, and infusion of ibogaine into this

Currently, there are only three synthetic routes to racemic ibogaine, region of the midbrain reduces alcohol-seeking behaviour in rodents³.

with longest linear sequences of between 9 and 16 steps and overall A more recent study demonstrated that ibogaine affects brain-derived

yields that range from 0.1 to 4.8%⁶. Second, the safety profile of ibogaine neurotrophic factor and GDNF signalling in several brain regions that

is unacceptable. It is very non-polar, which leads to its accumulation are implicated in the behavioural effects of addictive drugs⁴. Recently,

in adipose tissue⁷and contributes to its known cardiotoxicity through we demonstrated that noribogaine—an active metabolite of ibogaine¹²

—

the inhibition of hERG potassium channels^8,9. Several deaths have been is a potent psychoplastogen¹³that increases the complexity of cortical

linked to the cardiotoxicity of ibogaine^10,11, and it induces hallucinations neuron dendritic arbors¹⁴. Other psychoplastogens—such as lysergic

¹Neuroscience Graduate Program, University of California, Davis, Davis, CA, USA. ²Department of Chemistry, University of California, Davis, Davis, CA, USA. ³Department of Molecular, Cell and

Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA. ⁴Department of Neurology, University of California, San Francisco, San Francisco, CA, USA. ⁵Institute for

Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA. ⁶Quantitative Biosciences Consortium, University of California, San Francisco, San Francisco, CA,

USA. ⁷Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA. ⁸Department of Cell Biology, Neurobiology, and Anatomy, Medical

College of Wisconsin, Milwaukee, WI, USA. ⁹Department of Pharmacology, School of Medicine, University of California, Davis, Davis, CA, USA. ¹⁰West Coast Metabolomics Center, University of

California, Davis, Davis, CA, USA. ¹¹Department of Physiology, University of California, San Francisco, San Francisco, CA, USA. ¹²Department of Anesthesiology, University of Colorado Denver,

Anschutz Medical Campus, Aurora, CO, USA. ¹³Department of Pharmacology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA. ¹⁴Department of Biochemistry and

Molecular Medicine, School of Medicine, University of California, Davis, Sacramento, CA, USA. ¹⁵Center for Neuroscience, University of California, Davis, Davis, CA, USA. ¹⁶Delix Therapeutics,

✉

Inc., Palo Alto, CA, USA. e-mail: deolson@ucdavis.edu

Nature | www.nature.com | 1

Article

acid diethylamide (LSD) and psilocin (the active metabolite of psilo- psychoplastogenic performance to ibogaine despite its simplified

cybin)—have also been shown in anecdotal and open-label studies to chemical structure. We therefore prioritized IBG for further develop-

decrease drug use in the clinic, similar to ibogaine¹⁵. We suggest that ment, because we reasoned that its reduced lipophilicity (calculated

the ability of psychoplastogens to promote structural and functional logarithm of partition coefficient, clogP = 2.61) relative to that of ibo-

neural plasticity in addiction-related circuitry might explain their gaine (clogP = 4.27) would make formulation less challenging and would

ability to reduce drug-seeking behaviour for weeks to months after a also reduce the potential for cardiotoxicity, as lipophilicity is a known

single administration. Moreover, by modifying neural circuitry rather contributing factor to hERG channel inhibition. The attractiveness of

than simply blocking the targets of a particular addictive substance, IBG as a potential therapeutic was underscored by its improved central

psychoplastogens such as ibogaine could have the potential for broad nervous system (CNS) multiparameter optimization (MPO) score¹⁸

use as anti-addictive agents, applicable to the treatment of addiction (MPO IBG = 5.2; MPO ibogaine = 3.8; optimal MPO = 6.0) coupled with

to various substances.

the fact that it can be synthesized in a single step.

To develop simplified, and potentially safer, analogues of ibogaine we

first needed to understand which of its structural features were critical

for its psychoplastogenic effects. Our approach was similar to seminal

TBG is a safer, non-hallucinogenic 5-HT_2Aagonist

function-oriented synthesis studies of the structurally complex marine The structure of IBG is similar to that of the potent hallucino-

natural product bryostatin 1¹⁶. Here we report our efforts to engineer gen and serotonin 2A receptor (5-HT_2A) agonist 5-methoxy-

simplified analogues of iboga alkaloids (ibogalogs) that lack the toxic- N,N-dimethyltryptamine (5-MeO-DMT). Previous structure–activity

ity and hallucinogenic effects of ibogaine but maintain its behavioural relationship studies demonstrated that, unlike 5-MeO-DMT and other

effects in rodent models of drug self-administration and relapse.

known hallucinogens, 6-MeO-DMT did not substitute for the hallucino-

gen 2,5-dimethoxy-4-methylamphetamine at any dose in rodents that

were trained to discriminate this compound from saline¹⁹. Moreover,

our group has previously shown that 6-MeO-DMT does not produce a

Function-oriented synthesis of ibogalogs

Characteristic structural features of ibogaine include an indole, a head-twitch response²⁰—a well-established rodent behavioural proxy

7-membered tetrahydroazepine and a bicyclic isoquinuclidine (Fig. 1). for hallucinations induced by 5-HT_2Aagonists²¹. We therefore synthe-

We reasoned that systematic deletion of these key structural elements sized the 6-methoxyindole-fused tetrahydroazepine 18. Because 18

would reveal the essential features of the psychoplastogenic pharma- resembles the iboga alkaloid tabernanthine, we refer to this compound

cophore of ibogaine. By adapting previously developed chemistry¹⁷, we as tabernanthalog (TBG). Like IBG, TBG can be synthesized in a single

were able to access a series of isoquinuclidine-containing compounds step, enabling large quantities (more than 1 g) to be rapidly produced.

(8–11) that lacked the tetrahydroazepine and/or indole moieties that

To evaluate the hallucinogenic potential of IBG and TBG, we tested

are characteristic of ibogaine (Extended Data Fig. 1a). Ibogalog 8a them in the head-twitch response assay using 5-MeO-DMT (10 mg kg⁻¹

)

lacks both the indole and the tetrahydroazepine rings of ibogaine, as a positive control (Fig. 2a). Whereas 5-MeO-DMT produces a robust

whereas 9a–11a lack only the tetrahydroazepine. Ibogamine, ibogaine head-twitch response, its conformationally restricted analogue IBG

and noribogaine differ from 9a, 10a and 11a only by the presence of exhibits significantly reduced hallucinogenic potential. As hypoth-

the C2–C16 bond (using the LeMen and Taylor conventions for atom esized, the 6-methoxy substituent of TBG rendered it devoid of halluci-

numbering), respectively.

nogenic potential as measured by this assay. For these in vivo studies, we

used the fumarate salts of IBG and TBG; unlike ibogaine hydrochloride,

theyarereadilysolublein0.9%salineatconcentrationsofupto40mgml⁻¹

(Extended Data Table 1).

Psychoplastogenic pharmacophore of ibogaine

Except for 11a, ibogalogs containing the isoquinuclidine but lacking

The lipophilicity of ibogaine not only poses practical issues for its

administration, but is also likely to be a major contributory factor to its

the tetrahydroazepine ring (8–10 and 11b) were either weak psychop

-

lastogens or did not promote neuronal growth compared to the vehicle toxicity and adverse cardiac effects. Ibogaine inhibits hERG channels

control (Extended Data Fig. 2). By contrast, the majority of ibogalogs with a half-maximal inhibitory concentration (IC₅₀) of 1 μM (Fig. 2b). By

lacking the isoquinuclidine but retaining the tetrahydroazepine (12– contrast, IBG and TBG are approximately 10- and 100-fold less potent

16) were efficacious (Extended Data Fig. 2). Substitution at C5 of the than ibogaine, respectively, indicating a lower potential for cardio-

indole with either fluorine (15) or chlorine (16) was tolerated, but a toxicity. Administration of ibogaine to immobilized larval zebrafish

more sterically demanding bromine substituent (17) in this position decreased heart rate (Extended Data Fig. 3a, Supplementary Video 1)

was not. We found that ibogainalog (IBG; 13) showed comparable and increased the likelihood of inducing arrhythmias, as measured by

the ratio of atrial to ventricular beats per minute (Fig. 2c). Neither IBG

Tetrahydroazepine

nor TBG induced these undesirable phenotypes.

R¹

R¹, R²= H, H

Ibogamine

Ibogaine

Noribogaine

Tabernanthine

To compare the acute behavioural effects of ibogaine, IBG and TBG,

we treated larval zebrafish with these compounds across a range of

doses (1–200 μM), recorded their behavioural responses to a series of

light and acoustic stimuli^20,22and analysed the resulting behavioural

profiles (Extended Data Fig. 3b). Treatment with ibogaine and nori-

bogaine produced behavioural profiles similar to that of the lethal

control (Extended Data Fig. 3b, c); by contrast, treatment with IBG

and TBG produced behavioural profiles more similar to that of the

vehicle control. Zebrafish treated with increasing concentrations of

ibogaine, noribogaine and the hERG inhibitors haloperidol, sertindole

and terfenadine become more phenotypically distinct from the vehicle

control, while more closely resembling the lethal control (eugenol, 100

μM) (Extended Data Fig. 3d). By contrast, treatment with IBG and TBG

did not produce this phenotype.

R¹, R²= OMe, H

R¹, R²= OH, H

R¹, R²= H, OMe

N

R²

Indole

16

2

N

H

R¹

Tetrahydroazepine

Isoquinuclidine

R¹

Scaffold 1

Scaffold 2

R²

Me

N

X

Indole

FOS

N

H

Indole

N

H

Y

Isoquinuclidine

Scaffold 1: isoquinuclidine

9a: R¹= H, X = Et, Y = H

Scaffold 2: tetrahydroazepine

¹, R²= H, H (ibogaminalog)

N

12: R

10a: R¹= OMe, X = Et, Y = H

11a: R¹= OH, X = Et, Y = H

9b: R¹= H, X = H, Y = Et

10c: R¹= OMe, X = H, Y = Et

11d: R¹= OH, X = H, Y = Et

13: R¹, R²= OMe, H (ibogainalog)

14: R¹, R²= OH, H (noribogainalog)

15: R¹, R²= F, H

X

Y

17: R¹, R²= Br, H

8a: X = Et, Y = H

8b: X = H, Y = Et

16: R¹, R²= Cl, H

18: R¹, R²= H, OMe (tabernanthalog)

It is currently unclear whether or not ibogaine causes seizures at very

Fig. 1 | Function-oriented synthesis of ibogalogs. The key structural features

of ibogaine, related alkaloids and ibogalogs.

high doses^1,23. We therefore used larval zebrafish expressing GCaMP5

2 | Nature | www.nature.com

a

b

c

d

0 h

1 h

2.00

1.75

1.50

1.25

1.00

0.75

0.50

Veh.

IBO

35

30

25

20

15

10

5

120

100

80

60

40

20

0

IBO

IC₅₀= 1.09 μM

****

IBG

IC₅₀= 19.3 μM

*

***

NOR

IBG

TBG

IC₅₀= 148 μM

*

0

TBG

Veh.

IBO

IBG

TBG

SI

–8 –7 –6 –5 –4

log([Compound], M)

e

Veh.

NOR

IBG

TBG

f

IBG + 5-HT

TBG + 5-HT

5-HT

5-MeO-DMT

6-MeO-DMT

IBO

5-HT_2A

5-HT_2B

100

50

0

100

80

60

40

20

0

IBG

TBG

50

0

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5

2

3

4

5

1

2

3

4 5

dpf

–12 –11 –10 –9 –8 –7 –6 –5 –4 –3

Malformed

Dead

log([Compound], M)

Fig. 2 | TBG is a safer analogue of iboga alkaloids. a, Mouse head-twitch

e, Compound-induced malformations and death over time (n = 48 zebrafish for

all treatment groups). f, Activities at 5-HT_2A(left) and 5-HT_2B(right) receptors as

measured by G_q-mediated calcium flux. Data show the calcium flux response as

a percentage of the serotonin-induced maximal response. Exact n values for

each experimental condition are reported in the Source Data and in

Supplementary Table 1. *P < 0.05, ***P < 0.001, ****P < 0.0001; specific statistical

tests, information on reproducibility, and exact P values are reported in

Methods and in Supplementary Table 1.

response assays demonstrate that TBG is not hallucinogenic. The doses (mg

kg⁻¹) of IBG and TBG are indicated. The positive control (+Ctrl) is 5-MeO-DMT

(10 mg kg⁻¹). Veh., vehicle. b, Inhibition of hERG channels expressed in HEK293

cells. Data are mean s.d. IBO, ibogaine. c, Unlike ibogaine, IBG and TBG do not

increase the risk of arrhythmias in larval zebrafish. Sertindole (SI) was used as a

positive control. bpm, beats per minute. d, Representative images of zebrafish

treated with compounds (100 μM) for 2 dpf. NOR, noribogaine. Scale bar, 1 mm.

to assess the seizurogenic potential of ibogaine and TBG. Unlike the

To determine whether TBG has rewarding effects, we performed

known seizure-inducing compound pentylenetetrazole, we found a conditioned place preference assay in mice (Extended Data Fig. 6).

that treatment with ibogaine and TBG did not induce excessive neural A low dose of TBG (1 mg kg⁻¹) did not have any effect on place pref-

activity (Extended Data Fig. 3e, Supplementary Video 2).

erence (P = 0.8972, comparing pre- and post-conditioning). Higher

Finally, we compared the morphological effects of ibogaine, IBG doses produced a modest conditioned place aversion (P = 0.0199 for

and TBG using a well-established zebrafish developmental toxicity 10 mg kg⁻¹; P = 0.0489 for 50 mg kg⁻¹), which suggests that TBG has a

assay²⁴. Ibogaine (100 μM) significantly increased malformations and low potential for abuse.

mortality at 2 and 5 days post-fertilization (dpf), respectively (Fig. 2d, e).

At both time points, the proportion of viable to non-viable fish was

Effect of TBG on neural plasticity

significantly different from that in the vehicle-treated control group

(P < 0.0001). Ibogaine-treated zebrafish suffered from numerous mal- Having demonstrated the improved safety profile of TBG relative to that

formations. Treatment with noribogaine resulted in greater survival, of ibogaine, we next assessed its effects on structural plasticity. Treat

-

but the majority of zebrafish exhibited yolk sac and/or pericardial oede- ment of rat embryonic cortical neurons with TBG increased dendritic

mas. By contrast, both IBG and TBG treatment (100 μM) resulted in arbor complexity, as measured by Sholl analysis (Fig. 3a); this effect

significantly fewer non-viable fish than ibogaine treatment (P < 0.0001 seems to be dependent on the 5-HT_2Areceptor, as it was blocked by

for ibogaine versus IBG treatment and ibogaine versus TBG treatment pretreatment with the 5-HT_2Aantagonist ketanserin (Fig. 3b).

at both 2 and 5 dpf). Notably, reducing the concentration of TBG from

In addition to promoting dendritic growth, TBG also increases den-

100 to 66 μM resulted in a proportion of viable to non-viable fish that dritic spine density—to an extent comparable to that of ibogaine—in

was statistically indistinguishable from that of the vehicle control after mature (20 days in vitro, DIV20) cortical cultures (Fig. 3c, d). Next, we

5 dpf (P = 0.3864, Extended Data Fig. 3f).

used transcranial two-photon imaging (Fig. 3e) to assess the effects

To validate the targets of IBG and TBG, we performed a panel of sero- of TBG on spine dynamics in the mouse sensory cortex. An increase

tonin (5-HT) and opioid receptor functional assays to assess canonical in spine formation, with no effect on spine elimination, was observed

GPCR signalling. Unlike noribogaine, IBG and TBG showed weak or no 24 hours after treatment with either TBG or the hallucinogenic 5-HT_2A

opioid agonist activity (Extended Data Figs. 4, 5). However, IBG and TBG agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) (Fig. 3f, g). Similar

demonstrated potent agonist activity at human (Fig. 2f) and mouse results were observed in more anterior parts of the cortex, and were

(Extended Data Fig. 4) 5-HT_2Areceptors. Many 5-HT_2Aagonists—such consistent with the previously reported effects of ketamine²⁶

as 5-MeO-DMT—are also agonists of 5-HT_2Breceptors, which can lead

.

to cardiac valvulopathy²⁵. By contrast, IBG and TBG act as antagonists

Effect of TBG on forced swim test behaviour

at 5-HT_2Breceptors (Fig. 2f). When profiled across the 5-HT recepto-

rome, both IBG and TBG displayed more selective, and potentially Increased structural plasticity in the anterior parts of the brain

safer, profiles as compared to the less conformationally restricted (for example, the prefrontal cortex) have been shown to medi-

5-MeO-DMT (Extended Data Figs. 4, 5). A full safety screen across 81 ate the sustained (more than 24 h) antidepressant-like effects of

potential targets revealed that TBG exhibits high selectivity for 5-HT₂ketamine²⁷in rodent models and are suggested to have a key role

receptors (Extended Data Table 2).

in the therapeutic effects of 5-HT_2Aagonists^14,28. We therefore

Nature | www.nature.com | 3

Article

a

b

Because ketamine produces antidepressant-like effects in the

forced swim test even in the absence of unpredictable mild stress,

we performed a direct comparison of the effects of ketamine and

TBG (Fig. 4a, b). Drugs were administered 24 h after a pre-test, and the

forced swim test was then performed 24 h and 7 d after drug admin-

istration. Both ketamine and TBG significantly reduced immobil-

ity in mice 24 h after drug administration; however, the effects of

ketamine seemed to be more durable. Notably, TBG had no effect

on locomotion 24 h after administration (Extended Data Fig. 6c). As

anticipated, treatment with a 5-HT_2Aantagonist, ketanserin, blocked

the antidepressant-like effect of TBG (Fig. 4b). The efficacy exhibited

by TBG in the forced swim test is consistent with the fact that other

5-HT_2Aagonists have demonstrated potential for the treatment of

depression^13,28. However, future studies should evaluate the effects of

TBG on other behaviours that are relevant to depression, particularly

those that measure anhedonia.

8

7

6

5

7.5

****

***

**

6.5

5.5

4.5

4

Veh. Veh. IBO IBO IBG IBG TBG TBG

Veh. TBG

KETSN

–

+

–

+

–

+

–

+

c

d

20

15

10

5

**

*

**

0

Veh.

IBO

IBG

TBG

Veh. KET IBO IBG TBG

Day 0 Day 1 Day 0 Day 1

TBG reduces alcohol- and heroin-seeking behaviour

e

f Day 0 Day 1

Two-photon

imaging

To assess the effect of TBG on alcohol (ethanol) intake, we used an

intermittent-access, two-bottle choice experiment (20% ethanol (v/v)

versus water) that models binge-drinking behaviour in humans²⁹. Mice

underwent repeated cycles of binge drinking and withdrawal over the

course of 7 weeks (Fig. 4c), which resulted in high ethanol consump-

tion (11.44 0.76 g per kg per 24 h) and binge-drinking-like behaviour

(3.89 0.33 g per kg per 4 h) and generated a blood-alcohol content

equivalent to that of humans with alcohol use disorder. Systemic injec-

tions of TBG 3 hours before a drinking session reduced binge drinking

during the first 4 hours of the session without affecting water intake

(Fig. 4d). Alcohol consumption was lower for at least 2 days after TBG

administration (Fig. 4e). Similar results have been observed previously

for ibogaine³. Notably, administration of TBG before giving mice access

to both water and a 5% sucrose solution did not decrease sucrose prefer-

ence (Fig. 4f) or total fluid consumption (Fig. 4g), indicating that TBG

selectively reduced alcohol intake.

As with alcohol consumption, there are anecdotal reports to suggest

that ibogaine can reduce opioid use in humans^1,6. In rodent models

of opioid self-administration³⁰, the greatest decrease is seen when

ibogaine is administered at a dose of 40 mg kg⁻¹. Here, we used a rat

model of heroin self-administration (Fig. 4h) to assess the effects of

TBG (40 mg kg⁻¹) administered during three distinct periods: during

self-administration, before the first day of extinction, and immediately

before cued reinstatement. As a control, vehicle was administered at

these same time points whenever TBG was not (Fig. 4i); each group

therefore received a total of three injections. When administered

during self-administration (Fig. 4j, k), immediately before extinc-

tion (Fig. 4l) or before cued reinstatement (Fig. 4m), TBG acutely

reduced heroin-seeking behaviour. Although TBG did not induce any

acute locomotor deficits (Extended Data Fig. 8a), the acute effects

of TBG should be interpreted with caution. Similar to heroin, acute

TBG administration strongly reduced sucrose self-administration

at all three time points (Extended Data Fig. 8b–e), indicating that

the acute effects of TBG may be due to non-selective disruption of

operant responding.

Administer TBG

in the homecage,

then wait 24 h

Veh.

DOI

TBG

g

10

*

Two-photon

imaging

**

8

6

4

2

0

(same dendrites)

Veh. DOI TBG

Elimination

Veh. DOI TBG

Formation

Fig. 3 | TBG promotes neural plasticity. a, Maximum numbers of crossings

(N_max) of Sholl plots obtained from rat embryonic cortical neurons (DIV6).

b, The effects of TBG on dendritic growth are blocked by the 5-HT_2Aantagonist

ketanserin (KETSN). c, Representative images of secondary branches of rat

embryonic cortical neurons (DIV20) after treatment with ibogaine, IBG and

TBG for 24 h. Scale bar, 2 μm. d, TBG increases dendritic spine density on rat

embryonic cortical neurons (DIV20) after treatment for 24 h. KET, ketamine.

e, Schematic of the design of transcranial two-photon imaging experiments.

f, Representative images of the same dendritic segments from the mouse

primary sensory cortex before (day 0) and after (day 1) treatment. Blue, red and

white arrowheads represent newly formed spines, eliminated spines and

filopodia, respectively. Scale bar, 2 μm. g, 2,5-Dimethoxy-4-iodoamphetamine

(DOI) and TBG increase spine formation but have no effect on spine

elimination. Exact n values for each experimental condition are reported in the

Source Data and in Supplementary Table 1. *P < 0.05, **P < 0.01, ***P < 0.001,

****P < 0.0001; specific statistical tests, information on reproducibility, and

exact P values are reported in Methods and in Supplementary Table 1.

Notably, cue-induced relapse was reduced in the groups that were

treated with TBG long before cued reinstatement (that is, during

evaluated the effect of TBG on the behaviour of mice in a forced self-administration and before the first day of extinction; Fig. 4m). By

swim test after 7 days of unpredictable mild stress (Extended Data contrast, TBG had no effect on cued reinstatement of sucrose-seeking

Fig. 7a, b). The amount of time spent immobile was significantly behaviour when it was administered 12–14 days previously (Extended

increased after stress. This effect was rescued by a 50 mg kg⁻¹, Data Fig. 8e). Therefore, a single administration of TBG elicited

but not a 10 mg kg⁻¹, dose of TBG. Preliminary pharmacokinetic anti-addictive effects lasting up to 12–14 days. Long-lasting protec-

studies revealed that a 50 mg kg⁻¹dose of TBG produced signifi- tion against relapse after a single treatment has rarely been reported,

cantly higher concentrations in the brain than did a 10 mg kg⁻¹but is reminiscent of results in a cocaine self-administration model

dose (Extended Data Fig. 7c). Therefore, the 50 mg kg⁻¹dose was used after the direct injection of brain-derived neurotrophic factor into

in subsequent mouse behavioural experiments.

the prefrontal cortex³¹

.

4 | Nature | www.nature.com

a

b

24 h

1 week

220

200

180

160

140

220

200

180

160

140

*

****

Day 1

Day 2

Day 3

Day 4–8

Day 9

FST

pre-test

Administer

TBG or veh.

FST

24 h

Rest in

homecage

FST

1 week

120

Veh. KET TBG TBG

Veh. KET TBG

KETSN

–

+

KETSN

–

c

Establishment of binge drinking (7 weeks)

d

f

**

Veh.

NS

TBG

NS

1.5

8

6

60

40

20

1.00

NS

****

1.0

0.5

0.75

0.50

0.25

0.00

1.00

0.75

0.50

0.25

0.00

0.0

4

–0.5

–1.0

–1.5

EtOH or H₂O

24 h

H₂O Only

24–48 h

EtOH or H₂O

24 h

2

0

4 h

24 h

Veh. TBG

Testing of alcohol consumption (5 days)

e

g

20

15

10

5

150

15

*

NS

***

100

50

0

10

5

Dose then

wait 3 h

48 h

24 h

NS

Veh.

Initial binge

Day 1

Day 2

TBG

0

4 h

Veh.

24 h

TBG

1

2

5

1

2

5

1

2

5

Day 5

Days

h

j

l

Heroin taking and seeking

Extintinction training

250

400

300

200

100

0

Self-administration

Cue reinstatement

200

150

100

50

Veh.

SA

EXT

CUE

***

****

Cue

test

i

600

500

400

300

200

100

0

Active

Inactive

Active

Inactive

Veh.

SA

k

m

Veh.

SA

EXT

CUE

800

600

400

200

0

2.0

1.5

1.0

0.5

0.0

***

Extinction

EXT

CUE

*

VR15

***

VR5

FR1

****

Sessions

TBG

Active lever

Inactive lever

Veh. SA

Fig. 4 | Effects of TBG on animal behaviours relevant to depression, alcohol

use disorder and substance use disorder. a, Schematic of the design of forced

swim test (FST) experiments conducted without unpredictable mild stress.

b, The antidepressant-like effects of TBG are blocked by ketanserin. c, Timeline

of alcohol binge-drinking experiment. White and blue droplets represent 20%

ethanol (EtOH) and H₂O, respectively. d, TBG acutely reduced ethanol

consumption and preference during a binge-drinking session without

affecting H₂O intake. e, Acute TBG administration decreased ethanol

consumption for at least 48 h. f, g, TBG did not decrease sucrose preference (f)

or reduce total liquid consumption (g) in a two-bottle choice experiment.

h, Schematic of the design of the heroin self-administration experiments.

i, Heroin seeking over time. Coloured arrows indicate when each group

received TBG. Vehicle was administered at all other time points to each group.

SA, self-administration; EXT, extinction; CUE, cued reinstatement; FR1, fixed

ratio 1; VR5, variable ratio 5; VR15, variable ratio 15. j, k, TBG acutely reduced

heroin self-administration—both lever pressing ( j) and heroin intake (k). l, TBG

acutely reduced heroin-seeking when administered immediately before the

first extinction session. The cued reinstatement (injection 1, vehicle; injection

2, vehicle) and extinction (injection 1, vehicle; injection 2, TBG) groups were

compared, as they were matched for the number of withdrawal days between

the last self-administration and first extinction session. m, Acute TBG

administration completely blocked cued reinstatement (purple bar). A single

previous (12–14 d) administration of TBG during heroin self-administration or

on the first day of extinction (blue and red bars, respectively) inhibited cued

reinstatement. Exact n values for each experimental condition are reported in

the Source Data and in Supplementary Table 1. *P < 0.05, **P < 0.01, ***P < 0.001,

****P < 0.0001; specific statistical tests, information on reproducibility, and

exact P values are reported in Methods and in Supplementary Table 1.

disorders including depression, post-traumatic stress disorder and sub-

stance use disorder¹³. In principle, such psychoplastogenic medicines

Discussion

Compounds that are capable of modifying neural circuits that control could produce sustained therapeutic effects by rectifying underlying

motivation, anxiety and drug-seeking behaviour have been proposed pathological changes in circuitry rather than by masking symptoms of

to be effective treatments for a diverse range of neuropsychiatric the disorder. Psychedelic compounds could prove useful in this regard

Nature | www.nature.com | 5

Article

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because they promote structural and functional neural plasticity in

the prefrontal cortex of rodents¹⁴. Although their putative therapeutic

mechanisms of action are unknown, anecdotal reports and small clinical

trials suggest that they might produce sustained therapeutic responses

in several neuropsychiatric disorders after a single administration.

It has been suggested that psychedelic compounds and ketamine

share a common antidepressant mechanism of action that is related

to cortical neuron growth in the prefrontal cortex; however, a causal

link between psychedelic-induced neuronal growth and behaviour

has yet to be established in either humans or rodents. By contrast, a

synapse-targeting photoactivatable RAC1 has recently been used to

demonstrate that the sustained antidepressant-like effects of ketamine

in mice are mediated in part by spinogenesis in the prefrontal cortex²⁷.

Here we used the principles of function-oriented synthesis to iden-

tify the indole-fused tetrahydroazepine as the key psychoplastogenic

pharmacophore of ibogaine. This information enabled us to develop a

one-step synthesis of ibogaine analogues that are capable of promoting

structural neural plasticity both in cell culture and in vivo. Simplifica-

tion of the architecture of ibogaine to produce TBG not only enhanced

synthetic tractability but also improved physicochemical properties

and safety. Compounds lacking the isoquinuclidine moiety were sig-

nificantly less potent inhibitors of hERG channels and did not induce

bradycardia or show signs of toxicity in zebrafish. With the exception

of 18-methoxycoronaridine (18-MC), which is currently in phase II clini-

cal trials, very few ibogaine analogues have demonstrated this level

of safety while also producing therapeutic effects³². It is not known

if 18-MC is psychoplastogenic; however, unlike ibogaine, it does not

increase the expression of GDNF in SH-SY5Y cells or reduce alcohol

self-administration after direct infusion into the ventral tegmental area,

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which suggests that their mechanisms of action are probably distinct³³

.

In addition, whereas the synthesis of 18-MC requires 13 steps³⁴, TBG can

be synthesized in a single step.

Not only does TBG potently promote neuronal growth, it also pro-

duces antidepressant-like behavioural responses and reduces alcohol—

but not sucrose—consumption in mice. In rats, acute administration

of TBG strongly inhibits both heroin- and sucrose-seeking behaviour,

probably by disrupting operant responding. However, when admin-

istered days in advance, TBG prevents only cued reinstatement of

heroin-seeking behaviour and not sucrose-seeking behaviour. Future

work is required to further characterize the optimal dosing regimen

and time course to produce these behavioural effects in rodents, and

to determine whether structural plasticity has a causal role.

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Online content

Any methods, additional references, Nature Research reporting sum-

maries, source data, extended data, supplementary information,

acknowledgements, peer review information; details of author con-

tributions and competing interests; and statements of data and code

availability are available at https://doi.org/10.1038/s41586-020-3008-z.

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6 | Nature | www.nature.com

logP and total polar surface area were predicted using Molinspira-

tion (https://www.molinspiration.com/). logD (the logarithm of the

distribution coefficient) was calculated using the following equation

Methods

Data analysis and statistics

Treatments were randomized, and data were analysed by experimenters logD = logP − log₁₀(1 + 10^{(pKa − 7.4)}).

blinded to treatment conditions. Statistical analyses were performed

using GraphPad Prism (v.8.1.2) unless noted otherwise. All comparisons

Dendritogenesis experiments

were planned before performing each experiment. Statistical methods For the dendritogenesis experiments conducted using cultured cor-

were not used to predetermine sample sizes, with the exception that tical neurons, timed pregnant Sprague Dawley rats were obtained

power analyses were conducted to estimate animal numbers for in vivo from Charles River Laboratories. Full culturing, staining and analysis

experiments. Data are represented as mean s.e.m., unless otherwise experiments were performed as previously described²⁰. Dendrites

noted, with asterisks indicating *P < 0.05, **P < 0.01, ***P < 0.001 and were visualized using a chicken anti-MAP2 antibody (1:10,000; EnCor,

****P < 0.0001. Box plots depict the three quartile values of the distri- CPCA-MAP2) as previously reported²⁰

.

bution with whiskers extending to points that lie within 1.5 IQRs (inter-

quartile range) of the lower and upper quartile. Observations falling

Head-twitch response

outside this range are displayed independently. For Figs. 2a, 3a, b, d, g, The head-twitch response assay was performed as described previ-

4b, Extended Data Fig. 2b and Extended Data Fig. 7b, compound treat- ously²⁰using both male and female C57BL/6J mice (2 each per treat-

ments were compared to the vehicle control using a one-way ANOVA ment). The mice were obtained from Jackson Laboratory and were

with Dunnett’s post hoc test. For Extended Data Fig. 3a and Fig. 2c, time approximately 8 weeks old at the time of the experiments. Compounds

0 h and time 1 h (before and after drug administration, respectively) data were administered via intraperitoneal (i.p.) injection (5 ml kg⁻¹) using

were compared using a paired t-test. For Fig. 2e and Extended Data Fig. 3f, 0.9% saline as the vehicle. As a positive control, we used 5-MeO-DMT

compound treatments were compared using Fisher’s exact test and fumarate (2:1 amine/acid), which was synthesized as described pre-

P values are indicated in the text. For Fig. 4d and Extended Data Fig. 6b, viously²⁰. Behaviour was videotaped, later scored by two blinded

data were analysed using a two-tailed paired t-test. Data in Fig. 4e–g observers, and the results were averaged (Pearson correlation coef-

and Extended Data Fig. 8a (distance and velocity) were analysed using a ficient = 0.93).

two-way ANOVA with Sidak’s post hoc test. For Fig. 4j, l–m and Extended

Data Fig. 8c–e, two-way repeated measures (RM) ANOVAs with treatment

hERG inhibition studies

group as the between-subject factor and lever type (active versus inac- All experiments were conducted manually using a HEKA EPC-10 ampli-

tive) as the within-subject factor were used. Sidak post hoc tests were fier at room temperature in the whole-cell mode of the patch-clamp

conducted as appropriate. For Extended Data Fig. 6c, a one-way ANOVA technique. HEK293 cells stably expressing hKv11.1 (hERG) under G418

with Tukey’s post hoc test was used. For Fig. 4k and Extended Data Fig. 8a selection were a gift from C. January (University of Wisconsin, Madison).

(thigmotaxis) two-tailed unpaired t-tests were used.

Cells were cultured in DMEM containing 10% fetal bovine serum, 2 mM

glutamine, 1 mM sodium pyruvate, 100 U ml⁻¹penicillin, 100 μg ml⁻¹

streptomycin, and 500 mg ml⁻¹G418. The cell line was not authenticated

Drugs

The NIDA Drug Supply Program provided ibogaine hydrochloride, nori- or tested for mycoplasma contamination. Before experiments, cells

bogaine, heroin (diamorphine hydrochloride) and cocaine hydrochlo- were grown to 60–80% confluency, lifted using TrypLE, and plated onto

ride. Other chemicals were purchased from commercial sources as poly-L-lysine-coated coverslips. Patch pipettes were pulled from soda

follows:ketaminehydrochloride(Fagron),ketanserin(ApexBio),eugenol lime glass (micro-haematocrit tubes) and had resistances of 2–4 MΩ.

(Tokyo Chemical Industries) and 5-hydroxytryptamine (Sigma-Aldrich). For the external solution, normal sodium Ringer was used (160 mM

The fumarate salt of 5-methoxy-N,N-dimethyltryptamine (2:1, NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4 and

5-MeO-DMT:fumaric acid) was synthesized in-house as described 290–310 mOsm). The internal solution used was potassium fluoride

previously²⁰and judged to be analytically pure on the basis of nuclear with ATP (160 mM KF, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, 4 mM

magnetic resonance (NMR) and liquid chromatography–mass spec- NaATP, pH = 7.2 and 300–320 mOsm). A two-step pulse (applied every

trometry (LC–MS) data. For cell culture experiments, the vehicle was 10 s) from −80 mV initially to 40 mV for 2 s and then to −60 mV for 4 s,

a 0.1% (agonist studies) or 0.2% (antagonist studies) molecular biology was used to elicit hERG currents. The percentage reduction of tail cur-

grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich) solution. For in vivo rent amplitude by the drugs was determined and data are shown as

experiments, the vehicle was USP grade saline (0.9%). Free bases were mean s.d. (n = 3–4 per data point). For all experiments, solutions of

used for all cellular experiments and the fumarate salts of ibogainalog the drugs were prepared fresh from 10 mM stocks in DMSO. The final

and tabernanthalog were used for the in vivo studies.

DMSO concentration never exceeded 1%.

Animals

Larval zebrafish heart rate experiments

All experimental procedures involving animals were approved by either Zebrafish express Zerg, an orthologue of hERG, and many hERG inhibi-

the University of California, Davis (UCD), the University of California, tors induce bradycardia and arrhythmia in zebrafish³⁵. Heart rate was

San Francisco (UCSF), the University of California, Santa Cruz (UCSC), recorded and calculated as reported previously³⁶with slight modifica-

or the University of Colorado Denver, Anschutz Medical Campus (CU tions (n = 3–9). In brief, 7 dpf zebrafish larvae were anaesthetized with

Anschutz) Institutional Animal Care and Use Committee (IACUC) and tricaine (Acros Organics) and immobilized in a lateral orientation using

adhered to principles described in the National Institutes of Health 1% low melt agarose (LMA, Gene Mate) dissolved in egg water³⁷. Tricaine

Guide for the Care and Use of Laboratory Animals. Power analyses was washed out and the drug was added to 4 ml embryo media in a 6-well

were conducted to ensure appropriate sample size for all experiments plate (final concentration 50 μM). Videos were collected at 30 frames

involving animals. UCD, UCSF, UCSC and CU Anschutz are accredited per second (fps) using a Leica M80 scope with an ACHRO 1× nosepiece

by the Association for Assessment and Accreditation of Laboratory attachment and a Leica IC80 HD camera. Regions of interest (ROIs)

Animal Care International (AAALAC).

were drawn around the atrium and ventricle of individual zebrafish

and average pixel dynamics were calculated using the ImageJ plugin

Time Series Analyzer V3. This pixel change oscillation was graphically

smoothed using the Savgol filter in SciPy. Peaks were detected using

Calculation of CNS MPO score

CNS MPO scores were calculated using a previously published method¹⁸

.

Predicted pK_avalues were determined using Marvin Sketch (19.25.0). the SciPy package “find_peaks”. Peak time interval and heart rate (bpm)

Article

were calculated using custom code. The arrythmia score was calculated supplemented with 0.1% bovine serum albumin and 0.01% ascorbic

as the ratio of atrial bpm to ventricular bpm (n = 6–18).

acid) and dispensed into 384-well assay plates using a FLIPR Tetra

automated dispenser head (Molecular Devices). Every plate included

a positive control such as 5-HT (for all 5-HT receptors), DADLE (DOR),

Larval zebrafish behavioural experiments

Behavioural data were collected and analysed as described previ- salvinorin A (KOR), and DAMGO (MOR). For measurements of 5-HT_2A

,

ously^20,22, with slight modifications. Locomotion of 7 dpf zebrafish was 5-HT_2B, and 5-HT_2CG_q-mediated calcium flux function, HEK Flp-In 293

recorded at 100 fps under a 17-min series of acoustic and light-based T-Rex stable cell lines (Invitrogen) were loaded with Fluo-4 dye for

stimuli 1 h after treatment. Data were collected in a concentration– one hour, stimulated with compounds and read for baseline (0–10 s)

response format on eight 96-well plates with 8 zebrafish per well. Each and peak fold-over-basal fluorescence (5 min) at 25ꢀ°C on the FLIPR

plate contained all 10 compounds at 8 concentrations plus 8 DMSO Tetra system. For measurement of 5-HT₆and 5-HT_7afunctional assays,

(vehicle) and 8 eugenol (lethal) control wells. Treatments and well G_s-mediated cAMP accumulation was detected using the split-luciferase

positions were scrambled according to two randomized plate lay- GloSensor assay in HEKT cells measuring luminescence on a Microbeta

outs. Motion in Extended Data Fig. 3a was smoothed using the mean Trilux (Perkin Elmer) with a 15 min drug incubation at 25ꢀ°C. For 5-HT_1A

,

over a 10-frame sliding window. Classifiers were trained as previously 5-HT_1B, 5-HT_1F, MOR, KOR and DOR functional assays, G_i/o-mediated

described²². For Extended Data Fig. 3d, 6 repeat vs-solvent and 6 repeat cAMP inhibition was measured using the split-luciferase GloSensor

vs-lethal classifiers (3,000 trees) were trained per comparison, each to assay in HEKT cells, conducted similarly to that above, but in combina

-

classify 8 treatment wells and 8 randomly subsampled control wells. tion with either 0.3 μM isoproterenol (5-HT_1A, 5-HT_1B, 5-HT_1F) or 1 μM

Responses were calculated from the out-of-bag accuracy values as forskolin (MOR, KOR and DOR) to stimulate endogenous cAMP accu-

r = (a − 0.4)/(1 − 0.4). 95% confidence intervals were calculated over mulation. For measurement of 5-HT_1D, 5-HT_1E, 5-HT₄, and 5-HT_5Afunc-

1,000 bootstrap samples per comparison. For Extended Data Fig. 3b, tional assays, β-arrestin2 recruitment was measured by the Tango assay

a single classifier (10,000 trees) was trained on all data for the treat- using HTLA cells expressing tobacco etch virus (TEV) fused-β-arrestin2,

ments shown, and the out-of-bag predictions were used.

as described previously³⁹with minor modifications. Cell lines were

not authenticated, but they were purchased mycoplasma-free and

tested for mycoplasma contamination. Data for all assays were plot-

Larval zebrafish seizure experiments

At 6 dpf, transgenic zebrafish larvae (Tg(elavl3:GCaMP5G)a4598)³⁸ted and nonlinear regression was performed using “log(agonist) vs.

were anaesthetized with tricaine and immobilized in a dorsal orienta- response” in GraphPad Prism to yield estimates of the efficacy (E_max

tion using 1% LMA dissolved in egg water. Tricaine was washed out and and half-maximal effective concentration (EC₅₀).

zebrafish were treated for 1 h with compounds (50 μM for IBO and TBG;

)

Safety pharmacology profiling panel

15 mM for pentylenetetrazole (PTZ)). Videos were acquired using a

Zeiss Axiozoom.V16, and GCaMP5G fluorescence was induced using a TBG (10 μM) was screened by Eurofins Discovery against their Safe-

Lumencor sola light engine. Zen software V2 blue edition controlled tyScreen87 Panel and in their VMAT (non-selective) human vesicular

an Axiocam 506 mono camera set to 33 fps. Short videos (1–3 min) monoamine transporter binding assay.

were acquired per condition. Change in fluorescence intensity (F) was

Conditioned place preference assay

calculated using ImageJ from an ROI drawn in the cerebellar region, and

∆F/F was calculated and visualized using custom functions.

The conditioned place preference apparatus consisted of two cham-

bers (18 cm L × 20 cm W × 35 cm H) connected by a corridor (10 cm L ×

20 cm W × 35 cm H). One chamber had a smooth floor and black walls

Larval zebrafish toxicity

Tropical 5D wild-type larval zebrafish were obtained from the Sinnhuber whereas the second chamber had a mesh floor and patterned walls. A

Aquatic Research Laboratory (SARL) at Oregon State University, and block was placed in the corridor to restrict mice to a particular cham-

subsequent generations were raised at UCD. Zebrafish husbandry, ber. On day 1 (pre-conditioning), male C57/BL6J mice (9–10 weeks old)

spawning, dechorionation of embryos and exposures were performed were allowed to explore the entire apparatus for 30 min. Mice were

as described previously²⁴. Chemical stocks were prepared at 100 mM randomly sorted into treatment groups (TBG at 50 mg kg⁻¹, 10 mg kg⁻¹

in DMSO and diluted to 200 μM with embryo media. This solution and 1 mg kg⁻¹), ensuring that their initial preferences for what would

was diluted twofold into individual wells of 96-well plates housing become the TBG-paired side were approximately equal. Next, the mice

larval zebrafish. The final compound and DMSO concentrations were were administered an i.p. injection of either vehicle (saline) or TBG

100 μM and 0.1% (v/v), respectively. Wells were covered with Parafilm (counterbalanced) immediately before being confined to one of the

M (Bemis) then covered with the plate lid. Plates were maintained in two chambers for 30 min. The following day, the other treatment was

an incubator at 28.5ꢀ°C with a 14 h light (~300 lx)/10 h dark cycle. Fish administered, and the mice were confined to the opposite chamber for

were statically exposed to compounds 6 hours post-fertilization (hpf) 30 min. This sequence was repeated twice, such that all mice received 3

through 5 dpf. All compounds were tested for mortality or teratology vehicle-side pairings and 3 TBG-side pairings. The mice were returned

in triplicate experiments (three experiments conducted on independ- to their homecages in between treatment-side pairings. On day 8 (post

ent days using fish from independent spawns). For each experiment, conditioning), the mice were allowed to explore the entire apparatus

16 fish were tested per concentration per compound (n = 48 fish per for 30 min, and the time spent on the vehicle- and TBG-paired sides

condition). At 1, 2, 3, 4, and 5 dpf, fish were examined for mortality and was quantified using ANYmaze software (v.6.2). The apparatus was

developmental malformations using a Leica Stereo Microscope Model cleaned with 70% ethanol between trials. Drug solutions were prepared

S6D (Leica) up to 4.5× magnification.

fresh daily.

Serotonin and opioid receptor functional assays

Pharmacokinetic studies

Functional assay screens at 5-HT and opioid receptors were performed Male and female C57/BL6J mice (12 weeks old) were administered TBG

in parallel using the same compound dilutions and 384-well-format via i.p. injection at doses of either 50 mg kg⁻¹, 10 mg kg⁻¹or 1 mg kg⁻¹

.

high-throughput assay platforms. Assays assessed activity at all human Mice were euthanized 15 min or 3 h after injection by cervical disloca-

isoforms of the receptors, except where noted for the mouse 5-HT_2Ation. Two males and two females were used per dose and time point.

receptor. Receptor constructs in pcDNA vectors were generated from Brain and liver were collected, flash-frozen in liquid nitrogen, and stored

the Presto-Tango GPCR library³⁹with minor modifications. All com- at −80ꢀ°C until metabolomic processing. Metabolites were extracted

pounds were serially diluted in drug buffer (HBSS, 20 mM HEPES, pH 7.4 from tissue as described previously⁴⁰. In brief, whole brain and liver

sections were lyophilized overnight to complete dryness, then homog- stressors were used: day 1: light phase = 30 min of restraint stress × 2;

enized with 3.2-mm diameter stainless-steel beads using a GenoGrinder dark phase = homecage space reduction. Day 2: light phase = expo-

for 50 s at 1,500 rpm. Ground tissue was then extracted using 225 μl cold sure to a new room + 30 min on the orbital shaker, sudden loud noise

methanol, 190 μl water, 750 μl methyl tert-butyl ether (MTBE). Seven × 5, tail suspension for 6 min; dark phase = wet bedding. Day 3: light

method blanks and seven quality-control samples (pooled human phase = exposure to new mice; dark phase = exposure to light. Day

serum, BioIVT) were extracted at the same time as the samples. The non- 4: light phase = social isolation; dark phase = tilted cage. Day 5: light

polar fraction of MTBE was dried under vacuum and reconstituted in phase = tilted cage, island isolation; dark phase = no bedding. Day 6:

60 μl of 90:10 (v/v) methanol: toluene containing 1-cyclohexyl- light phase = no bedding, random puff of air × 5–10; dark phase = for-

dodecanoic acid urea as an internal standard. Samples were then vor- eign objects. Day 7: light phase = foreign objects, food deprivation;

texed, sonicated and centrifuged before analysis. For analysis of TBG in dark phase = food deprivation, continual exposure to loud music.

liver and brain, samples were randomized before injection with method Immediately following UMS, TBG or vehicle were administered via

blanks and quality-control samples were analysed between every ten i.p. injection, and 24 h later the mice were subjected to a FST using the

study samples. A six-point calibration curve was analysed after column same procedure as described below.

equilibration using blank injections, and then after all study samples.

Blanks were injected after the calibration curve to ensure no that no

Forced swim test in the absence of UMS

TBG was retained on the column and carried over to samples. Recon- Male C57/BL6J mice (9–10 weeks old at the time of the experiment) were

stituted sample (5 μl) was injected onto a Waters Acquity UPLC CSH obtained from the Jackson Laboratory and housed 4–5 mice per cage

C18 column (100 mm × 2.1 mm, 1.7 μm particle size) with an Acquity in a vivarium at UCD following an IACUC-approved protocol. After 1

UPLC CSH C18 VanGuard precolumn (Waters) using a Vanquish UHPLC week in the vivarium, each mouse was handled for approximately 1 min

coupled to a TSQ Altis triple quadrupole mass spectrometer (Thermo by a male experimenter for 3 consecutive days leading up to the first

Fisher Scientific). Mobile phase A consisted of 60:40 v/v acetonitrile/ FST. All experiments were carried out by the same male experimenter

water with 10 mM ammonium formate and 0.1% formic acid. Mobile who performed handling. During the FST, mice underwent a 6-min

phase B was 90:10 v/v isopropanol/acetonitrile with 10 mM ammonium swim session in a clear Plexiglas cylinder 40 cm tall, 20 cm in diameter,

formate and 0.1% formic acid. Gradients were run from 0–2 min at 15% and filled with 30 cm of 24 1ꢀ°C water. Fresh water was used for each

B; 2–2.5 min 30% B; 2.5–4.5 min 48% B; 4.5–7.3 min 99% B; 7.3–10 min mouse. After handling and habituation to the experimenter, drug-naive

15% B. The flow rate was 0.600 ml min⁻¹and the column was heated to mice first underwent a pre-test swim to more reliably induce a depres-

65ꢀ°C. Mass spectrometer conditions were optimized for TBG by direct sive phenotype in the subsequent FST sessions. Immobility scores for

infusion. Selected reaction monitoring was performed for the top five all mice were determined after the pre-test and mice were randomly

ions, with collision energy, source fragmentation, and radiofrequency assigned to treatment groups to generate groups with similar average

optimized for TBG. Data were processed with TraceFinder 4.1 (Thermo immobility scores to be used for the following two FST sessions. The

Fisher Scientific). Organ weights were recorded. The concentration in next day, the mice received i.p. injections of TBG (50 mg kg⁻¹), a positive

the brain was calculated using the experimentally determined number control (ketamine, 3 mg kg⁻¹), or vehicle (saline). One additional group

of moles of TBG in the whole organ divided by the weight of the organ. received ketanserin (4 mg kg⁻¹i.p.) 10 min before i.p. administration

of TBG (50 mg kg⁻¹). The following day, the mice were subjected to the

Spinogenesis experiments

FST and then returned to their homecages. One week later, the FST was

Spinogenesis experiments were performed as previously described¹⁴, performed again to assess the sustained effects of the drugs. All FSTs

with the exception that cells were treated on DIV19 and fixed 24 h after were performed between the hours of 8:00 and 13:00. Experiments

treatment on DIV20. The images were taken on a Nikon HCA Confocal were video-recorded and manually scored offline. Immobility time—

microscope a with a 100×/NA 1.45 oil objective. DMSO and ketamine defined as passive floating or remaining motionless with no activity

(10 μM) were used as vehicle and positive controls, respectively.

other than that needed to keep the head above water—was scored for

the last 4 min of the 6 min trial.

In vivo spine dynamics

Male and female Thy1-GFP-M line mice⁴¹(n = 5 per condition) were pur-

Alcohol consumption

chased from The Jackson Laboratory ( JAX 007788) and maintained in Male C57/BL6J mice (6–8 weeks old) were obtained from The Jack-

the animal facilities at UCSC according to an IACUC-approved protocol. son Laboratory and were individually housed in a reverse light/dark

In vivo transcranial two-photon imaging and data analysis were per- cycle room (lights on 22:00–10:00). Temperature was kept constant

formed as previously described⁴². In brief, mice were anaesthetized at 22 2ꢀ°C, and relative humidity was maintained at 50 5%. Mice

with an i.p. injection of a mixture of ketamine (87 mg kg⁻¹) and xylazine were given access to food and tap water ad libitum. After one week of

(8.7 mg kg⁻¹). A small region of the exposed skull was manually thinned habituation to the vivarium, the two-bottle-choice alcohol-drinking

down to 20–30 μm for optical access. Spines on apical dendrites in experiment was conducted as described previously²⁹. For 7 weeks,

mouse primary sensory cortices were imaged using a Bruker Ultima IV mice were given intermittent access to alcohol in their homecage. On

two-photon microscope equipped with an Olympus water-immersion Mondays, Wednesdays and Fridays, two bottles were made available

objective (40×, NA = 0.8) and a Ti:sapphire laser (Spectra-Physics Mai for 24 h: one containing 20% ethanol and another containing only

Tai, excitation wavelength 920 nm). Images were taken at a zoom of 4.0 water. On Tuesdays, Thursdays, Saturdays and Sundays, the mice were

(pixel size 0.143 × 0.143 μm) and Z-step size of 0.7 μm. The mice received given access only to water. After 7 weeks, mice were administered TBG

an i.p. injection of DOI (10 mg kg⁻¹) or TBG (50 mg kg⁻¹) immediately after (50 mg kg⁻¹) or vehicle (saline) via i.p. injection 3 h before the begin-

they recovered from the anaesthesia of the first imaging session. The ning of a drinking session. Ethanol (g kg⁻¹) and water (ml kg⁻¹) intake

mice were re-imaged 24 h after drug administration. Dendritic spine were monitored during the first 4 h (initial binge), the first 24 h and

dynamics were analysed using ImageJ. Spine formation and elimination the second 24 h. Next, the mice were given only water for 48 h before

were quantified as percentages of spine numbers on day 0.

the start of another drinking session, during which ethanol and water

consumption were monitored. The placement (right or left) of the bot-

tles was altered in each session to control for side preference. Spillage

was monitored using an additional bottle in a nearby unused cage.

Antidepressant-like response following unpredictable mild

stress

Male and female mice (8 weeks old) were subjected to 7 d of unpredict- Alcohol preference was calculated as alcohol/(water + alcohol). Mice

able mild stress (UMS), as described previously⁴³. In brief, the following were tested using a counterbalanced, within-subject design with one

Article

week of drug-free alcohol drinking regimen between treatments. One consisted of vehicle, SA (2.5), SA (10), SA (40), EXT (40) and CUE (40).

mouse was excluded because a bottle from which it was drinking was The number of rats in each group was 7, 6, 7, 7, 7 and 6, respectively (40

found to be leaking.

rats total). At least one week after arrival and acclimatization to the ani-

mal facility, sucrose self-administration training began on a fixed ratio

1 (FR1) schedule of reinforcement. Operant chambers were equipped

Sucrose preference

Male C57/BL6J mice were individually housed and subjected to a with an active (sucrose-delivering) and inactive lever, and each sucrose

two-bottle choice experiment. First, mice were administered TBG reward (45 mg pellet; Bio-Serv F0023) was coupled with the same tone

(50 mg kg⁻¹) or vehicle (saline) via i.p. injection 3 h before the beginning + light cues used for the heroin study. Levers retracted upon pellet

of a two-bottle choice session. During this 3-h period, mice were not delivery and remained retracted during cue presentation (5 s). After six

given access to water in an attempt to increase their thirst. At the start self-administration sessions (2 h) on FR1, rats progressed to a variable

of the experiment, mice were given one bottle of water and one bottle ratio 5 (VR5) for three sessions and continued to the final variable ratio

of water containing 5% sucrose. Sucrose solution and water intake were 15 (VR15) for five sessions. Rats then began extinction training. Extinc-

monitored during the first 4 h and the first 24 h. Sucrose preference tion training sessions (1 h) were conducted in the same operant cham-

was calculated as the amount of sucrose solution consumed minus bers (context) in which animals previously self-administered sucrose,

the amount of water consumed, divided by the total amount of liquid but neither sucrose nor the sucrose cues were available. Responding

consumed.

on both levers was recorded during each session, but produced no

consequence. After completing 7 extinction sessions, rats underwent

a cued reinstatement test (1 h). During the cue test, the sucrose tone

Heroin self-administration behaviour

Subjects were age-matched male (n = 16) and female (n = 16) Wistar + light cues were available but sucrose was not. The first active lever

rats (Charles River). Rats were single-housed in a temperature- and press resulted in presentation of the sucrose cues, and then cues were

humidity-controlled room with a 12 h light/dark cycle (7:00 lights available on a VR5 schedule (active lever only) for the remainder of the

on) with free access to standard laboratory chow and water. Two rats test. Lever retraction occurred during cue presentation (as during

(one male and one female) were excluded from the final dataset owing self-administration). Injections were administered on the third VR15

to defective catheters to give a final n = 30 rats. Rats were surgically session, the first extinction session, and the cued reinstatement test.

implanted with an intravenous catheter as previously described⁴⁴. For each of these tests, TBG (2.5 mg kg⁻¹, 10 mg kg⁻¹, or 40 mg kg⁻¹i.p.)

Heroin self-administration training began at least one week after or vehicle was injected 30 min before placement in the chamber. The

surgery on a fixed ratio 1 (FR1) schedule of reinforcement. Operant low (2.5 mg kg⁻¹) and intermediate (10 mg kg⁻¹) doses of TBG were tested

chambers were equipped with both an active (heroin-delivering) and only on the third VR15 sucrose self-administration session. The high

an inactive lever, and each heroin infusion (0.04 mg, 50 μl, 2.85 s) was dose (40 mg kg⁻¹) was tested at all three test time points. Statistical

coupled with delivery of a light cue located above the active lever and tests were performed in Prism (GraphPad Prism, RRID:SCR_002798;

a 3.5 kHz tone (5 s). Both levers retracted upon initiation of a heroin V8.0) software.

infusion and remained retracted during the tone + light heroin cue

presentation. After six self-administration sessions (2.5 h) on FR1, rats

Open-field test

progressed to a variable ratio 5 (VR5) for three sessions and continued Naive male (n = 7) and female (n = 6) Wistar rats (Charles River) were

to the final variable ratio 15 (VR15) for five sessions. Rats then began allowed to acclimatize to the animal facility for at least one week after

extinction training. Extinction training sessions (1 h) were conducted arrival. Spontaneous locomotion in response to a novel open field

in the same operant chambers (context) in which rats previously (44 cm long × 36 cm wide × 43 cm tall) was assessed 30 min after injec-

self-administered heroin, but in the absence of heroin and its tone + tion of vehicle or TBG (40 mg kg⁻¹, i.p.). Videos were recorded with an

light cue. Both levers remained extended throughout the session, and overhead camera connected to the tracking software EthoVision XT

responding was recorded but produced no consequence. After com- (Noldus) for subsequent offline analysis. Rats were allowed to move

pleting a total of 7 extinction sessions, rats underwent a cued reinstate- freely in the open field for 30 min, then they were briefly removed from

ment test (1 h, withdrawal day 10–12). During the cue test, the heroin the apparatus to receive an injection of cocaine (15 mg kg⁻¹, i.p.). The

tone + light cues were available but heroin was not. The first active rats were immediately returned to the open field for an additional

lever press resulted in presentation of the heroin cues, and then cues hour to assess cocaine-induced locomotion. Each open-field cham-

were available on a VR5 schedule (active lever only) for the remainder ber was cleaned with Clidox-S in between sessions. Locomotion was

of the test. Lever retraction occurred during cue presentation (as dur- tracked using EthoVision XT to assess the velocity (cm s⁻¹) and total dis-

ing self-administration). Injections of TBG (40 mg kg⁻¹i.p.) or vehicle tance travelled (m) during the baseline (first 30 min) and cocaine (last

were administered on the third VR15 session, the first extinction ses

-

60 min) phases separately. Thigmotaxis—assessed as the percentage

sion and the cued reinstatement test. For each of these time points, of time spent in the centre of the apparatus (26 cm long × 18 cm wide;

TBG or vehicle was injected 30 min before placement in the chamber. that is, 9-cm perimeters)—was also analysed during the baseline period

Treatment groups were balanced on the basis of response rates, heroin to determine whether TBG alters anxiety in the open field.

intake and sex. Behavioural sessions were conducted daily (on weekdays

Reporting summary

only). Catheters were flushed after each self-administration session

with cefazolin and taurolidine citrate solution to prevent infection Further information on research design is available in the Nature

and/or catheter occlusion. Statistical tests were performed in Prism Research Reporting Summary linked to this paper.

(GraphPad Prism, RRID:SCR_002798; V8.0) software.

Data availability

Sucrose self-administration behaviour

Sucrose self-administration procedures were designed to mimic Data are available at https://doi.org/10.6084/m9.figshare.11634795.

heroin self-administration conditions. Subjects were age-matched Source data are provided with this paper.

male (n = 24) and female (n = 24) Wistar rats (Charles River). Rats were

single-housed and had free access to standard laboratory chow and

water throughout the experiment. Eight rats (seven males and one

Code availability

female) were excluded from the final dataset due to failure to acquire Custom-written data analysis codes are available upon request from

sucrose self-administration to give a final n = 40 rats. The final groups the corresponding author.

TR001860 and linked award TL1 TR001861. Delix Therapeutics funded the large receptor

screen conducted at Eurofins Discovery. We thank F. F. Wagner for help in coordinating with

Eurofins Discovery. This project used the Biological Analysis Core of the UC Davis MIND

Institute Intellectual and Development Disabilities Research Center (U54 HD079125). The

Olympus FV1000 confocal microscope used in this study was purchased using NIH Shared

Instrumentation Grant 1S10RR019266-01. We thank the MCB Light Microscopy Imaging Facility,

which is a UC Davis Campus Core Research Facility, for the use of this microscope. Several of

the drugs used in this study were provided by the NIDA Drug Supply Program. We thank D. R.

Carty for assistance with larval zebrafish toxicity assays.

35. Langheinrich, U., Vacun, G. & Wagner, T. Zebrafish embryos express an orthologue of

HERG and are sensitive toward a range of QT-prolonging drugs inducing severe

arrhythmia. Toxicol. Appl. Pharmacol. 193, 370–382 (2003).

36. Sampurna, B. P., Audira, G., Juniardi, S., Lai, Y.-H. & Hsiao, C.-D. A simple ImageJ-based

method to measure cardiac rhythm in zebrafish embryos. Inventions 3, 21 (2018).

37. Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)

5th edn (Univ. Oregon Press, 2007).

38. Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional

imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420

(2013).

39. Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the

druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).

40. Barupal, D. K. et al. A comprehensive plasma metabolomics dataset for a cohort of mouse

knockouts within the international mouse phenotyping consortium. Metabolites 9, 101

(2019).

41. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral

variants of GFP. Neuron 28, 41–51 (2000).

42. Xu, T. et al. Rapid formation and selective stabilization of synapses for enduring motor

memories. Nature 462, 915–919 (2009).

43. Chen, C. C., Lu, J., Yang, R., Ding, J. B. & Zuo, Y. Selective activation of parvalbumin

interneurons prevents stress-induced synapse loss and perceptual defects. Mol.

Psychiatry 23, 1614–1625 (2018).

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freely cycling female rats regulates distinct aspects of heroin seeking. Learn. Mem. 27,

6–11 (2020).

Author contributions A.J.P., Z.Q.H. and G.Z. synthesized the ibogalogs. L.E.D. synthesized

5-MeO-DMT fumarate and performed the CNS MPO calculations. L.P.C. performed the

dendritogenesis and spinogenesis assays. L.P.C. and J.V. performed the head-twitch response

experiments. M.N.M. performed the zebrafish heart-rate and seizure experiments. J.C.T., D.M.-T.

and R.J.T. performed the zebrafish behavioural experiments. R.J.T. and B.Y. performed the

zebrafish toxicity assays. B.M.B. and L.P.C. performed the hERG inhibition studies. L.P.C.

performed the solubility studies and conditioned place preference experiments. J.L., T.L. and

L.P.C. performed the experiments assessing in vivo spine dynamics. L.J.L., E.I.A. and J.D.M.

performed the receptor functional assays. J.L. and M.T. performed the forced swim test

following UMS. M.V.V. performed the forced swim test study without UMS with assistance from

L.E.D. Z.T.R. and L.P.C. performed the pharmacokinetic studies. J.P. performed the heroin

self-administration experiments. Y.E. performed the alcohol consumption assays. L.P.C.

performed the sucrose preference assay. O.F., H.W., J.D.M., P.J.L., D.K., D.R., J.P., Y.Z. and D.E.O.

supervised various aspects of this project and assisted with data analysis. D.E.O. conceived the

project and wrote the manuscript with input from all authors.

Competing interests D.E.O. is the president and chief scientific officer of Delix Therapeutics.

Delix Therapeutics has licensed TBG-related technology from the University of California,

Davis.

Acknowledgements This work was supported by funds from the National Institutes of Health

(NIH) (R01GM128997 to D.E.O.; R37AA01684 to D.R.; R01AA022583 to D.K.; R01MH109475,

R01MH104227 and R01NS104950 to Y.Z.; R01DA045836 to J.P.; and U19AG023122 to O.F.),

a Hellman Fellowship (D.E.O.), UC Davis STAIR and STAIR Plus grants (D.E.O.), a Max Planck

Fellowship at MPFI (Y.Z.), four NIH training grants (T32GM113770 to R.J.T., T32MH112507 to

L.P.C., 5T32GM099608 to M.V.V., and 4T32GM6754714 to D.M.-T.), two UC Davis Provost’s

Undergraduate Fellowships (to J.V. and A.J.P.), the Paul G. Allen Family Foundation (M.N.M. and

D.K.), the Genentech Fellowship Program (D.M.-T.), and a Medical College of Wisconsin

Research Affairs Counsel Pilot Grant (J.D.M.). B.M.B. was supported by the National Center for

Advancing Translational Sciences, National Institutes of Health, through grant number UL1

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-

3008-z.

Correspondence and requests for materials should be addressed to D.E.O.

Peer review information Nature thanks Amy Newman, Yavin Shaham and the other,

anonymous, reviewer(s) for their contribution to the peer review of this work.

Reprints and permissions information is available at http://www.nature.com/reprints.

Article

Extended Data Fig. 1 | Synthesis of ibogalogs. a, Ibogalogs lacking the

tetrahydroazepine of ibogaine were synthesized in only a few steps. In brief,

acylation of pyridine 1 under reductive conditions yielded the carboxybenzyl

(Cbz)-protected dihydropyridine 2, which was immediately subjected to a

Diels–Alder reaction with methyl vinyl ketone (3) followed by an in situ

epimerization with NaOMe to afford an inseparable 1:1 mixture of exo (4a) and

endo (4b) isomers (73% over 3 steps). Reaction of 4a and 4b with tosylhydrazide

yielded the hydrazones 5a and 5b, which were separable via a combination of

selective crystallization and chromatography (total yield of the two

isomers, 75%). Caglioti reduction of the tosylhydrazones yielded 6a or 6b,

which were readily converted to a variety of analogues via reaction sequences

involving hydrogenolysis of the Cbz group, hydrogenation of the olefin, and

C–N bond formation (Supplementary Information). b, Ibogalogs lacking the

isoquinuclidine of ibogaine were synthesized in a single step through Fischer

indole cyclization. See Supplementary Information for details.

Extended Data Fig. 2 | The effects of ibogalogs on dendritogenesis.

a, Representative images of rat embryonic cortical neurons (DIV6) treated with

the indicated compounds. Scale bar, 10 μm. b, Maximum numbers of crossings

(N_max) of the Sholl plots demonstrate that tetrahydroazepine-containing

ibogalogs are more effective at increasing dendritic arbor complexity than are

isoquinuclicine-containing ibogalogs. c, Sholl analysis (circle radii, 1.34-μm

increments) demonstrates that cultured cortical neurons treated with several

ibogalogs have more complex dendritic arbors compared to vehicle control

(n = 52–83 neurons per treatment). The shaded area surrounding each line

represents 95% confidence intervals. Control compounds, isoquinuclidines

and tetrahydroazepines are shown in blue, purple and red, respectively. Exact n

values for each experimental condition are reported in Supplementary Table 1.

Specific statistical tests, information on reproducibility, and exact P values are

reported in Methods and Supplementary Table 1.

Article

Extended Data Fig. 3 | TBG is safer than ibogaine. a, Unlike ibogaine, IBG and

TBG do not induce bradycardia in larval zebrafish. Sertindole (SI) was used as a

positive control. b, Heat maps are shown representing aggregate larval

zebrafish locomotor activity per well compared to vehicle controls (pseudo-Z-

score). Red and blue indicate higher and lower activity than the mean of vehicle

controls, respectively, while white indicates activity within 1 s.d. of the

control. Stimuli applied over time are indicated under the heat maps. Colours

indicate bright LED light of respective colours, black traces represent the

waveforms of acoustic stimuli, and grey vertical lines indicate physical tapping

as secondary acoustic stimuli. c, Confusion matrix for classification of

compounds (200 μM) plus vehicle and lethal controls. d, Concentration–

response curves are shown for treated zebrafish subjected to the series of

stimuli depicted in b. Lower percentages indicate treatments that were more

often classified as vehicle (blue) or lethal (red). The solid line denotes the

median and the shading denotes a 95th percentile confidence interval

calculated by bootstrap. n = 8 wells per condition (64 zebrafish per condition).

Blue lines indicate that all compounds produce behavioural phenotypes more

distinct from vehicle at higher concentrations. Red lines indicate that known

toxins (for example, PTZ, picrotoxin, endosulfan), known hERG inhibitors

(sertindole, haloperidol, terfenadine) and iboga alkaloids (IBO, NOR) produce

behavioural phenotypes more closely resembling a lethal phenotype as their

concentrations are increased. Increasing concentrations of IBG or TBG do not

produce lethal-like behavioural phenotypes. e, Transgenic larval zebrafish

expressing GCaMP5G were immobilized in agarose, treated with compounds,

and imaged over time. The known seizure-inducing compound PTZ was used as

a positive control. Ibogaine and TBG were treated at 50 μM (n = 2 per condition).

f, Proportion of viable and non-viable (malformed + dead) zebrafish following

treatment with vehicle and TBG (66 μM) for 5 dpf (Fisher’s exact test:

P = 0.3864). Representative images of zebrafish treated with vehicle and TBG

(66 μM) for 2 and 5 dpf are shown. Scale bar, 2 mm. Exact n values for each

experimental condition are reported in Supplementary Table 1. Specific

statistical tests, information on reproducibility and exact P values are reported

in the Methods and Supplementary Table 1.

Extended Data Fig. 4 | Concentration–response curves demonstrating the

abilities of ibogalogs and related compounds to activate 5-HT and opioid

receptors. All compounds were assayed in parallel using the same drug

dilutions. Graphs reflect representative concentration–response curves

plotting mean and s.e.m. of data points performed in duplicate or triplicate.

Assay details are described in Methods. Exact n values for each experimental

condition are reported in Supplementary Table 1. Specific statistical tests,

information on reproducibility and exact P values are reported in Methods and

in Supplementary Table 1.

Article

Extended Data Fig. 5 | Pharmacological profiles of ibogalogs and related

compounds. EC₅₀and E_maxestimates from at least two independent

concentration-response curves performed in duplicate or triplicate. log(E_max

EC₅₀) activity relative to the system E_max. Inactive, inactive in agonist mode;

N.D., not determined; blue boxes indicate that the compound exhibits

agonist mode but not tested in antagonist mode; orange boxes indicate that

the compound is an inverse agonist. Ibogalogs are more selective 5-HT_2A

agonists than is 5-MeO-DMT. Exact n values for each experimental condition

are reported in Supplementary Table 1. Specific statistical tests, information

on reproducibility and exact P values are reported in Methods and in

Supplementary Table 1.

/

antagonist activity; dark grey boxes indicate that the compound is inactive in

Extended Data Fig. 6 | High doses of TBG do not produce a conditioned

place preference. a, Schematic of the design of the conditioned place

preference experiments. On day 1, the amount of time the mice spent in each

distinct side of a two-chamber apparatus was recorded. Next, vehicle and TBG

were administered to mice on alternating days while they were confined to

chamber A (white box) or chamber B (grey parallel lines), respectively.

Conditioning lasted for a total of 6 days. On day 8, preference for each distinct

side of the two-chamber apparatus was assessed. b, A low dose of TBG

(1 mg kg⁻¹) did not produce conditioned place preference or conditioned

place aversion. Higher doses (10 and 50 mg kg⁻¹) produce a modest conditioned

place aversion. c, TBG does not produce any long-lasting (>24 h) effects on

locomotion. There is no statistical difference in locomotion between any pre-

or post-conditioning groups (P = 0.9985, one-way ANOVA). White bars indicate

groups before receiving TBG (that is, pre-conditioning), and blue bars indicate

groups 24 h after the last TBG administration (that is, post-conditioning). Exact

n values for each experimental condition are reported in Supplementary

Table 1. Specific statistical tests, information on reproducibility and exact

P values are reported in Methods and in Supplementary Table 1.

Article

Extended Data Fig. 7 | TBG produces antidepressant effects in mice.

a, Schematic illustrating the stressors used as part of the 7-day UMS protocol.

White and grey boxes represent the light and dark phases of the light cycle,

respectively. b, TBG rescues the effects of UMS on immobility. c, TBG (50 mg kg⁻¹

reaches high brain concentrations and is rapidly eliminated from the body.

Mice were administered 3 different doses of TBG via i.p. injection and

euthanized either 15 min or 3 h later. Whole brains and livers were collected,

dried, homogenized and extracted with MTBE. Quantification was

accomplished using LC–MS and concentrations of TBG in the two organs were

calculated. Several samples for the 10 and 1 mg kg⁻¹doses at the 3 h time point

had TBG at levels below the limit of quantification (around 5 nmol g⁻¹). In those

cases, the values were recorded as 0. Exact n numbers for each experimental

condition are reported in Supplementary Table 1. Specific statistical tests,

information on reproducibility and exact P values are reported in Methods and

in Supplementary Table 1.

)

Extended Data Fig. 8 | Effects of TBG on locomotion and sucrose-seeking

behaviour in rats. a, Acute administration of TBG does not impair locomotion

in the open field. Rats were subjected to novelty-induced locomotion

(baseline) for 30 min. At that time, cocaine was administered and

dose-dependent manner when administered during self-administration.

d, TBG acutely reduces sucrose seeking when administered immediately

before the first extinction session. The CUE (injection 1 = vehicle; injection

2, vehicle) and EXT (injection 1, vehicle; injection 2, TBG) groups were

compared, as they were matched for the number of withdrawal days between

the last self-administration and first extinction session. e, TBG does not have

long-lasting effects on sucrose-seeking behaviour, as it does not reduce active

lever pressing during the cued reinstatement when administered 12–14 days

previously during self-administration (SA) or immediately before extinction

(EXT). Exact n values for each experimental condition are reported in

Supplementary Table 1. Specific statistical tests, information on

reproducibility and exact P values are reported in Methods and in

Supplementary Table 1.

psychostimulant-induced locomotion (+cocaine) was assessed for 60 min.

There were no differences between the vehicle- and TBG-treated groups with

respect to total distance travelled or average velocity. Furthermore, there was

no difference in thigmotaxis measured during the baseline period (that is, the

percentage of time in the centre of the open field). b–e, A sucrose

self-administration experiment was conducted in a similar manner to the

heroin self-administration experiment in Fig. 4. Doses in mg kg⁻¹are shown in

parentheses. b, Sucrose seeking over time is shown. Coloured arrows indicate

when each group received TBG. VEH was administered at all other time points

to each group. c, TBG acutely reduces sucrose-seeking behaviour in a

Article

Extended Data Table 1 | TBG and IBG are more soluble than ibogaine

a, The fumarate salts of TBG and IBG are readily soluble in saline (0.9%), whereas ibogaine hydrochloride is not.

b, Ibogaine hydrochloride exhibits limited solubility in various saline-based vehicles. Solutions of saline (0.9%) containing various percentages of co-solvents/additives were added to finely

crushed ibogaine hydrochloride. All of our attempts to improve its solubility through pulverizing, sonication, and mild heating (<50ꢀ°C) were unsuccessful. Moreover, the addition of co-solvents

(ethanol, dimethyl sulfoxide, glycerol), surfactants (Kolliphor), or hydrotropes (ATP) to the vehicle did not substantially improve its solubility. We confirmed the purity and identity of the ibogaine

hydrochloride used in these studies through a combination of NMR, LC–MS, and X-ray crystallography experiments. Exact n values for each experimental condition are reported in Supplemen-

tary Table 1. Specific statistical tests, information on reproducibility and exact P values are reported in Methods and in Supplementary Table 1.

Extended Data Table 2 | Safety pharmacology screen

The effects of TBG (10 μM) on a wide range of targets was assessed by Eurofins Discovery. Assays were conducted in duplicate and the results were averaged. Targets with ≥50% inhibition

are highlighted in blue. Exact n values for each experimental condition are reported in Supplementary Table 1. Specific statistical tests, information on reproducibility and exact P values are

reported in Methods and in Supplementary Table 1.

Corresponding author(s):

Last updated by author(s):

David E. Olson

10/12/2020

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