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Published in final edited form as: Pharmacol Res. 2017 Nov 20;129:475–481. doi: 10.1016/j.phrs.2017.11.019

Enantiomer-specific positive allosteric modulation of CB1 signaling in autaptic hippocampal neurons

Jose Mitjavila 1, Danielle Yin 1, Pushkar M Kulkarni 2, Chiara Zanato 3, Ganesh A Thakur 2, Ruth Ross 4, Iain Greig 3, Ken Mackie 1, Alex Straiker 1
PMCID: PMC5828971  NIHMSID: NIHMS923098  PMID: 29158048

Abstract

The cannabinoid signaling system is found throughout the CNS and its involvement in several pathological processes makes it an attractive therapeutic target. Because orthosteric CB1 cannabinoid receptor ligands have undesirable adverse effects there has been great interest in the development of allosteric modulators – both negative (NAMs) and positive (PAMs) – of these receptors. NAMs of CB1 appeared first on the scene, followed more recently by PAMs. Because allosteric modulation can vary depending on the orthosteric ligand it is important to study their function in a system that employs endogenous cannabinoids. We have recently surveyed first generation NAMs using cultured autaptic hippocampal neurons. These neurons ex depolarization induced suppression of excitation (DSE), a form of synaptic plasticity that is mediated by CB1 and 2-arachidonoyl glycerol (2-AG); they are therefore an excellent neuronal model of endogenous cannabinoid signaling in which to test CB1 modulators.

Keywords: Allosteric, orthosteric, G protein-coupled receptor, cannabinoid receptor, CB1

Graphical Abstract

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INTRODUCTION

Cannabinoid receptors mediate most of the salient effects of marijuana consumption [1]. First identified in the late-1980s [2, 3] these receptors are now known to be key components of an endogenous signaling system that is found throughout the body. Orthosteric agonists (i.e. WIN55212-2 and CP55940) and antagonists (i.e. SR141716, AM251) were quickly identified and have been studied extensively. There is, however, a strong interest in developing allosteric modulators of CB1 signaling, partly due to the sometimes unfavorable therapeutic profiles of orthosteric ligands. Negative allosteric modulators (NAMs) act by inhibiting binding and/or signaling, while positive allosteric modulators (PAMs) act by potentiating binding and/or signaling, by an orthosteric agonist [4]. NAMs/PAMs may act in various means but the essential concept is that some receptors have one or more secondary ‘allosteric’ sites. When engaged by allosteric ligands these sites modulate orthosteric signaling. This generally occurs via alteration of binding kinetics of the orthosteric ligand and/or potentiation/inhibition of receptor signaling. One attractive quality of allosteric modulators is the potential to modulate only those receptors that are being actively engaged by endogenous ligands. For the broadly distributed cannabinoid CB1 receptors the risk of off-target action is considerable, making such selectivity especially desirable.

We recently reported on first-generation CB1 allosteric modulators on synaptic transmission in autaptic hippocampal neurons [5]. These neurons serve as a well-characterized model of endogenous cannabinoid signaling that possesses the machinery to synthesize, release and metabolize endogenous cannabinoids and presynaptic CB1 receptors, the activation of which inhibits neurotransmitter release [6–8]. Depolarization of these neurons yields depolarization induced suppression of excitation (DSE) a form of plasticity that involves retrograde signaling via 2-arachidonoyl glycerol (2-AG) that act on presynaptic CB1 receptors [6, 9]. Because this model is well-characterized and DSE is mediated by an endogenous cannabinoid, this makes autaptic neurons well-suited to an examination of allosterism with endocannabinoids. In our recent test of allosteric modulators (five negative and one positive allosteric modulator) we found that three acted in a manner consistent with allosteric modulation of CB1 receptor function.

ZCZ011 was recently reported to serve as a PAM albeit with a mixed pharmacological profile that included agonist properties [10] (Fig. 1A). This compound is derived from 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole [11]; CAS Registry Number: 102704-40-5; F-0870-0064) referred to here as GAT211 (Fig. 1B). GAT 211, a racemic compound, has been recently characterized as a CB1-selective ago-PAM with its allosteric agonist activity residing in the R-(+)-enantiomer GAT228 and PAM activity in S-(−)-enantiomer GAT229 [12]. We now report our study of these candidate positive allosteric modulators in a neuronal model of endogenous CB1 signaling.

Figure 1.

Figure 1

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Structures of ZCZ011 and GAT211 and its enantiomers GAT228 and GAT229

METHODS

Hippocampal culture preparation

All procedures used in this study were approved by the Animal Care Committee of Indiana University and conform to the Guidelines of the National Institutes of Health on the Care and Use of Animals. Mouse hippocampal neurons isolated from the CA1-CA3 region were cultured on microislands as described previously [13, 14]. Neurons were obtained from mice (C57Bl/6, postnatal day 0–2, of indeterminate sex) and plated onto a feeder layer of hippocampal astrocytes that had been laid down previously [15]. Cultures were grown in high-glucose (20 mM) DMEM containing 10% horse serum, without mitotic inhibitors and used for recordings after 8 days in culture and for no more than three hours after removal from culture medium.

Electrophysiology

When a single neuron is grown on a small island of permissive substrate, it forms synapses—or “autapses”—onto itself. All experiments were performed on isolated autaptic neurons. Whole cell voltage-clamp recordings from autaptic neurons were carried out at room temperature using an Axopatch 200A amplifier (Axon Instruments, Burlingame, CA). The extracellular solution contained (in mM) 119 NaCl, 5 KCl, 2.5 CaCl2, 1.5 MgCl2, 30 glucose, and 20 HEPES. Continuous flow of solution through the bath chamber (~2 ml/min) ensured rapid drug application and clearance. Drugs were typically prepared as stocks, and then diluted into extracellular solution at their final concentration and used on the same day.

Recording pipettes of 1.8–3 MΩ were filled with (in mM) 121.5 KGluconate, 17.5 KCl, 9 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 MgATP, and 0.5 LiGTP. Access resistance and holding current were monitored and only cells with both stable access resistance and holding current were included for data analysis. Conventional stimulus protocol: the membrane potential was held at −70 mV and excitatory postsynaptic currents (EPSCs) were evoked every 20 seconds by triggering an unclamped action current with a 1.0 ms depolarizing step. The resultant evoked waveform consisted of a brief stimulus artifact and a large downward spike representing inward sodium currents, followed by the slower EPSC. The size of the recorded EPSCs was calculated by integrating the evoked current to yield a charge value (in pC). Calculating the charge value in this manner yields an indirect measure of the amount of neurotransmitter released while minimizing the effects of cable distortion on currents generated far from the site of the recording electrode (the soma). Data were acquired at a sampling rate of 5 kHz.

DSE stimuli: After establishing a 10–20 second 0.5 Hz baseline, DSE was evoked by depolarizing to 0 mV for 50 msec, 100 msec, 300 msec, 500 msec, 1 sec, 3 sec and 10 sec, followed in each case by resumption of a 0.5 Hz stimulus protocol for 20–80+ seconds, allowing EPSCs to recover to baseline values. This approach allowed us to determine the sensitivity of the synapses to DSE induction. To allow comparison, baseline values (prior to the DSE stimulus) are normalized to one. DSE inhibition values are presented as fractions of 1, i.e. a 50% inhibition from the baseline response is 0.50 ± standard error of the mean. The x-axis of DSE depolarization-response curves are log-scale seconds of the duration of the depolarization used to elicit DSE. Depolarization response curves are obtained to determine pharmacological properties of endogenous 2-AG signaling by depolarizing neurons for progressively longer durations (50 msec, 100 msec, 300 msec, 500 msec, 1 sec, 3 sec and 10 sec). The data are fitted with a nonlinear regression, allowing calculation of an ED50, the effective dose or duration of depolarization at which a 50% inhibition is achieved. Statistical significance in these curves is taken as non-overlapping 95% confidence intervals.

RESULTS

GAT229 has PAM-like response in autaptic hippocampal neurons

Depolarization of excitatory autaptic hippocampal neurons elicits depolarization induced suppression of excitation, a form of retrograde inhibition that is mediated by 2-AG and that requires CB1 receptors (DSE; [6]). Stimulating neurons with a series of progressively longer depolarizations (50ms, 100ms, 300ms, 500ms, 1 sec, 3 sec, 10 sec) results in successively greater inhibitions of neurotransmission via enhanced DSE [16]. The resultant “depolarization-response curve” allows characterization of certain pharmacological properties of cannabinoid signaling. This includes the calculation of an effective-dose 50 (ED50, duration of depolarization that gives 50%-maximal response). Because we do not see evidence for tonic activity via CB1 in autaptic neurons [6], a positive allosteric modulator is not expected to directly inhibit synaptic transmission but should shift the depolarization response curve down and/or to the left by enhancing the action of the 2-AG released by depolarization at CB1 receptors. We found that GAT211 directly inhibited excitatory postsynaptic currents (EPSCs) in autaptic neurons cultured from wild type (WT) but not CB1−/− mice (Fig. 2, Relative EPSC charge after GAT211 (1μM) in WT: 0.62 ± 0.08, n=14; in CB1−/−: 1.00 ± 0.02, n=5; p<0.005, unpaired t test vs. CB1−/−). This substantial inhibition of EPSCs in a CB1-dependent fashion by GAT211 is inconsistent with pure positive allosteric modulation.

Figure 2. GAT211 inhibits EPSCs via CB1.

Figure 2

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A) Sample time course shows GAT211 (1μM) inhibits integrated EPSC current (charge, pC). B) Summarized data shows that GAT211 inhibits EPSCs in WT (n=14) but not CB1−/− neurons (n=6). **, p < 0.005, unpaired t-test vs. CB1−/−.

Because GAT211 is a racemic mixture of two enantiomers, we tested each enantiomer separately. One of these, GAT228, the R-(+)-enantiomer affected EPSCs bi-modally, inhibiting only a subset of CB1-expressing neurons (Fig. 3A,B, Relative EPSC charge after GAT228 (1μM) in WT: 0.81 ± 0.08, n=10; in CB1−/− : 0.99 ± 0.03, n=5). No inhibition by GAT228 was seen in any CB1−/− neurons tested, indicating that when inhibition was present it likely occurred via CB1 activation. In cells that were not directly inhibited by GAT228, there was no net effect of GAT228 on the depolarization response curve (Fig. 3C: ED50 before GAT228: 1.34 (1.19–1.50) sec; with GAT228: 1.00 (0.77–1.29) sec, n=5; 95% CI overlapping). However the DSE recovery time courses were slowed (Fig. 3D: DSE t1/2 baseline: 6.0 (4.4–9.1) sec; with GAT228: 17.7 (13.6–25.4) sec, n=10; 95% CI non-overlapping). The response profile for GAT228 is therefore consistent with modest agonism.

Figure 3. GAT228 inhibits EPSCs in some neurons.

Figure 3

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A) Sample time course shows GAT228 (1μM) inhibits EPSCs in some neurons (circles) but not others (squares). B) Summarized data as bar graph (left) and scatter plot (right) shows a bimodal inhibition of EPSCs in WT (n=10) but not CB1−/− (n=5) neurons by GAT228. C) The DSE depolarization response profile in cells not directly inhibited by GAT228 treatment is unaltered (n=5). D) A summarized DSE (3 sec depolarization) time course before and after treatment shows that GAT228 does not change the peak inhibition but does slow recovery (n=10).

In contrast, the other S-(−)-enantiomer, GAT229 did not inhibit EPSCs when applied on its own (Fig. 4AB: relative EPSC charge GAT229 (1μM) in WT: 1.0 ± 0.05, n=10; in CB1−/−: 0.97 ± 0.04, n=6; p>0.05, unpaired t test). It did however enhance DSE. Figure 4C shows a DSE depolarization series before and after 1μM GAT229; DSE was more pronounced after treatment, particularly for longer depolarizations. These data are summarized in Figure 4D. In contrast to GAT228, GAT229 did not significantly slow recovery from DSE (Fig. 4: t1/2 baseline: 9.8 (7.0–16.5) sec; with GAT229: 17.1 (11.2–36.6) sec; n=10, overlapping 95%CI).

Figure 4. GAT229 acts in a manner consistent with positive allosteric modulation of CB1 in autaptic neurons.

Figure 4

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A) Average time course shows GAT229 (1μM) does not inhibit EPSCs. B) Summarized data from A) n=10, 6. C) Series of progressively longer depolarizations shows that treatment with GAT229 results in greater EPSC inhibition. D) DSE depolarization response curve is shifted down and to the left after GAT229 treatment (n=10). E) Summary of the 3 sec depolarization DSE time course before and after treatment with GAT229 (n=5).

ZCZ011 has both agonist and PAM-like properties in autaptic hippocampal neurons

We also tested the structurally related ZCZ011 compound for PAM-like properties. We found that as with GAT211, ZCZ011 directly inhibited EPSCs in most wild type (Fig. 5A,C; 0.65 ± 0.08, n=9; p<0.005 unpaired t-test vs. CB1−/−) but not CB1−/− knockout neurons (Fig 5C; 1.06 ± 0.02, n=4). But in contrast to GAT211, some neurons did not see a direct inhibition of EPSCs in response to ZCZ011 (Fig. 5B,D). Direct inhibition of currents is consistent with this compound possessing some orthosteric activity and makes PAM-like properties difficult to discern, but as shown in the example in Figure 5E, in some neurons that did not see a direct inhibition, ZCZ011 did however potentiate their DSE responses in a manner similar to that seen for GAT229. Furthermore, the DSE recovery time courses in the five neurons with the least agonist effect were slowed with ZCZ011 (Fig.5E and 5F: DSE t1/2 baseline: 24.5 (21.9–27.2) sec; with ZCZ011: 59.5 (50.2–72.8) sec; 95% CI non-overlapping).

Figure 5. ZCZ011 exhibits both agonist and PAM-like properties in autaptic neurons.

Figure 5

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A–B) sample time courses where ZCZ011 (1μM) inhibits (A) or does not inhibit (B) EPSCs. C) Summary data for direct inhibition of neurotransmission in wild type (WT) and CB1−/− neurons. D) Scatterplot of wild type data in C shows distribution of responses. E) Sample DSE series time course (50ms-1sec) in neuron that did not exhibit a direct inhibition in response to ZCZ011. F) Sample DSE time courses in neurons that saw limited or no inhibition in response to ZCZ011. **, p<.005, unpaired t-test vs. CB1−/−

DISCUSSION

The potential of allosteric modulators of cannabinoid receptor signaling has generated considerable interest. We have recently surveyed the function of ‘first generation’ NAMs in a neuronal model that uses 2-AG as the endocannabinoid in an endogenous retrograde signaling system [5] and now report the function of two structurally related candidate PAMs, using an autaptic model of endogenous cannabinoid signaling. Our chief findings are that these compounds can exhibit PAM-like responses in this system but that their pharmacology is complex, even in the architecturally simple autaptic model. Most intriguingly however we found that enantiomers of the racemic GAT211 were pharmacologically distinct and effectively separated out the ‘undesired’ direct (orthosteric) actions of GAT211 from the ‘desired’ PAM-like properties. Of the compounds we tested, therefore, GAT229 appears to act as a PAM in this model. We have additionally found that the related ZCZ011, recently described as a PAM with agonist properties [10] behaves in a similar manner (i.e. as a PAM with agonist properties) in autaptic neurons.

As we have recently noted [5], our experiments can ‘rule out’ a candidate allosteric modulator with respect to 2-AG modulation of synaptic transmission, but our experiments cannot definitively identify a compound as an allosteric agent; this typically requires binding studies that evaluate the compound’s effect on the orthosteric ligand’s binding kinetics. We do not therefore claim that the ZCZ011 and GAT compounds are or are not allosteric modulators. However we have shown whether they act in a manner consistent with allosteric modulation in their CB1-dependent inhibition of glutamatergic transmission.

Our synaptic physiology studies suggest that GAT228 directly inhibits EPSCs via CB1 in a manner that is inconsistent with “pure” positive allosteric modulation. GAT228 was recently shown to act as a partial agonist through allosteric site [12]. As noted above, we have previously shown that these neurons do not exhibit detectable CB1-dependent tonic activity and do not therefore expect that sufficient 2-AG is present under baseline conditions to serve as a substrate for allosteric modulation. But since GAT228 directly inhibits EPSCs via CB1, one would expect this to shift the depolarization response curve to the right. Instead we see no shift at all. This may be explained as resulting from a dual action since GAT228 also slows the DSE recovery time course. This slowing may result from slowing the dissociation of 2-AG from CB1, perhaps allosterically. If so GAT228 may be an ago-PAM in this neuronal setting though this does not explain the differential responses in some neurons versus others. Because GAT211 consists of both GAT228 and GAT229, the issues associated with GAT228 extend to GAT211. In contrast, GAT229 behaved in a manner consistent with positive allosteric modulation in this assay.

Our findings for the structurally similar ZCZ011 are similar to those for the GAT211/228/229 compounds but are also distinct in important ways. Like GAT229, ZCZ011 exhibited a PAM-like response in some neurons, but like GAT228 it also directly inhibited neurotransmission in many autaptic neurons. And unlike GAT211, ZCZ011 did not directly inhibit EPSCs in all neurons. Indeed this points up a puzzling attribute of GAT211 in that the racemic mixture appears to behave differently than one would predict based on the properties of the enantiomers when taken alone. That profile would look more like ZCZ011 – PAM properties in some neurons, agonist properties in others – but instead GAT211 appears to routinely exhibit agonist properties. The reasons for this are unclear but may involve a synergistic action of the enantiomers.

As noted above ZCZ011 was developed as a derivative of 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole, referred to here as GAT211 but also known as F087 [11]. The PAM-like properties of this compound were first described in a conference abstract in 2007 (“AZ-4” [17]). However a thorough biochemical characterization of GAT211 and its enantiomers was reported only recently [12]. The structure of ZCZ011 suggests that it is also racemic. It would therefore be of interest to test whether the enantiomers also effectively separate the PAM and agonist properties we have seen for ZCZ011. These enantiomers are not however currently available. Furthermore there may be a limit to the value of studying ZCZ011 enantiomers: although these compounds represent an exciting development in terms of CB1 allosteric pharmacology, they all suffer from an important shortcoming that greatly limits their application as therapeutic agents. The structures of GAT211/228/229 and ZCZ011 all include an aliphatic nitro moiety that is generally avoided in the design of pharmacotherapeutics. The challenge now will be to develop PAMs that offer similar pharmacological properties without the structural properties that limit their therapeutic relevance.

In summary, we have found that GAT211 behaves as an agonist at cannabinoid CB1 receptors in autaptic hippocampal neurons. Importantly the racemic GAT211 exhibits enantiomer-specific signaling; it is possible to dissect out the PAM-like responses in one enantiomer while the other enantiomer exhibits the agonist properties. In the related compound, ZCZ011, also likely to be racemic, we similarly see agonist and PAM-like properties. These are the first compounds that we have observed to behave as PAMs in this model system. Our results indicate that PAMs can effectively enhance endogenous cannabinoid signaling via CB1 and the characterization of this class of PAMs may pave the way for further development of clinically significant allosteric modulators and therefore expand upon the considerable clinical potential of cannabinoid-based therapies.

Acknowledgments

Research was supported by NIH grants DA-009789 (RR), DA-026449 (RR), DA-003672 (RR), CIHR Proof of Principal Phase 1 grant 288645 (RR), EY24625 (AS), EY24717 (GT & AS), DA011322 (KM) and DA021696 (KM).

RR and IRG are inventors on patent applications filed by the Universities of Toronto and Aberdeen, which disclose pharmaceutical agents targeting molecular pathways described in the present article. RAR and IRG have an equity share in Signal Pharma Ltd, a University spin-out company developing CB1-positive allosteric modulators.

In this study we find that while two related compounds, GAT211 and ZCZ011, each show PAM-like responses in autaptic hippocampal neurons, they also exhibit complex pharmacology. Notably we were able to separate the PAM- and agonist-like responses of GAT211 by examining the enantiomers of this racemic compound: GAT228 and GAT229. We find that GAT229 exhibits PAM-like behavior while GAT228 appears to directly activate the CB1 receptor.

Both GAT229 and ZCZ011 represent the first PAMs that we have found to be effective in using this 2-AG utilizing neuronal model system. Because these compounds may exhibit both probe selectivity and biased signaling it will be important to test them with anandamide as well as other signaling pathways.

Abbreviations

DSE

depolarization-induced suppression of excitation

2-AG

2-arachidonoyl glycerol

NAM

negative allosteric modulator

PAM

positive allosteric modulator

EPSC

excitatory postsynaptic current

Footnotes

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Conflict of Interest Statement

The remaining authors declare no competing financial interests.

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