Synthesis and biological evaluation of indole-2-carboxamides bearing photoactivatable functionalities as novel allosteric modulators for the cannabinoid CB1 receptor

Article abstract of DOI:10.1021/acschemneuro.6b00041

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569, 1) is a prototypical allosteric modulator for the cannabinoid CB1 receptor. Based on this indole-2-carboxamide scaffold, we designed and synthesized novel CB1 allosteric m

Full text of DOI:10.1021/acschemneuro.6b00041

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Article
MAPPING CANNABINOID RECEPTOR 1 ALLOSTERIC SITE(S):
CRITICAL MOLECULAR DETERMINANT AND SIGNALING PROFILE OF
GAT100 - A NOVEL, POTENT AND IRREVERSIBLY BINDING PROBE
Robert B. Laprairie, Abhijit R Kulkarni, Pushkar M Kulkarni, Dow P. Hurst, Diane
L. Lynch, Patricia H. Reggio, David R. Janero, Roger G. Pertwee, Lesley A.
Stevenson, Melanie Kelly, Eileen M. Denovan-Wright, and Ganesh A Thakur
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00041 • Publication Date (Web): 04 Apr 2016
Downloaded from http://pubs.acs.org on April 5, 2016
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MAPPING CANNABINOID RECEPTOR 1 ALLOSTERIC SITE(S): CRITICAL
MOLECULAR DETERMINANT AND SIGNALING PROFILE OF GAT100 ꢀ A NOVEL,
POTENT AND IRREVERSIBLY BINDING PROBE
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Robert B. Laprairie,† Abhijit R. Kulkarni,‡ Pushkar M. Kulkarni,‡ Dow P. Hurst,§ Diane Lynch,§
Patricia H. Reggio,§ David R. Janero,‡,║ Roger G. Pertwee,♯ Lesley A. Stevenson,♯ Melanie E.
M. Kelly,†,$ Eileen M. DenovanꢀWright,†, and Ganesh A. Thakur‡*
$
†Departments of Pharmacology and Ophthalmology and Visual Sciences, Dalhousie University,
Halifax NS Canada B3H 4R2
‡Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health
Sciences, Northeastern University, Boston, MA 02115
§Center for Drug Discovery, University of North Carolina at Greensboro, Greensboro, NC 27402
║Center for Drug Discovery, Northeastern University, Boston MA 02115
♯School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, AB25 2ZD, Scotland.
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KEYWORDS:
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Allosteric covalent probe, allosteric site, biased signaling, binding domain, cannabinoid 1
receptor, cysteine, functional selectivity, G protein-coupled receptors (GPCRs), homology
modeling, isothiocyanate, ligand bias, ligand-binding motif, negative allosteric modulator, 7-
transmembrane receptor, signal transduction, therapeutics discovery
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ABSTRACT:
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One of the most abundant Gꢀprotein coupled receptors (GPCRs) in brain, the cannabinoid 1
receptor (CB1R) is a tractable therapeutic target for treating diverse psychobehavioral and
somatic disorders. Adverse onꢀtarget effects associated with smallꢀmolecule CB1R orthosteric
agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric
CB1R modulators offer a potentially safer modality through which CB1R signaling may be
directed for therapeutic benefit. Rational design of candidate, drugꢀlike CB1R allosteric
modulators requires greater understanding of the architecture of the CB1R allosteric
endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor’s information
output. We have recently reported the synthesis of a focused library of rationally designed,
covalent analogs of Org27569 and PSNCBAMꢀ1, two prototypic CB1R negative allosteric
modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the
isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the
[35S]GTPγS and βꢀarrestin signaling assays and its ability to label CB1R as a covalent allosteric
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probe with significantly reduced inverse agonism in the [35S]GTPγS assay as compared to
Org27569. We report here a comprehensive functional profiling of GAT100 across an array of
important downstream cellꢀsignaling pathways and analysis of its potential orthosteric probeꢀ
dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the
orthosteric CB1R agonist CP55,940 and the endocannabinoids 2ꢀarachidonylglycerol and
anandamide for βꢀarrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP
accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in
Neuro2a and STHdhQ7/Q7 cells endogenously expressing CB1R. Distinctively, GAT100 was a
more potent and efficacious CB1R NAM than Org27569 and PSNCBAMꢀ1 in all signaling
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assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAMꢀ1.
Computational docking studies implicate C7.38(382) as a key feature of GAT100’s ligandꢀ
binding motif. These data help inform the engineering of newerꢀgeneration, druggable CB1R
allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping
structureꢀfunction correlates characteristic of the druggable CB1R allosteric space.
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INTRODUCTION
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The ubiquitous mammalian endocannabinoid biosignaling system includes the
cannabinoid 1 and (CB1R) and 2 (CB2R) Gꢀprotein coupled receptors (GPCRs), their
endogenous agonist ligands (endocannabinoids) anandamide (AEA) and 2ꢀarachidonoylglycerol
(2ꢀAG), and enzymes responsible for endocannabinoid synthesis and inactivation.1, 2 As the
mostꢀexpressed GPCR in brain, the CB1R’s principal neurobiological function is to transduce
endocannabinoid signaling and thereby inhibit neurotransmitter release.3 On the other hand,
CB2R is mainly expressed in peripheral tissues and to a little extent in CNS and has been
pursued for treating pain and inflammation.4-10 In the periphery, CB1R activity helps regulate
metabolic processes including substrate storage and mobilization. Dysregulation of CB1Rꢀ
mediated signaling is implicated in the etiology of several disorders, making CB1R an attractive
target of smallꢀmolecule pharmacotherapeutics for controlling pain and nausea and treating
diseases including obesity, type 2 diabetes, glaucoma, and substance abuse.11, 12
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Structurally diverse CB1R orthosteric ligands have been identified with varying
chemotypes, potencies, and selectivities.12-19 These include agonists such as the natural
phytocannabinoid ꢀ9ꢀtetrahydrocannabinol and the synthetic agent CP55,940 ([2ꢀ[(1R,2R,5R)ꢀ5ꢀ
hydroxyꢀ2ꢀ(3ꢀhydroxypropyl)cyclohexyl]ꢀ5ꢀ(2ꢀmethyloctanꢀ2ꢀyl)phenol])]
and
the
antagonist/inverse agonist SR141716A ([5ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(2,4ꢀdichloroꢀphenyl)ꢀ4ꢀmethylꢀ
Nꢀ(piperidinꢀ1ꢀyl)ꢀ1Hꢀpyrazoleꢀ3ꢀcarboxamide]). These and other CB1R orthosteric agonists and
antagonists/inverse agonists can exert therapeutic effects in preclinical disease models and, as
demonstrated to a more limited extent, in humans.20 However, typical orthosteric CB1R agonists
carry risks of abuse potential, euphoria, anxiety, panic, and impaired cognition and motor
function.14 CB1R orthosteric antagonists/inverse agonists are associated with serious negative
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psychobehavioral (e.g., depression, social aversion, suicidal ideation) and somatic (e.g.,
gastrointestinal) effects, making for an unacceptable adverseꢀevent profile that has been
attributed to their inverseꢀagonist property.21, 22
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Nonꢀselective (de)activation of downstream CB1R signaling pathways by typical CB1R
orthosteric ligands is believed to contribute to the unwanted side effects that have impeded their
therapeutic application as drugs. Although CB1R preferentially couples to Gαi/oꢀtype G proteins,
it may also interact with Gαs or Gαq.23, 24 The CB1Rꢀmediated signaling network involves
diverse G proteinꢀdependent (e.g., adenylate cyclase) and ꢀindependent (e.g., βꢀarrestin)
pathways and modulation of complex communication networks such as mitogenꢀactivated
protein kinase (MAPK) cascades.25-27 The variegated nature of the CB1Rꢀassociated signaling
network is indicative of multiple, finely controlled informationꢀtransducing mechanisms that
could be selectively exploited for therapeutic gain with the potential of reduced adverseꢀevent
risk as compared to typical orthosteric CB1R agonists antagonists/inverse agonists. Mounting
evidence over the past decade indicates that CB1R contains (an) allosteric binding site(s)
topographically distinct from the receptor’s orthosteric site that can engage endogenous and
synthetic ligands.28-32 Allostericꢀbased drugs represent a pharmacological approach to therapeutic
CB1R modulation that could offer distinct translational advantages over ligands that bind at the
receptor’s orthosteric site, as implied by the extended ternary complex model of GPCR
multistate structureꢀfunction dynamics.33, 34 This model posits that engagement of an allosteric
modulator by a GPCR alters the receptor’s affinity for an orthosteric ligand and/or the orthosteric
ligand’s efficacy by inducing cooperative conformational transitions in the receptor that affect
both orthostericꢀligand (dis)association rates and downstream functional effects.33, 34 In this
manner, an allosteric modulator may stabilize a GPCR in conformations not readily attainable
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with orthosteric ligands so as to “fineꢀtune” the pharmacological activity of the orthosteric ligand
and elicit unique, therapeutically advantageous downstream signaling patterns. The enhanced
selectivity, reduced interꢀreceptor promiscuity, and higherꢀresolution control of information
transmission characteristic of GPCR allosteric modulators are also believed to contribute to an
improved safety profile as compared to typical orthosteric ligands.33, 34
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The two classical CB1R allosteric ligands, Org2756928 (5ꢀchloroꢀ3ꢀethylꢀNꢀ(4ꢀ(piperidinꢀ
1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide) and PSNCBAMꢀ129 (1ꢀ(4ꢀchlorophenyl)ꢀ3ꢀ(3ꢀ(6ꢀ
(pyrrolidinꢀ1ꢀyl)pyridineꢀ2ꢀyl)phenyl)urea) (Figure 1), each behave as both a negative allosteric
modulator (NAM) of CP55,940 potency and efficacy in Gꢀprotein mediated signaling and a
positive allosteric modulator (PAM) of CP55,940 binding affinity. Additionally, few endogenous
ligands (Lipoxin A4, pregnenolone and pepcans; see Figure 1 for representative structures) have
also been shown to act as CB1 allosteric modulators.28, 31, 32, 35 Org27569 and PSNCBAMꢀ1
displace the CB1R orthosteric antagonist/inverse agonist SR141716A in competitive binding
assays, suggesting that these three ligands may partially share a binding region.28, 29, 36, 37
Although Org27569 and PSNCBAMꢀ1 have been intensively studied over the past decade, their
therapeutic application has been hampered by the lack of suitably liganded CB1R crystal
structures for atomicꢀlevel structural characterization and, perhaps most critically, their inverseꢀ
agonist property whichꢀꢀ by extension from CB1R orthosteric antagonists/inverse agonistsꢀꢀ
could predispose to unacceptable adverse events.29, 38, 39 To inform rational drug design aimed at
therapeutic CB1R allosteric modulation and delineate the optimal structural and pharmacophore
requirements thereof, it thus becomes necessary to expand the currently limited repertoire of
CB1R allosteric ligands and detail their receptorꢀinteraction profile and effects on CB1R
structure and function.
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Although homology modeling and mutation studies have offered some insight into the
Org27569 and PSNCBAMꢀ1 binding domains at CB1R, inherent limitations associated with
these techniques do not allow direct experimental delineation of intermolecular interactions
between NAMs and functional, wildꢀtype CB1R and the consequent effect of NAM engagement
on CB1R signaling and trafficking.37, 40 Purposeꢀdesigned, pharmacologically active orthosteric
ligands bearing functionalities reactive with specific GPCR amino acid(s) are increasingly being
utilized as covalent molecular probes for GPCR activity profiling and orthosteric bindingꢀsite
mapping.41, 42 For instance, this approach has been successfully integrated with mutational, mass
spectrometry, and in silico modeling studies in an experimental paradigm called ligandꢀassisted
protein structure (LAPS) to provide unique information on the interaction of various CB1R
orthosteric ligands with biologicallyꢀactive receptor.41, 43-47
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In an effort to extend the application of covalent probes for experimental mapping of the
CB1R druggable allosteric space, we recently detailed the rational design and synthesis of novel
Org27569 and PSNCBAMꢀ1 analogs as the firstꢀreported designer CB1R allosteric probes.48 In
contrast to those two prototypical nonꢀcovalent CB1R allosteric ligands, the pharmacologicallyꢀ
active derivatives in our library, by virtue of their electrophilic or photoactivatable moiety, are
capable of reacting covalently and in a chemically defined, amino acid residueꢀspecific manner
within the allosteric ligandꢀbinding domain of functional, wildꢀtype CB1R, thus serving as
covalent molecular probes. Among those CB1R designer allosteric probes, GAT100 (Figure 1),
featuring an electrophilic NCS functionality at the Cꢀ5 position, emerged as the most potent
ligand in the two functional assays (GTPγS and βꢀarrestin) evaluated. GAT100 retained the
paradoxical effects of parent compound Org27569, behaving as a PAM of CP55,940 binding, but
with improved potency over Org27569.48 Distinctively, in the GTPγS assay, GAT100 did not
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exhibit the inverse agonism that is a feature of both Org2756928, 36, 37 and PSNCBAMꢀ1.29, 36
Since the association between inverse agonism at CB1R and undesirable psychological and
somatic side effects has limited the therapeutic application of orthosteric CB1R
antagonists/inverse agonists, GAT100’s lack of inverseꢀagonist property holds therapeutic
promise and underscores the importance of the Cꢀ5 site for enhancing Org27569’s druggability.48
The translational implications of those data are further emphasized by the resurgence of interest
in the therapeutic opportunities afforded by targeted covalent receptor ligands, especially with
respect to simplifying dosing regimens, widening therapeutic margins, and increasing potency,
duration, and specificity.49-51
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These considerations make it essential to characterize the signaling repertoire of GAT100
at CB1R as compared to that of Org27569 and PSNCBAMꢀ1 in the same cellular systems and
analyze the potential of GAT100 to display orthostericꢀprobe dependence [i.e., the effect of
different orthosteric ligands (“orthosteric probes”) on the potency and efficacy of a given
allosteric ligand] and functional selectivity/ligand bias (i.e., ligandꢀspecific selective modulation
of the activity of select signal transduction pathways over others through a given receptor).33, 34
For this purpose, we report here a comprehensive in vitro evaluation of GAT100 downstream
signaling in cell systems heterologously or endogenously expressing CB1R. To evaluate the
orthostericꢀprobe dependence of GAT100, we chose the highꢀaffinity, potent synthetic
cannabinoid CP55,940 and the endocannabinoids 2ꢀAG and AEA. Pharmacological profiling
encompassed quantification of CB1Rꢀmediated, G proteinꢀindependent βꢀarrestin1 recruitment,
Gαqꢀdependent phospholipase Cβ3 (PLCβ3) activation, Gαi/oꢀdependent extracellular signalꢀ
regulated kinase (ERK) activation and cAMP production, and CB1R internalization.
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The data demonstrate that GAT100 is a more potent NAM of CB1R signaling than
Org27569 or PSNCBAMꢀ1 and, notably, lacks inverseꢀagonist activity compared to Org27569
and PSNCBAMꢀ1 in cAMP assay. In radioligand equilibriumꢀbinding assays with hCB1Rꢀ
transfected CHO cells GAT100 acted as a NAM of CB1R antagonist/inverse agonist
([3H]SR141716A) binding and was more potent than Org27569. In silico molecular modeling
studies suggest that C7.38(382) is a key feature of GAT100’s ligandꢀbinding motif as the most
likely amino acid reactive with the probe’s isothiocyanate moiety. The data constitute the first
comprehensive characterization of the functional consequences of CB1R modulation by a potent
covalent CB1R allosteric NAM GAT100 and provide initial insight into that ligand’s binding
motif and receptorꢀinteraction profile.
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RESULTS AND DISCUSSION
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CB1Rꢀmediated βꢀArrestin1 Recruitment. A bioluminescence resonance energy
transfer (BRET) assay was used to evaluate the effect of GAT100 on βꢀarrestin1 recruitment by
CB1R as compared to that of the two prototypic CB1R NAMs, Org27569 and PSNCBAMꢀ1.
Three cell systems were used: HEK293A cells overexpressing human CB1R (hCB1R) (Figure 2,
panels A, D, and G) and two murine cells, Neuro2a neuroblastoma stem cells (Figure 2, panels
B, E, and H) and differentiated STHdhQ7/Q7 striatal progenitor cells (Figure 2, panels C, F, and I),
endogenously expressing CB1R. Cells were treated with 1 nM – 10 ꢁM 2ꢀAG (Figure 2, panels
AꢀC), AEA (Figure 2, panels DꢀF), or CP55,940 (Figure 2, panels GꢀI) with or without 1 ꢁM
GAT100, Org27569, or PSNCBAMꢀ1 for 30 min before quantifying the assay endpoint, BRET
efficiency of energy transfer (BRETEff) between hCB1R green fluorescent protein 2 (GFP2) and
βꢀarrestin1ꢀRenilla luciferase (Rluc), as a sensitive measure of βꢀarrestin1 recruitment. GAT100
treatment reduced the potency and decreased the maximum possible effect (i.e., efficacy) (Emax
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of all CB1R orthosteric ligands on CB1Rꢀmediated βꢀarrestin1 recruitment to a greater extent
than vehicle, Org27569, and PSNCBAMꢀ1 in all cell types studied (Figure 2). The decrease in
Emax observed in the presence of Org27569 or PSNCBAMꢀ1 is presented in Table 1. In the
absence of orthosteric agonists, neither GAT100, Org27569, nor PSNCBAMꢀ1 (1 nM – 10 ꢁM)
evidenced inverse agonism toward βꢀarrestin1 recruitment in HEK293A (Figure 2, panel J),
Neuro2a (Figure 2, panel K), and STHdhQ7/Q7 cells (Figure 2, panel L). Inhibition of orthosteric
agonistꢀmediated βꢀarrestin1 recruitment by Org27569, PSNCBAMꢀ1, and GAT100 was
observed here, as has been shown elsewhere.29, 48, 52, 53 Although Org27569 has been reported to
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promote the recruitment of βꢀarrestin1 in immunohistochemical coꢀlocalization assays54, βꢀ
arrestin1 recruitment was not observed here using the BRET assay.
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To quantify the concentrationꢀdependence of test compounds for βꢀarrestin1 recruitment,
BRETEff between hCB1RꢀGFP2 and βꢀarrestin1ꢀRluc was then measured in HEK293A (Figure
3, panels A, D, and G), Neuro2a (Figure 3, panels B, E, and H), and STHdhQ7/Q7 cells (Figure 3,
panels C, F, and I) treated with 1 nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 and 500
nM 2ꢀAG (Figure 3, panels AꢀC), AEA (Figure 3, panels DꢀF), or CP55,940 (Figure 3, panels Gꢀ
I) for 30 min. As summarized in Table 1, GAT100 was a more potent NAM of CB1Rꢀ βꢀ
arrestin1 recruitment than either Org27569 or PSNCBAMꢀ1 in all cell types tested and with all
orthosteric ligands. No difference in potency (IC50) or efficacy (Emax) was observed in cells
treated with GAT100 and 2ꢀAG, AEA, or CP55,940. In contrast, Org27569 and PSNCBAMꢀ1
displayed probe dependence in all cell types, since both were more potent and efficacious NAMs
in 2ꢀAGꢀtreated cells as compared to AEAꢀ and CP55,940ꢀtreated cells (Table 1).
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CB1Rꢀmediated PLCβ3 Phosphorylation. We previously reported that the Gαq effector
PLCβ3 is phosphorylated via CB1R following treatment of STHdhQ7/Q7 cells with 2ꢀAG, AEA,
or CP55,940.55 We have now measured CB1Rꢀdependent PLCβ3 phosphorylation in HEK293A
cells transiently transfected with hCB1RꢀGFP2ꢀexpressing plasmid (Figure 4, panels A, D, and
G) and in Neuro2a (Figure 4, panels B, E, and H), and STHdhQ7/Q7 cells (Figure 4, panels C, F,
and I) expressing endogenous CB1R upon cell treatment with 1 nM – 10 ꢁM 2ꢀAG (Figure 4,
panels AꢀC), AEA (Figure 4, panels DꢀF), or CP55,940 (Figure 4, panels GꢀI) with or without 1
ꢁM GAT100, Org27569, or PSNCBAMꢀ1 for 10 min. GAT100 treatment reduced the potency
(EC50) and efficacy (Emax) of agonistꢀdependent PLCβ3 phosphorylation to a greater extent than
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vehicle, Org27569, and PSNCBAMꢀ1 in all cell types and with all orthosteric ligands (Figure 4).
The decrease in Emax observed in the presence of Org27569 or PSNCBAMꢀ1 is presented in
Table 2. To assess possible intrinsic activity associated with these CB1R allosteric modulators,
PLCβ3 phosphorylation was also measured in HEK293A (Figure 4, panel J), Neuro2a (Figure 4,
panel K), and STHdhQ7/Q7 cells (Figure 4, panel L) treated with 1 nM – 10 ꢁM GAT100,
Org27569, or PSNCBAMꢀ1 for 10 min. GAT100, Org27569, and PSNCBAMꢀ1 had no effect on
basal PLCβ3 phosphorylation in the absence of orthosteric agonists, indicating a lack of intrinsic
activity in this assay (Figure 4, panels JꢀL).
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To obtain comparative potency (IC50) values for the effect of these allosteric ligands on
CB1Rꢀmediated PLCβ3 phosphorylation, we used HEK293A (Figure 5, panels A, D, and G),
Neuro2a (Figure 5, panels B, E, and H), and STHdhQ7/Q7 cells (Figure. 5, panels C, F, and I)
treated with 1 nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 and 500 nM 2ꢀAG (Figure 5,
panels AꢀC), AEA (Figure 5, panels DꢀF), or CP55,940 (Figure 5, panels GꢀI) for 10 min.
GAT100 was a more potent NAM of CB1Rꢀdependent PLCβ3 phosphorylation than either
Org27569 or PSNCBAMꢀ1 in all cell types and with all orthosteric ligands tested (Table 2).
GAT100 displayed marginal orthostericꢀprobe dependence in CP55,940ꢀtreated HEK293A and
STHdhQ7/Q7 cells, being less potent than in 2ꢀAGꢀ or AEAꢀtreated cells (Table 2). In contrast,
Org27569 displayed probeꢀdependence in HEK293A and Neuro2a cells as a more potent NAM
in 2ꢀAGꢀtreated HEK293A cells and in 2ꢀAGꢀ and CP55,940ꢀtreated Neuro2a cells (Table 2).
PSNCBAMꢀ1 was a more potent NAM in AEAꢀtreated HEK293A cells, CP55,940ꢀtreated
Neuro2a cells, and 2ꢀAGꢀtreated STHdhQ7/Q7 cells as compared to other orthosteric ligands
(Table 2). No orthostericꢀprobe dependence in efficacy (Emax) was observed among cells treated
with GAT100, Org27569, or PSNCBAMꢀ1 and 2ꢀAG, AEA, or CP55,940 (Table 2).
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CB1Rꢀmediated ERK1/2 Phosphorylation. Rapid, transient ERK1/2 phosphorylation
of the MAPK family member, ERK1/2, is a hallmark of CB1R activation via Gαi/o.56
Consequently, we evaluated CB1Rꢀdependent ERK1/2 phosphorylation in HEK293A cells
transiently transfected with hCB1RꢀGFP2ꢀexpressing plasmid (Figure 6, panels A, D, and G) and
in Neuro2a (Figure 6, panels B, E, and H) and STHdhQ7/Q7 cells (Figure 6, panels C, F, and I)
expressing endogenous CB1R in response to treatment with 1 nM – 10 ꢁM 2ꢀAG (Figure 6,
panels AꢀC), AEA (Figure 6, panels DꢀF), or CP55,940 (Figure 6, panels GꢀI) with or without 1
ꢁM GAT100, Org27569, or PSNCBAMꢀ1 for 10 min. GAT100 reduced the potency and efficacy
of orthosteric agonistꢀinduced ERK1/2 phosphorylation to a greater extent than vehicle,
Org27569, and PSNCBAMꢀ1 in all cell types and with all orthosteric ligands tested (Figure 6,
Table 3).
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We next determined the effect of allosteric ligands on CB1Rꢀdependent ERK1/2
phosphorylation. For this purpose, ERK1/2 phosphorylation was measured in HEK293A (Figure
6, panel J), Neuro2a (Figure 6, panel K), and STHdhQ7/Q7 cells (Figure 6, panel L) treated with 1
nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 for 10 min. GAT100, Org27569, and
PSNCBAMꢀ1 had no effect on basal ERK1/2 phosphorylation in the absence of orthosteric
agonists (Figure 6, panels JꢀL). ERK1/2 phosphorylation was then measured in HEK293A
(Figure 7, panels A,D, and G), Neuro2a (Figure 7, panels B, E, and H), and STHdhQ7/Q7 cells
(Figure 7, panels C, F, and I) treated with 1 nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1
and with 500 nM 2ꢀAG (Figure 7, panels AꢀC), AEA (Figure 7, panels DꢀF), or CP55,940
(Figure 7, panels GꢀI) for 10 min. GAT100 was a more potent NAM of CB1Rꢀdependent
ERK1/2 phosphorylation than either Org27569 or PSNCBAMꢀ1 in all cell types and with all
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orthosteric ligands tested (Table 3). GAT100 only displayed probeꢀdependence in CP55,940ꢀ
treated HEK293A cells, where GAT100 was less potent compared to 2ꢀAGꢀ or AEAꢀtreated cells
(Table 3). In contrast, Org27569 was most potent in the presence of 2ꢀAG in HEK293A cells,
AEA in Neuro2a cells, and CP55,940 in STHdhQ7/Q7 cells (Table 3). PSNCBAMꢀ1 was most
potent in the presence of 2ꢀAG in HEK293A cells and AEA in Neuro2a cells, but did not display
orthostericꢀprobe dependence in STHdhQ7/Q7 cells (Table 3). No probe dependence was observed
in terms of efficacy for any of the three allosteric ligands examined (Emax). Inhibition of
orthosteric agonistꢀmediated Gαi/o signaling by Org27569, PSNCBAMꢀ1, and GAT100 was
observed here, as has been shown elsewhere.29, 48, 52, 53 In contrast, Org27569 has been reported
to promote the ERK1/2 phosphorylation via βꢀarrestin1 in the absence of orthosteric ligand in
western blot experiments54, Org27569ꢀdependent ERK1/2 phosphorylation was not observed in
the Inꢀcell western assay used here.
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CB1Rꢀmediated Adenylate Cyclase Activity. Cellular adenylate cyclase activity has
been used extensively to index Gαiꢀdependent signal modulation by CB1R allosteric ligands.27
We used a luciferaseꢀbased reporter assay to indirectly measure CB1Rꢀdependent modulation of
forskolinꢀstimulated adenylate cyclase activity (i.e., as modulation of cAMP accumulation) in
HEKꢀCRE cells transiently transfected with hCB1RꢀGFP2ꢀexpressing plasmid and treated with
10ꢁM forskolin and 1 nM – 10 ꢁM 2ꢀAG (Figure 8, panel A), AEA (Figure 8, panel B), or
CP55,940 (Figure 8, panel C) with or without 1 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 for 4
h. Relative to orthosteric ligand alone, GAT100 shifted the orthosteric ligandꢀcAMP
concentrationꢀresponse curves rightward to a greater extent than Org27569 or PSNCBAMꢀ1 in
the presence of 2ꢀAG, AEA, or CP55,940 (Figure 8, panels AꢀC). In HEKꢀCRE cells treated with
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1 nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 for 4 h, Org27569 and PSNCBAMꢀ1
alone, but not GAT100, increased cAMP levels significantly in the absence of orthosteric
agonists (Figure 8, panel D). Therefore, Org27569 and PSNCBAMꢀ1, but not GAT100,
demonstrated measurable inverse agonist activity in the cAMP assay, as has been shown
previously for Org27569 and PSNCBAMꢀ1.29, 36, 52, 53
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Accumulation of cAMP was also measured in HEKꢀCRE cells treated with 10 µM
forskolin in combination with 1 nM – 10 ꢁM GAT100, Org27569, or PSNCBAMꢀ1 and with
500 nM 2ꢀAG (Figure 9, panel A), AEA (Figure 9, panel B), or CP55,940 (Figure 9, panel C) for
4 h. In the absence of orthosteric agonist, GAT100 did not affect cAMP accumulation, whereas
Org27569 and PSNCBAMꢀ1 both increased cellular cAMP in a concentrationꢀdependent manner
(Figure 9). These results support the literature evidence that Org27569 and PSNCBAMꢀ1
possess inverseꢀagonist activity,28, 29, 37 whereas GAT100 does not.48, 57 Furthermore, GAT100
was the most potent and efficacious inhibitor of cAMP accumulation in the HEKꢀCRE cells
treated with orthosteric agonist (Figure 9). The data in Figure 9 also demonstrate that GAT100,
Org27569, and PSNCBAMꢀ1 did not display probe dependence in HEKꢀCRE cells with respect
to potency (IC50) or efficacy (Emax) in the cAMP assay. The data presented in Figure 8, panel 8,
and Figure 9 demonstrate that GAT100 did not display inverse agonist activity at Gαiꢀdependent
signal pathways.
CP55,940ꢀdependent CB1R Internalization. As with other GPCRs, CB1R
internalization is recognized as a temporal and spatial modulator of CB1Rꢀmediated signaling.58,
59
Accordingly, we determined the effect of allosteric ligands on CP55,940ꢀinduced CB1R
internalization in STHdhQ7/Q7 cells using Onꢀ and Inꢀcell™ Western analysis. CP55,940 alone
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resulted in sustained CB1R internalization beginning at 30 min after treatment inception (Figure
10). GAT100, Org27569, or PSNCBAMꢀ1 reversed this effect, increasing the fraction of CB1R
at the plasma membrane. GAT100 elicited a more rapid reversal of CB1R internalization than
Org27569 or PSNCBAMꢀ1.
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Summary of potency and efficacy for GAT100, Org27569, and PSNCBAMꢀ1. Table
5 summarizes the signaling profile of GAT100 across the activity assays, cell systems, and
orthosteric ligands utilized along with the parallel data for Org27569 and PSNCBAMꢀ1. All
three allosteric modulators profiled in this study were active in each of the signaling assays
conducted. The potencies observed for Org27569 and PSNCBAMꢀ1 were comparable to
published values for arrestin recruitment and G proteinꢀdependent [35S]GTPγS binding
conducted with cellular CB1R overexpression and isolatedꢀmembrane systems.28, 29, 36 However,
in the presence of CP55,940 or the endocannabinoids 2ꢀAG or AEA, GAT100 was more potent,
and efficacious NAM than Org27569 and PSNCBAMꢀ1.
To determine whether GAT100 displayed functional selectivity, the NAM activity of
GAT100 was characterized at several distinct pathways: βꢀarrestin1 (G proteinꢀindependent),
PLCβ3 (Gαqꢀdependent), ERK1/2 and adenylate cyclase (cAMP) (Gαi/oꢀdependent). GAT100
was a more potent inhibitor of βꢀarrestin1 recruitment compared to its inhibitory potency in the
ERK1/2, and cAMP assays. GAT100, Org27569, and PSNCBAMꢀ1 were generally more
efficacious inhibitors of adenylate cyclaseꢀmediated pathways as compared to their efficacy in
the βꢀarrestin1, PLCβ3, and ERK1/2 assays.
Potential signaling bias was further analyzed using to the global nonꢀlinear regression
operational model of Black and Leff (eq. 1).60 Concentrationꢀresponse data from Figures 3, 5, 7,
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and 9 were fit to that operational model in order to calculate the transduction coefficients [logR
(τ/ KA)] of each allosteric modulator and its relative activity (ꢂlogR) as compared to GAT100 in
the presence of 2ꢀAG (eq. 2) (Table S1). GAT100 in the presence of 500 nM 2ꢀAG was chosen
as the reference ligand because it was the most potent and efficacious compound in all assays
used; all other agents were compared to the transduction coefficient (logR) of GAT100. Relative
activity values were then used to calculate a bias factor (ꢂꢂlogR) (eq. 3) for each signaling
pathway relative to βꢀarrestin1 (Table 6). Within this model, GAT100 displayed signaling bias
for βꢀarrestin1 compared to PLCβ3 (in the presence of AEA in Neuro2a and STHdhQ7/Q7 cells)
and ERK1/2 (in the presence of AEA and CP55,940 in Neuro2a cells). Org27569 displayed
signaling bias against βꢀarrestin1 compared to PLCβ3 (in the presence of AEA in HEK293A
cells) and cAMP (in the presence of AEA in HEK293A cells). PSNCBAMꢀ1 displayed signaling
bias against βꢀarrestin1 compared to PLCβ3 (in the presence of AEA and CP55,940 in
HEK293A cells) and ERK1/2 (in the presence of AEA in HEK293A cells). The Black and Leff
operational model has been used to study CB1R agonist bias.52, 61 This study is the first to apply
the Black and Leff operational model to CB1R NAMs.
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The present study is the first evaluation of Org27569 and PSCNBAMꢀ1 for potential
PLCβ3ꢀrelated pathway bias.36, 52, 54, 62 Previous observations have been made that Org27569 is
biased against both Gαi/o and βꢀarrestin signaling.52, 54 Such differences among studies likley
reflect the wellꢀrecognized difficulty in extrapolating ligand bias across different assay
64
systems/experimental conditons, especially when comparing ligands.63
An important
advantage of using the operational model to estimate the relative activity and ligand bias is that
this model negates the effects of differences in receptor density.65 Therefore, differences in bias
between cell types are not the result of changes in agonist potency or efficacy.65, 66
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Recognition of CP55,940 as a standard reference synthetic cannabinoid and 2ꢀAG and
AEA as the principal endocannabinoids justified their use in this study to determine if GAT100
displayed orthostericꢀprobe dependence.1 When data from different cell models and assays were
pooled to calculate arithmetic means, GAT100, Org27569, and PSNCBAMꢀ1 did not display
significant orthostericꢀprobe dependence among CP55,940, 2ꢀAG, and AEA (Table 5). Baillie et
al.36 reported that the Org27569 and PSNCBAMꢀ1 both showed probeꢀdependence as more
potent modulators of CP55,940 activity as compared to WIN 55,212ꢀ2. Khajehali et al.52
observed that Org27569 fully inhibits CP55,940, only weakly inhibits 2ꢀAG, and does not inhibit
AEA activity. Neither of these cited studies examined PSNCBAMꢀ1 probeꢀdependence with the
endocannabinoids 2ꢀAG or AEA.
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Furthermore, GAT100, Org27569, and PSNCBAMꢀ1 did not display cellꢀdependence
among HEK293A, Neuro2a, and STHdhQ7/Q7 cells (Table 5). The lack of orthostericꢀprobe and
cellꢀdependence suggests that all three CB1R allosteric modulators could be active in distinct
biological environments.
Notably, GAT100 could be differentiated pharmacologically from both Org27569 and
PSNCBAMꢀ1 according to therapeuticallyꢀrelevant characteristics. As compared to those
prototypic CB1R allosteric ligands across all cell types and information pathways examined,
GAT100 was a more potent and efficacious NAM of endocannabinoid activity (Table 5) and was
devoid of the appreciable inverseꢀagonist activity48 we and others have observed to be a hallmark
of both Org27569 and PSNCBAMꢀ1.28, 29, 36 By directly evaluating the downstream signaling
profiles of these three CB1R NAMs in parallel across three distinct cell systems with both
overexpressed and native CB1R and assessing multiple Gꢀproteinꢀdependent and ꢀindependent
signaling pathways, we have countered the possibility that the GAT100 signaling pattern
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described and the pharmacological differences uncovered between GAT100 vs. Org27569 and
PSNCBAMꢀ1 reflect assay or cellꢀcontext dependent factors rather than the multidimensional
information network of CB1R itself.63, 64
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Effect of GAT100 on [3H]SR141716A Equilibrium Binding by hCB1R.
GAT100, Org27569, and PSNCBAMꢀ1 increase CP55,940 equilibrium binding by CB1R,
29, 48
GAT100 being the most potent and efficacious enhancer.28,
Both Org27569 and
PSNCBAMꢀ1 can displace the CB1R antagonist/inverse agonist SR141716A, suggesting that the
binding domains of these three ligands may overlap.37 These combined data invited examination
of the effect of GAT100 on equilibrium binding of SR141716A in order to gain some insight into
the CB1R GAT100 binding domain. At 1 and 10 µM, both GAT100 and Org27569 reduced
[3H]SR141716A specific binding to membranes obtained from CHO cells transfected with
hCB1R in a concentrationꢀrelated manner (Figure 11). Unlike Org27569, GAT100 did not
completely abrogate [3H]SR141716A binding at a concentration of 10 ꢁM. GAT100 displayed ~
6ꢀfold greater potency (EC50 = 409.8 nM) than Org27569 (EC50 = 2522 nM) as an inhibitor of
[3H]SR141716A binding.
Computational Modeling of the Interaction Profile between GAT100 and
Intermediate State hCB1R (hCB1R**). The absence of a reported CB1R crystal structure led
us to apply computational methods for gaining insight into the GAT100 binding site within
hCB1R and potential GAT100ꢀamino acid interactions critical to the allosteric ligand’s
functional pharmacology. Four considerations guided to this effort: (a) Ligand binding to CB1R
and other class A GPCRs is widely accepted to occur within transmembrane helices (TMHs) and
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extracellular loops (ECLs) with lipophilic ligands predisposed to accessing the TMH bundle via
the membrane lipid bilayer.43, 67(b) GAT100’s reactive isothiocyanate functionality is a
conservative modification of the parent Org27569 structure at the critical Cꢀ5 position such that
Org27569 can be used as direct comparator to GAT100 (Figure 1).48 (c) Under physiological
incubation/reaction conditions as used in our GAT100ꢀCB1R studies (i.e., aqueous milieu and
physiological pH), the nucleophilic thiol moiety of cysteine residues renders them by far the
most reactive nucleophilic amino acid toward various electrophiles, including isothiocyanates68-
70, making cysteine the most likely participant in a nucleophilic addition reaction with the
isothiocyanate GAT100. Indeed, under these conditions, isothiocyanates have been recognized
as “sulfhydrylꢀreactive affinity ligands,” and as such useful probes “to investigate ligandꢀ
receptor interactions.”69 The avidity and facile reactivity of designer isothiocyanate probes for
GPCR cysteine residues has been previously exploited by us and others to help map critical
ligandꢀinteraction domains.41, 43-45 Given that GAT100 is an isothiocyanate, the above contextꢀꢀ
in addition to prior in silico and experimental descriptions of CB1R next discussedꢀꢀ provides a
sound rationale for focusing the modeling studies on cysteine and no other nucleophilic amino
acids. (d) Of the 13 total hCB1R cysteine residues, two ECL cysteines (C257 and C264) are
esential to hCB1R highꢀlevel expression and function by virtue of the disulfide bridge between
them that would preclude their reaction with GAT100.43 Six other hCB1R cysteines may
likewise be eliminated as potential covalent attachment sites for GAT100 because of their
location outside of hCB1R TMHs. The five remaining hCB1R cysteine residues [C1.55(139),
C4.47(238), C6.47(355), C7.38(382), and C7.42(386)] are located in hCB1R TMHs. C1.55(139)
and C4.47(238) are on the intracellular side of the CB1R 7ꢀTMH bundle, and face outward
toward bilayer lipid71, 72, rendering them potential targets for GAT100 molecules in the inner
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leaflet of the bilayer. In the CB1R inactive state, C7.42(386) is oriented toward the binding site
crevice, whereas C6.47(355) and C7.38(382) are oriented toward lipid with C6.47(355) deeper in
the lipid bilayer, making these latter two residues potential targets for GAT100 in the outer
bilayer leaflet.
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We used molecular dynamics (MD) simulation to identify the CB1R TMH cysteine
residue most likely to react with GAT100. For this purpose, a model was employed representing
a receptor conformation (hCB1R**) in a lipid bilayer intermediate between inactive (R) and
active (R*) states that is promoted by Org27569 in the presence of CP55,940 and that
preferentially binds agonist but cannot signal in G proteinꢀmediated pathways, as elaborated
elsewhere.37 Given that GAT100 is a direct Org27569 analog, we first analyzed MD profiles for
Org27569ꢀhCB1R** in a 1ꢀpalmitoylꢀ2ꢀoleoylꢀphosphatidylcholine (POPC) bilayer across
fourteen Org27569 trajectories. As illustrated by the MD snapshot in Figure 12, Org27569
contoured at its Van der Walls radii is horizontal to the POPC membrane acyl chains and sits two
turns down from the extracellular TMH termini. In this figure, the phosphorus atoms of the
phospholipid head groups are shown contoured at their Van der Waals radii, but the rest of the
lipid, including acyl chains, have been turned off for clarity. The Org27569 indole ring is
located at the level of C7.38(382) and is higher in the bilayer than either C7.42(386) or
C6.47(355). Over both 1ꢁs MD trajectories, only C1.55(139) and C7.38(382) achieved a
distance of less than 5 Å from the Org27569 chlorine atom, a proximity making them the most
likely cysteine residues to participate in a nucleophilicꢀaddition reaction with GAT100.
C1.55(139) is part of a cholesterol binding site73 formed by 44% of Class A GPCRs at the
intracellular end of the receptor in the TMH1ꢀ2ꢀ3ꢀ4. Although CB1 does not have the exact
sequence motif for this binding site, recent covalent labeling studies identified this region as a
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possible site for Org27569 interaction with CB1.74 However, binding at this site does not explain
the positive allosteric effects that Org27569 has upon CP55,940 binding, negative allosteric
effects upon CP55,940 signaling and partial displacement of SR141716A binding rendering
C1.55(139) an unlikely candidate for interaction with GAT100.28, 74
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Previous studies have identified CB1R C6.47(355) as the site of CB1R covalent labeling
by analogs of classical cannabinoids43, 44 and endocannabinoids46 derivatized with reactive
isothiocyanate functionalities. However, our MD analysis indicates that Org27569 resides, on
average, higher in the lipid bilayer from C6.47(355), nearer to C7.38(382) (Figure 12). These
aggregate data with parent compound Org27569 led us to focus subsequent modeling studies on
covalent GAT100ꢀC7.38(382) interaction in the outer leaflet of the membrane.
GAT100 Covalent Labeling C7.38(382). Modeling studies revealed that if GAT100
labels C7.38(382), it can enter the CB1R** bundle above CP55,940 such that it can interact with
CP55,940, while simultaneously interacting with binding site residues, including I6.54, D6.58,
F7.35, P(269) and F(268). CP55,940 retains all of its interactions in CB1R** including the
crucial hydrogen bond with K3.28.37 Figure 13 shows GAT100 (purple) covalently attached to
C7.38(382) interacting with CP55,940 (green) in the CB1R** binding site. The conformational
energy expense for GAT100 to assume this position in the binding pocket was 1.00 kcal/mol.
Figure S2 shows an overlay of the global minimum energy conformer of Org27569 to the
conformation of GAT100. It is clear here that the two compounds overlay very well. The
interaction energy between GAT100 and CP55,940 in this dock was calculated to be ꢀ2.08
kcal/mol. The total interaction energy for GAT100 in Figure 13 is ꢀ41.2 kcal/mol. The
interaction energies with individual receptor residues are given in Table S2. CP55,940 in this
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dock (Figure 13) has a total interaction energy of ꢀ61.8 kcal/mol. The CP55,940 interaction
energies with individual receptor residues are provided in Table S3.
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GAT100 as Positive Allosteric Modulator of CP55,940 Binding. Tables S1 and S2 show
that CP55,940 has a favorable interaction energy (ꢀ2.08 kcal/mol) with GAT100. In addition,
GAT100 is located above CP55,940, blocking CP55,940 exit from the binding pocket. Both
effects will increase CP55,940 binding affinity. Thus, GAT100 functions here as a positive
allosteric modulator of CP55,940 binding.
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GAT100 as a Negative Allosteric Modulator of CP55,940 Signaling. Class A GPCR
activation is accompanied by TMH conformational changes, as well as changes in IC and EC
loop conformations. We have previously reported that upon receptor activation, an important
ionic interaction forms between TMH2 and the ECꢀ3 loop (specifically, an interaction forms
between residues D2.63(176) and K373)75; this ionic interaction is necessary for signal
transduction and promotes a conformation of the ECꢀ3 loop that is pulled over the top
(extracellular face) of the receptor (see Figure 11A in reference37 ). We have shown previously
that Org27569 can impact CP55,940 signaling by interrupting this ECꢀ3 loop movement.37
Figure 14 illustrates that when GAT100 is bound in the CB1 R** binding pocket, it sits high
enough to prevent the D2.63(176) / K373 interaction. In this way, GAT100 can prevent
CP55,940 signaling.
GAT100 Displacement of SR141716A. Figure 15A provides an extracellular view of
SR141716A (orange) docked in CB1 R**.76 Figures 15B (view from extracellular) and 4C
(view from lipid bilayer) show that when GAT100 (purple) is docked in CB1 R**, it partially
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overlaps the SR141716A binding site. This overlap would account for the partial displacement of
SR141716A binding by GAT100.
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It remains for future studies to determine whether GAT100’s superior potency and
efficacy; its Gꢀproteinꢀindependent, βꢀarrestin1ꢀmediated signaling bias; and its freedom from
significant inverseꢀagonist activity engender (pre)clinical therapeutic advantages in vivo. In this
regard, it is noteworthy that the onꢀtarget specificity and selectivity associated with GPCR
covalent ligands have been considered to hold promise of improved drug safety, tolerance, and
efficacy77, and arrestinꢀbiased GPCR ligands can promote salutary effects and simultaneously
dampen deleterious ones in vivo.64 Arguably of greatest translational relevance, the propensity
of Org27569 and PSNCBAMꢀ1 to interfere with constitutive CB1R signaling through their
inverseꢀagonist action is not shared by GAT100, making it tempting to speculate that GAT100
might have less potential to induce the adverse events associated with typical orthosteric CB1R
antagonists/inverse agonists and suggesting more generally that the binding sites for NAM and
inverse agonist activity can be separated. By extension, NAMs such as GAT100 might represent
a tenable route toward resolving the beneficial and deleterious routes of CB1R information
output and reducing the sideꢀeffect risk of modulating/attenuating CB1R signaling for
therapeutic exploitation. Furthermore, GAT100’s more rapid reversal of agonistꢀinduced CB1R
internalization may beneficially attenuate net receptor desensitization, such as that induced by
increased orthosteric ligand binding to the receptor. The unique pharmacological distinctions
between GAT100 and parent NAM Org27569 in our signaling assays suggest that (non)covalent
GAT100 derivatives with alternative modifications at the Cꢀ5 site are worthy of synthesis and
cellꢀbased profiling. Such derivatives could be purposeꢀdesigned to react with amino acids other
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than cysteine and validated as probes for more global experimental identification of critical
ligandꢀinteraction sites within the allosteric hCB1R binding domain(s).41
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CONCLUSION
Biologicallyꢀactive allosteric ligands capable of fineꢀtuning GPCR information output are
being increasingly sought for their potential advantages over orthostericallyꢀdirected drugs,
specifically in terms of improved onꢀtarget potency, efficacy, and safety.78, 79 In this regard,
allosteric modulation of the principal GPCR in mammalian brain, CB1R, has gained particularly
intense interest as a therapeutic modality from the unacceptable adverseꢀevent risk associated
with typical orthosteric CB1R agonists and antagonists/inverse agonists that has plagued their
development as drugs. For those GPCRs such as CB1R for which crystal structures remain
elusive, molecular probes have assumed increasing importance for interrogating receptor ligandꢀ
binding domains and elucidating the functional consequences of ligand engagement in order to
guide rational drug design.42 The availability of the first, purposeꢀdesigned CB1R covalent
allosteric probe and Org27569 analog, GAT100, has enabled initial application of such an
experimental approach to this wellꢀrecognized drug target.
The present work constitutes the detailing of GAT100’s downstream signaling profile
and the application of computational methods to gain insight into the structureꢀfunction
correlates of GAT100 engagement by CB1R. Our ligandꢀdocking and in silico analysis of the
GAT100ꢀhCB1R** interaction profile identifies C7.38(382) as the hCB1R** amino acid with
which GAT100 is most likely to participate in a covalent interaction by virtue of its
isothiocyanate moiety. The availability of GAT100 as a functional, allostericꢀsite covalent probe
enables additional experimental studies aimed at further characterization of CB1R allosteric
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ligandꢀbinding motifs and the pharmacology of CB1R functional modulation by such a ligand.
Determination of the effect of targeted CB1R mutations on GAT100ꢀCB1R complex formation
and the application of peptideꢀlevel tandem mass spectrometry to define directly the allostericꢀ
site amino acid(s) reacting with this covalent ligand are also made possible. GAT100 may be
used to stabilize allosteric CB1R conformations to aid their crystallization as prerequisite to
atomicꢀlevel Xꢀray structure analysis of the liganded receptor. Proofꢀofꢀconcept animal studies
aimed at defining the effects of GAT100 in vivo and interrogating the extent to which GAT100ꢀ
sensitive signaling pathways identified herein are responsible for those effects could help in
realizing the therapeutic promise and purported translational advantages of covalent allosteric
GPCR modulators.
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MATERIALS AND METHODS
Compounds. 2ꢀAG, AEA, and CP55,940 were purchased from Tocris Bioscience
(Bristol, UK). Org27569, PSNCBAMꢀ1, and GAT100 were synthesized in the laboratory of Dr.
Ganesh A Thakur (Northeastern University, Boston, MA). All compounds were dissolved in
DMSO (final concentration, 0.1% in culture media for all cellꢀbased assays) and added directly
to the media at concentrations and times indicated.
Amino Acid Descriptor. The BallesterosꢀWeinstein formalism is used to designate the
loci of specific amino acids as derived from the hCB1R sequence numbers.37, 80 In this system,
the most highly conserved amino acid residue in each TMH is assigned a locant of 0.5. This
number is preceded by the helix number followed in parentheses by the sequence number. All
other residues in a TMH are numbered relative to that residue.
Cell Culture. HEK293A cells were a generous gift from Dr. Denis J Dupré (Dalhousie
University, Nova Scotia, Canada) and were originally obtained from the American Type Culture
Collection (ATCC, Manaassas, VA). HEK293A were transfected with 400 ng of the CB1ꢀ
GFP2ꢀexpressing plasmid described below using Lipofectamine 2000 according to the
manufacturer’s instructions (Invitrogen, Burlington, ON, Canada). HEK293A cells were
maintained at 37°C, 5% CO2ꢀ95% air in Dulbecco’s minimal essential medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 104 U/mL penicillin (Pen)/streptomycin
(Strep). HEK293A Cignal Lenti CRE (HEKꢀCRE) reporter cells were a generous gift by Dr.
Christopher J Sinal (Dalhousie University, Nova Scotia, Canada). HEKꢀCRE cells stably express
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the firefly luciferase gene driven by tandem repeat elements of the cAMP transcriptional
response element (Qiagen, Toronto, Ontario, Canada). Thus, the luciferase signal is directly
proportional to cAMP/ protein kinase A (PKA) pathway activity. HEKꢀCRE cells were
maintained at 37°C, 5% CO2ꢀ95% air in DMEM supplemented with 10% FBS, 104 U/mL
Pen/Strep, and 200 ꢁg/mL puromycin. Neuro2a mouse neuroblastoma stem cells were purchased
from ATCC and maintained 37°C, 5% CO2ꢀ95% air in Eagle’s minimal essential medium
(MEM) supplemented with 10% FBS and 104 U/mL Pen/Strep.81 STHdhQ7/Q7 cells are derived
from the conditionally immortalized striatal progenitor cells of embryonic day 14 C57BlJ/6 mice
(Coriell Institute, Camden, NJ) were maintained at 33°C, 5% CO2ꢀ95% air in DMEM
supplemented with 10% FBS, 2 mM Lꢀglutamine, 104 U/mL Pen/Strep, and 400 ꢁg/mL
geneticin.82 Cells were serumꢀdeprived for 24 h prior to experiments to promote
differentiation.55, 82
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Plasmids. Human CB1ꢀgreen fluorescent protein2 (GFP2) Cꢀterminal fusion protein was
generated using the pGFP2ꢀN3 (PerkinElmer, Waltham, MA) plasmid, as described previously.83
Human arrestin2 (βꢀarrestin1)ꢀRenilla luciferase (Rluc) Cꢀterminal fusion protein was generated
using the pcDNA3.1 plasmid and provided by Dr. Denis J Dupré (Dalhousie University, NS,
CAN). The GFP2ꢀRluc fusion construct, and Rluc plasmids have also been described.83
CHO cells. Chinese hamster ovary (CHO) cells transfected with cDNA encoding human
cannabinoid CB1 receptors were maintained at 37°C in Dulbecco’s modified Eagle’s medium
nutrient mixture Fꢀ12 HAM, supplemented with 1 mM Lꢀglutamine, 10% fetal bovine serum,
0.6% penicillin–streptomycin and G418 (400 mg/ml). All cells were exposed to 5% CO2 in their
media, and were passaged twice a week using nonꢀenzymatic cell dissociation solution. For
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