Human Cannabinoid Receptor 2 Ligand-Interaction Motif

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Human Cannabinoid Receptor 2 Ligand-interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose Han Zhou, Yan Peng, Aneetha Halikhedkar, Pusheng Fan, David R. Janero, Ganesh A Thakur, Richard Warren Mercier, Xin Sun, Xiaoyu Ma, and Alexandros Makriyannis ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00003 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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ACS Chemical Neuroscience

1

Human Cannabinoid Receptor 2 Ligand-interaction Motif: Transmembrane

Helix

2

Cysteine,

C2.59(89),

as

Determinant of Classical Cannabinoid Agonist Activity and Binding Pose

Han Zhou, Yan Peng, Aneetha Halikhedkar, Pusheng Fan, David R. Janero, Ganesh A. Thakur, Richard W. Mercier, Xin Sun, Xiaoyu Ma, and Alexandros Makriyannis*

Center for Drug Discovery and Departments of Chemistry and Chemical Biology, Pharmaceutical Sciences, and Bioengineering; College of Science, Bouvé College of Health Sciences, and College of Engineering; Northeastern University, Boston, MA 02115-5000, USA

Please address all editorial correspondence regarding this manuscript to: David R. Janero, Ph.D. Northeastern University Center for Drug Discovery; MUGAR 116 360 Huntington Avenue; Boston, MA 02115-5000 USA Direct-dial phone: 617-266-4234; FAX: 617-373-7493 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate drug-like hCB2R agonists. transmembrane

helix

2

(TMH2)

cysteine

Prior demonstration that hCB2R

C2.59(89)

reacts

with

small-molecule

methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents.

We now report the design and application of two novel, pharmacologically active,

high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ~ 60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine.

In contrast, AM841, an exceptionally potent hCB2R megagonist and

direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257), and not C2.59(89).

Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89)

orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in


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the receptor’s hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7.

These data constitute initial evidence

that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

KEYWORDS: binding motif; cannabinoid receptor 2, covalent chemical probe, cysteine, G-protein coupled receptor, isothiocyanate, ligand-binding domain, molecular probe, signal transduction, transmembrane helix



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INTRODUCTION The endocannabinoid signaling system is a ubiquitous information-transducing network that 1

helps regulate diverse aspects of mammalian (patho)physiology. Activation of two principal cell-surface G protein-coupled receptors (GPCRs), designated cannabinoid receptor 1 (CB1R) and 2 (CB2R), is the primary means of signal transduction from the cannabinergic lipid 2

mediators they engage.

As a regulator of synaptic activity, CB1R is among the most highly

expressed GPCRs in the central nervous system (CNS), and its activation by agonists including (−)-trans-∆9-tetrahydrocannabinol (THC), a “classical” cannabinoid and the main psychoactive 3

ingredient of marijuana (Figure 1A), is associated with psychotrophic effects.

Although the 4,5

extent of CB2R expression in neurons and brain endothelium remains a matter of debate,

consensus evidence supports high levels of functional CB2R expression in activated microglia, a resident population of macrophage-like cells in the CNS that plays a critical role in central 6

immune surveillance.

CB2R upregulation/activation in brain microglia and astrocytes is

considered tissue-protective against brain injury, attenuating proinflammatory cytokine release 7-10

and increasing anti-inflammatory cytokine production by these cells to favor neuron survival.

The etiologies of both acute CNS injury (e.g., cerebral trauma, ischemia) and several age-related degenerative neuropathies with uniformly poor prognosis (e.g., Alzheimer’s, Huntington’s, and Parkinson’s diseases) have well-recognized inflammatory components, and at least some features of their usually severe phenotypes are ameliorated by pharmacological CB2R activation in 11-15

laboratory disease models.

CB2R agonists can also reduce CNS inflammation and rescue

impaired neurogenesis in murine models of human immunodeficiency virus (HIV) insult and


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16-19

exert analgesic activity in preclinical models of acute, inflammatory, and neuropathic pain.

In man, low-function CB2R haplotypes arising from human CNR2 gene polymorphisms are associated with susceptibility to neuropsychiatric problems including schizophrenia and 20-22

substance-use, eating, and affective disorders.

Furthermore, the 1p36.11 chromosome

region to which CNR2 maps plays a key role in several autoimmune diseases such that CN2R 23

variants can increase the risk of susceptibility to celiac disease.

These considerations have

made orthosteric CB2R agonists potential pharmacotherapeutics for treating several 24-27

neurological/CNS conditions, managing pain, and reducing inflammation.

This therapeutic

modality gains additional interest from the fact that selective CB2R agonists should circumvent the risk of CNS-related psychological and behavioral adverse events associated with orthosteric CB1R agonists.25,28 Rational design of drug-like human CB2R (hCB2R) orthosteric ligands as pharmacological agonists necessitates thorough understanding of the determinants of ligand binding by the receptor and the functional consequences of ligand engagement.

Atomic-level protein X-ray

analyses have provided information on the conformational transitions of select GPCRs, usually in more stable inactive/liganded conformations and as modified crystal forms.29 However, a crystallized hCB2R construct is not currently available, and, in any event, its inherently static nature would not allow direct experimental interrogation of the conformational features of hCB2R activation in the functional (i.e., ligand binding- and signaling-competent) GPCR.

For

this purpose, a powerful experimental strategy has been adopted that utilizes functionalized probes as chemical reporters to label and identify amino acid residues important to ligand



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engagement and activity in intact, functional GPCRs.

30

An early application of this approach to

hCB2R demonstrated that the receptor’s cysteine residue in transmembrane helix 2 (TMH2), 32

C2.59(89), (Figure 2) is accessible to sulfhydryl-reactive methanethiosulfonate reagents.

However, this result cannot address structure-function correlates of classical cannabinoid agonists such as THC, given the pronounced differences in chemical structure between the 31-33

methanethiosulfonate tags used and both natural and synthetic cannabinergic ligands. To

address

this

issue,

we

designed

the

classical

(-)-7'-isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol

cannabinoid

(AM841),

an

electrophilic affinity label that incorporates an isothiocyanate (NCS) substituent at the terminus 34

of its C3 alkyl side chain (Figure 1B).

We subsequently demonstrated that AM841 not only

displays drug-like properties including high hCB2R affinity and unprecedented potency as a hCB2R “megagonist” in vitro and in vivo, but can also serve as a molecular probe for site-labeling hCB2R by virtue of its electrophilic NCS group.

35-38

Mass spectrometry-based

proteomics and mutational studies with an experimental paradigm termed “ligand-assisted 35,36

protein structure” (LAPS)

indicated that, under physiological conditions, AM841 reacts

selectively with C6.47(257), the most deeply located TMH cysteine residue within the receptor and a constituent of hCB2R’s CWFP “hinge”, an embodiment of the highly conserved CWxP 39

motif important to the conformational change underlying class-A GPCR activation (Figure 2).

In silico docking studies visualized the AM841 binding pose in a refined, active-state hCB2R (hCB2R*) homology model with the ligand’s tricyclic ring region oriented toward the upper face of the hCB2R* binding pocket and its long alkyl chain accommodated nearly perpendicularly to


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the receptor’s transmembrane helices (TMHs), positioned lengthwise, parallel to the TMHs of 35

the helical bundle and disposed toward the TMH6-TMH7 interface.

The behavior of AM841

as a sulfhydryl-reactive cannabinergic affinity ligand is congruent with data that, under physiological incubation/reaction conditions (i.e., aqueous milieu and physiological pH), the thiol moiety of protein cysteine residues renders them the most reactive nucleophilic amino acid 40-42

toward electrophiles, including isothiocyanates.

These collective data implicate TMH6, and

specifically C6.47(257), in the hCB2R ligand-interaction domain for classical cannabinoid agonists. Structure-activity relationship (SAR) studies demonstrate that, in addition to the C3 side chain of classical cannabinoids, the C9/C11 position at the cyclohexenyl C-ring is another prime pharmacophoric determinant of classical cannabinoid recognition and binding to CB1R/CB2R (Figure 1).

33,43

In this report, we apply site-directed labeling with novel, high-affinity designer

probes to interrogate the interaction profile of the C9/C11 region of classical cannabinoids with hCB2R.

For this purpose, we used AM841 as a template, given its exceptional potency as a

hCB2R agonist and validation as a molecular reporter that covalently reacts with hCB2R TMH6 35,36

cysteine C6.47(257).

We designed and synthesized two novel AM841 congeners:

AM4073, a classical cannabinoid that incorporates an NCS moiety at the C9 northern head group of its C-ring, and AM4099, a hybrid of both AM841 and AM4073 that features an NCS functionality at both the terminus of its C3 side chain and at its northern C9/C11 head-group (Figure 1B). We demonstrate that AM4073 and AM4099 are competitive, high-affinity hCB2R agonists, AM4073 more potent and less efficacious than AM4099 without the exceptional



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megagonist potency of AM841. Both AM4073 and AM4099 interact irreversibly with wild-type and hCB2R cysteine-mutant variants that retain TMH2 cysteine C2.59(89).

Competitive

binding data with hCB2R TMH cysteine mutants suggest the potential for covalent interaction of AM4073 and AM4099 with TMH2 cysteine C2.59(89), but not with C6.47(257), despite the presence of a terminal C3 side-chain NCS in AM4073 analogous to that in AM841 (Figure 1B). Molecular modeling orients these AM841 congeners toward the TMH2-TMH3 interface within the hCB2R* binding pocket, in contrast to AM841’s inter-helical orientation between TMH6 and TMH7.

This study is the first to implicate TMH2 residue C2.59(89) as a structural feature of

hCB2R activation by classical cannabinoid agonists and highlights the importance of cysteine residue C6.47(257) in orienting classical cannabinoids within the hCB2R binding pocket to afford exceptional “megagonist” potency in vitro and in vivo.

35-38

RESULTS AND DISCUSSION Characterization of Heterologously Expressed Wild-type and Mutant hCB2Rs. Stably transfected human embryonic kidney 293 (HEK293) cell lines expressing either the non-mutated wild type (WT) hCB2R or hCB2R variants with amino acid substitutions at (a) specific TMH cysteine residue(s) have been generated by this laboratory.35

Five hCB2R TMH cysteines were

modified, either individually or in combination, to yield hCB2R variants with the following conservative TMH cysteine-to-serine substitutions: C1.39(40)S, C2.59(89)S, C6.47(257)S, C7.38(284)S,

C7.42(288)S,

or

C7.38(284)7.42(288)S,

Ballesteros-Weinstein numbering scheme for

as

denoted

by

a

modified

class-A GPCR residues (Figure 2).44,45 Two


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extracellular-loop (ECL) cysteines, C174 and C179, were preserved, since they form a disulfide bridge essential to orthosteric ligand binding by hCB2R.31,43,46

Congruent with previous data,36

all six hCB2R cysteine-mutant receptors were capable of saturable [3H]CP55,940 binding with expression levels at the cell surface (as Bmax) in the low pmol/mg range and ligand affinities (as Kd) in the very low nM range, the magnitudes of these values within the range of those of WT hCB2R (Table 1).

[3H]CP55,940 specific binding in all cases constituted >70% of total

radioligand binding (data not shown). Membranes prepared from non-transfected HEK293 cells evidenced negligible specific [3H]CP55,940 binding (data not shown), demonstrating a lack of gross, nonselective ligand-membrane interaction. Furthermore, both the WT and cysteine-mutant hCB2Rs expressed in HEK293 cells are functionally coupled via Gi/o to adenylyl cyclase.35 These results validate the suitability of these hCB2R mutant receptors to interrogate potential, cysteine-related ligand-binding motifs within hCB2R for classical cannabinoid ligands. The data further suggest that the cysteine-to-serine mutations did not significantly impact the receptor’s stability and function, a likely reflection of the conservative nature of the substitution. AM4073 and AM4099 Binding Affinities and Pharmacological Activities at hCB2R. We evaluated the properties of AM4073 and AM4099 as potential hCB2R ligands using well-established binding and functional (i.e., signaling) assays. Binding affinities of AM4073 and AM4099 for WT hCB2R were determined in competition binding assays with [3H]CP55,940 radioligand. Because of the potential for these agents to form a covalent adduct at cysteine residues, their binding affinities are reported as apparent Ki (Ki*) values: 3.3 (1.9-5.7) nM for AM4073 and 12.6 (9.0-17.5) nM for AM4099 [means with 95% confidence intervals (CIs), n =



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3].

The concentration-dependent inhibition of forskolin-stimulated cellular cAMP production

by CP55,940, AM4073, and AM4099 indicates that these ligands acted as agonists at hCB2R, since the receptor is negatively coupled to adenylyl cyclase through Gi/o proteins (Figure 2).35 In a cell-based cAMP functional assay, CP55,940 was the most potent agonist among the three, with the potency and efficacy of AM4073 some 2-fold greater than those of AM4099 (Table 2). Together, these results demonstrate that AM4073 and AM4099 are high-affinity hCB2R agonists with low-nM potencies for modulating hCB2R-, adenylyl cyclase-dependent cell signaling. However, these two AM841 congeners are over 100-fold less potent as hCB2R agonists when compared to the parent compound, whose IC50 as a megagonist in this cAMP assay has been reported to be 0.08 nM.36 hCB2R Labeling with AM4073 and AM4099.

A 1-h preincubation of WT

HEK293-hCB2R membranes with either 33 nM AM4073 or 126 nM AM4099 (i.e., concentrations 10-fold their respective Ki* value) followed by extensive washing to remove free ligand reduced the subsequent Bmax for specific [3H]CP55,940 binding by ~ 60% as compared to control membranes not pretreated with either isothiocyanate compound (Figure 4A, 4B). Similar results were obtained with hCB2R variants featuring targeted mutations in TMH1, TMH6, and TMH7 cysteines (Figure 4C, 4D).

In marked contrast, both AM4073 (Figure 4C)

and AM4099 (Figure 4D) completely displaced specifically-bound [3H]CP55,940 from the C2.59(89)S variant.

The observed irreversible nature of AM4073 and AM4099 binding to WT

hCB2R and only those cysteine-mutant hCB2R variants in which C2.59(89) is present could reflect a covalent interaction of these classical-cannabinoid electrophiles with the receptor at its


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nucleophilic TMH2 C2.59(89) cysteine residue. The aggregate displacement data across all hCB2R mutants studied further suggest that C2.59(89) can serve as a critical component of the ligand-binding and functional domains of classical cannabinoid agonists. It is noteworthy that, although the C3 side chains of both AM841 and AM4099 terminate with an electrophilic NCS moiety (Figure 1), AM841 requires the presence of TMH6 cysteine C6.47(257) to bind irreversibly to hCB2R,35,36 whereas AM4099 does not (Figure 4B, 4D), implying that the adduct formation between AM841 and hCB2R C6.47(257) previously observed36 did not occur with AM4099 and is therefore not an essential feature of hCB2R activation by AM4099. Rather, the similarity between AM4099 and AM4073 with respect to their patterns of irreversible binding to WT and cysteine-mutant hCB2R (Figure 4) and their northern head-group C9/C11 NCS functionality underscores the critical importance of TMH2 cysteine C2.59(89) to the engagement and agonist function of these two ligands. The comparative data further suggest the possibility that the hCB2R-AM841 interaction at C6.47(257) orients this ligand within the hCB2R binding pocket to favor a highly-active conformational ensemble, as reflected in AM841’s exceptional, sub-nM megagonist potency36 as compared to the respective agonist potencies of CP55,940, AM4073, or AM4099 in the cell-based cAMP assay (Figure 3, Table 2).

This proposition is further substantiated by the finding that the

reported35 potency of the direct AM841 analog devoid of an -NCS moiety, AM4056 (Figure 1), is similar to the potencies of of AM4073 and AM4099 (Table 2) in the cell-based cAMP assay of adenylyl cyclase-dependent signaling. Molecular Modeling of hCB2R*-AM4073 Complex. The location of the NCS group at



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the terminus of the C3 side-chain in hCB2R megagonist AM841 vs. at the C9/C11 position of the C ring in AM4073 along with the different potencies of these two classical-cannabinoid antagonist probes led us to perform interactive docking studies aimed at illustrating the AM4073 biding pose in the hCB2R orthosteric site as a supplement to the experimental data.

For this

purpose, protonated AM4073 in its global minimum energy conformation was docked into the advanced, active-state hCB2R* model31 we previously used to visualize the hCB2R*-AM841 interaction.35

As detailed elsewhere,35 this model positions the C6.47(257) residue involved in

AM841 covalent attachment facing outward toward membrane bilayer lipid when hCB2R is in an inactive state, changing its orientation deep within the binding pocket toward the TMH6-TMH7 interface upon receptor activation, whereas C2.59(89) is located within the tunnel between TMH2-TMH3 at the interface of these two TMHs in hCB2R*.

The hydrogen bond

between S2.60(90) and K3.28(109) predicted to be a feature of the hCB2R* state31 was included. In light of the binding data supporting a link between hCB2R* and AM4073 at C2.59(89) (Figure 4), we introduced an AM4073-C2.59(89) covalent bond into our construct. Energy minimization was then imposed upon this starting hCB2R*-AM4073 ensemble. In the energy-minimized hCB2R*-AM4073 complex, AM4073 covalently attached to C2.59(89) is positioned in the vicinity of A2.53(83), F2.57(87), V3.32(113), F3.36(117), and V7.43(289) within the hydrophobic binding pocket (Figure 5).

The phenyl ring of AM4073

evidences π–π stacking with F2.57(87), the ring’s hydroxyl group forming a hydrogen bond with the hydroxyl of S3.35(116) (d = 2.24 Å; O – H- - -O angle = 158°).

The bulky tricyclic region

of AM4073 is disposed more toward TMHs 2 and 3 rather than the interface between TMHs 6


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and 7, although parallel with TMH6-TMH7 in the pocket formed between those two helices (Figure 5), the AM4073-S3.35(116) hydrogen bond constituting a major determinant of this topology.

Given evidence presented herein that C2.59(89) is an interaction site for both

AM4073 and AM4099, whereas C6.47(257) covalently interacts with AM84136 but not these two congeners, it is tempting to speculate that the orientation of AM4099 in the hCB2R* binding pocket resembles that of AM4073, and not that of AM841. Our model of the hCB2R*-AM841 complex previously detailed35 indicates that AM841’s carbocyclic-ring CH2OH substituent at the γ position forms a hydrogen bond with S7.39(285) (d = 2.62A°; O – H- -O angle = 175o), a residue postulated from mutation studies to represent an important interaction site for classical cannabinoid engagement by hCB2R.47 We also found that the phenolic hydroxyl of AM841 could hydrogen-bond with S6.58(268) (d = 2.61A°; O – H- -O angle = 176o) and still maintain its interaction with S7.39(285) and covalent link to C6.47(257).35 These anchoring interactions supported a molecular model in which AM841 is positioned within hCB2R* with its C9-head group oriented toward TMHs 6 and 7, its C3-alkyl side chain penetrating the hydrophobic ligand-binding pocket, and its C3 side-chain terminal NCS group reacting with C6.47(257) located deep within the binding pocket.35

In contrast to AM841,

AM4073 is oriented more toward TMHs 2 and 3 and less toward the interface between TMHs 6 and 7 in the modeled hCB2R*-AM4073 complex, hindering interaction of AM4073 with TMH6/7 residues.

This difference likely accounts for the lack of reactivity of AM4099’s C3

side-chain NCS, despite the fact that this group formed a covalent bond with C6.47(257) when in AM841.36

These distinctions between the binding poses of AM4073/4099 and AM841, along



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with the ~100-fold greater “megagonist” potency of AM841 than either of these congeners, invite conclusion that the interaction between AM841 and hCB2R at C6.47(257) elicits an hCB2R*-AM841 conformational ensemble whose structural features enhance the agonist pharmacological activity of classical cannabinoids.

This proposition invites the view that

different ligands of the same classical cannabinoid class can elicit distinct active-state hCB2R conformations. As compared to AM4073/4099, an important aspect of the exceptional structure-function relationship of AM841 may reflect the fact that the hCB2R TMH6 cysteine with which AM841 reacts, C6.47(257), is a component of hCB2R’s conserved CWFP “hinge” motif important to the conformational change underlying receptor activation.39 This modeled hCB2R*-AM4073 docking prediction provides a precedent theoretical framework for further analysis of the binding mode of AM4073 and other classical cannabinoids should, for example, an active-state hCB2R crystal structure emerge with appropriately detailed orthosteric-site coordinates. Potential Molecular Determinants of Observed hCB2R-Ligand Interaction Profiles. We next considered the potential for intrinsic molecular properties of hCB2R and/or AM841, AM4073, and AM4099 to influence ligand-hCB2R TMH cysteine interaction.

Information on

the structure of the hCB2R binding pocket obtained from homology modeling, computational ligand docking, molecular dynamics simulations, and nuclear magnetic resonance spectroscopy indicates that TMH2 cysteine C2.59(89) is located at the margin of the ligand-binding site as one of the most accessible hCB2R TMH cysteines, whereas C6.47(257) is located considerably deeper in the receptor’s ligand-binding domain, within an ellipsoid hydrophobic pocket that is


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occupied by the linear hydrocarbon side chain of classical cannabinoids.31,49-51

Previous work

from this laboratory has shown that the most energetically favored, preferred conformation of an amphiphilic cannabinoid-receptor ligand has its aliphatic, hydrophobic side-chain almost perpendicular to the plane of its polar head-group phenyl ring and disposed within the membrane phospholipid bilayer, the head-group facing the polar side of the membrane phospholipid bilayer.52 Taken together, the topology of the hCB2R binding pocket and the intrinsic conformational property of amphipathic cannabinergic ligands make it tempting to speculate that AM4073 and AM4099 diffuse laterally within the cell membrane with their polar head group disposed toward the outer region of the membrane phospholipid bilayer, favoring a facile, kinetically preferred interaction between the electrophilic NCS moiety at their northern C9/C11 head-group and TMH2 cysteine C2.59(89) to generate irreversible, active AM4073- and AM4099-hCB2R conformational ensembles. This scenario might also help account for the predilection of the electrophilic NCS substituent at the C9/C11 head-group of AM4099 to interact with hCB2R rather than the NCS substituent at the terminus of the AM4099 alkyl side chain.

In contrast, AM841 contains only the terminal, C3 alkyl side-chain NCS,

well-positioned to react with TMH6 cysteine C6.47(257) deep in the receptor’s binding pocket as a megagonist to stabilize a conformational ensemble evidencing exceptional biological activity for adenylyl cyclase-mediated signaling.36

CONCLUSION



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In contrast to hCB2R residues in TMH6/7, particularly those involving the conformationally flexible, highly-conserved TMH6 CWFP microswitch modulating receptor activation, few studies have investigated the potential involvement of hCB2R TMH2 in the structural features of hCB2R agonist recognition and engagement.

Although hCB2R TMH2 residue C2.59(89) has

been reported to be accessible and sensitive to thiol-directed methanethiosulfonate reagents,31 this study is the first to implicate cysteine C2.59(89) as a component of the hCB2R ligand-binding motif for classical cannabinoids. The activity and molecular modeling data presented suggest that novel hCB2R classical orthosteric agonists designed to favor a binding pose similar to that of AM4073/4099 would act as an efficacious, high-affinity hCB2R agonists. In particular, such agents would feature an interaction profile with a classical cannabinoid characterized by orientation of the ligand’s bulky headgroup toward TMHs 2 and 3, disposed more toward the surface of the receptor’s helical bundle. The apparently irreversible nature of AM4073/4099 binding to hCB2R suggests another design feature for novel classical cannabinergic agonists with therapeutic implications. Covalent ligands targeted to therapeutic proteins (GPCRs, enzymes) have met with clinical success across varied indications.53,54 Among the unique pharmacological features of such irreversible agonists is their ability to prolong GPCR activation, even after internalization of the agonist-bound GPCR by endocytic membrane trafficking and its transit to intracellular compartments (e.g., endosomes), potentially simplifying the dosing regimen and reducing the dose required for a sustained therapeutic effect.55,56

Irreversible hCB2R agonists might also alter the dwell times of

the agonist-hCB2R complex at the plasma membrane and thereby bias the information output of


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the activated receptor toward particular downstream signaling pathways to elicit salutary effects and/or unique spatio-temporal signaling characteristics, as has been demonstrated for hCB1R57,58 Profiling of newer-generation, drug-like hCB2R agonists may also uncover novel hCB2R pharmacophore motifs, help dispel ambiguities regarding the distribution and role of this GPCR among the diverse cell types found in the healthy and diseased brain, and augment our understanding of CB2R neurobiology within the CNS.4,59

Experimentation required to address

these considerations is enabled by the availability of hCB2R ligands such as AM4073/4099 and the insights into their binding and functional domains reported herein.

METHODS Materials. General laboratory chemicals and reagents were purchased from Sigma (St. Louis, MO) at the highest purity/grade. AM4073 and AM4099 were synthesized in the Center for Drug Discovery, Northeastern University (Boston, MA). CP55,940 and [3H]CP55,940 were kindly supplied by the National Institutes of Health (Bethesda, MD). pcDNA 3.1+ was from Invitrogen (Carlsbad, CA). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). The DC colorimetric protein assay kit was from BioRad (Hercules, CA). Amino Acid Descriptor. The loci of specific hCB2R TMH amino acid residues are designated with a modified Ballesteros and Weinstein numbering system.44,45 The most highly conserved amino acid in a given TMH is assigned a locant value of 0.5. This number is preceded by the helix number followed in parentheses by the sequence number. All other amino acid



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residues in that TMH are then assigned a locant relative to that most conserved residue. For example, aspartic acid, residue 80, is the most highly conserved amino acid in hCB2R TMH2. Using the Ballesteros-Weinstein numbering system, this amino acid is designated D2.50(80) and the alanine preceding it would be called A2.49(79). The location of each specific hCB2R TMH cysteine residue discussed in this report is shown in the receptor’s serpentine plot in Figure 2, and the two cysteine residues that are the main focus of this study-- TMH2 C2.59(89) and TMH6 6.47(257)-- are explicitly pointed out therein. Site-Directed Mutagenesis, Cell Transfection, and Tissue Culture. Five transmembrane cysteine residues in hCB2R, one each in TMHs 1, 2, and 6, and two in TMH7, were modified to serine individually or in combination to generate the following six hCB2R variants: C1.39(40)S, C2.59(89)S, C6.47(257)S, C7.38(284)S, C7.42(288)S and C7.38(284)7.42(288)S. Protocols for mutant cell line generation and characterization with HEK293 cells have been detailed in the literature.35,36,60 hCB2R-HEK293 Membrane Isolation. HEK293 monolayers were harvested on ice into PBS containing 1 mM ethlyedediaminetetraacetic acid (EDTA), and the cells were washed three times with this buffer. Washed cell pellets were disrupted on ice by cavitation in a pressure cell at 65 Torr for 30 min. Membrane fractions were isolated by ultracentrifugation, and protein content was determined with a dye-binding protein assay.61,62 Radioligand Binding Assays. Saturation binding assays were performed in a 96-well format using [3H]CP55,940 as radioligand according to the detailed protocol published.35,60,61,63 Bound radioactivity was quantified by liquid scintillation spectrometry. Bmax and Kd values were


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determined by fitting the saturation binding data to a one-site binding equation.

Each data point

represents the mean ± SEM from three independent experiments performed in triplicate. Competition binding assays were performed in a 96-well format using [3H]CP55,940 as displaceable ligand, as described.59 Ki values for test ligands were determined by fitting the competition-binding data to a non-linear regression curve and presented as means with 95% confidence intervals from three independent experiments performed in triplicate.

GraphPad

Prism (GraphPad Software, San Diego, CA) was used for the calculations. For the irreversible ligands AM4073 and AM4099, the Ki values designate apparent affinities (Ki*).64 Affinity Labeling Assays. Labeling assays were carried out with hCB2R-HEK293 membranes prepared as described.36,58,60 In brief, for each sample, 5 mg membrane protein was diluted with 25 mM Tris HCl/5 mM MgCl2/1 mM EDTA, pH 7.4 (TME) containing 0.1% bovine serum albumin (BSA) (TME-BSA) to a volume of 5 mL. Test electrophilic ligand (AM4073 or AM4099) in a stock concentration of 10 mM in dimethyl sulfoxide (DMSO) was diluted to 10 µM with TME-BSA.

One hCB2R-HEK293 membrane sample was treated with test ligand at a

final concentration 10-fold its apparent Ki, and a parallel control sample devoid of the ligand was diluted in the same manner. After incubation at 37 °C in a water bath for 1 h with gentle agitation, both samples were repeatedly centrifuged (27 000g for 10 min, 25 °C) and homogenized thoroughly twice with TME containing 1% BSA to remove unbound ligand and once with TME alone to remove BSA. Saturation binding assays were then carried out on the washed membranes with [3H]CP55,940 as radioligand. Cellular cAMP Determination. Functional evaluation of hCB2R agonists was determined



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with a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay for cyclic AMP (cAMP), indexed as the inhibition (IC50) of forskolin-stimulated cAMP accumulation in a cell-based bioassay conducted in duplicate in three independent experiments.61 Molecular Modeling. AM4073 was first pronated at pH 7.0 ± 2.0, minimized by Schrödinger’s LigPrep Wizard, and then docked covalently into a refined hCB2R* model31 in Schrödinger 2015 with the covalent docking package.35,36

Based on the mutagenesis data

detailed in this study, the thiol of the TMH2 cysteine C2.59(89) residue was highlighted as an interactive residue with the C9 isothiocyanate group of the C-ring of AM4073 (Figure 1B). A binding box was focused on the centroid of C2.59(89) and C6.47(257) with outbox dimensions of 30 Å × 30 Å × 30 Å.

A pose prediction mode was used, and the covalent docking procedure

was performed twice. To adjust the conformation of C2.59(89), AM4073 was firstly irreversibly docked onto this residue. The co-structure with the lowest prime energy was selected, and the AM4073 molecule was removed to recover cysteine C2.59(89).

The ligand was re-docked into

the model, and the bound structure with the highest co-docking affinity and lowest binding energy was selected using the molecular mechanics, generalized Born model and solvent accessibility (MM/GBSA) approach.65 Energy minimization was performed in Macromodel with OPLS3 force field. An extended non-bonded 8.0-Å Van der Walls cutoff, a 20.0-Å electrostatic cutoff, and a 4.0-Å hydrogen-bond cutoff were used. The backbone atoms of hCB2R were fixed, and a Polak-Ribière conjugate gradient method66 was employed in each minimization step with a distance-dependent dielectric 2.0 until an energy gradient of 0.05 Kcal/mol was achieved. AUTHOR INFORMATION


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Corresponding Author *. Phone: 617-373-4200; FAX: 617-373-7493. E-mail: [email protected]. Author Contributions Designed research:

H.Z., A.H., R.W.M., and A.M.

Synthesized AM4073 and AM4099: Conducted experiments:

G.T.

H.Z., A.H., Y.P., and P.F.

Performed data and literature analysis: Performed simulations: Wrote the manuscript:

H.Z., A.H., D.R.J., and R.W.M.

X.S. and X.M. H.Z., D.R.J., X.S., R.W.M., and A.M.

Funding Sources This work was supported by National Institutes of Health grants DA3801, DA9152 and DA9158 (to A. M.)

Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS



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A portion of this work was submitted by H. Zhou in partial fulfillment of the thesis requirements for the Ph.D. degree from Northeastern University, Boston, MA, USA.

We thank Dr. Patricia H.

Reggio and colleagues (Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro) for providing details of the hCB2R* model. We dedicate this work in grateful remembrance of our late colleague, Torbjörn U.C. Järbe, Ph.D. (1946-2016), Research Professor, Center for Drug Discovery and Bouvé College of Health Sciences, Northeastern University, Boston, MA, USA.

ABBREVIATIONS: GPCR, G protein-coupled receptor; CB1R, cannabinoid receptor 1; CB2R, cannabinoid

receptor

2;

CNS,

central

nervous

system;

∆9-THC,

(−)-trans-∆9-

tetrahydrocannabinol; HIV, human immunodeficiency; hCB2R, human cannabinoid receptor 2; TMH,

transmembrane

helix;

AM841,

(6aR,9R,10aR)-9-(hydroxymethyl)-3-(8-isothiocyanato-2-methyloctan-2-yl)-6,6-dimethyl-6a,7,8 ,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol;

AM4056,

(6aR,9R,10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahy dro-6H-benzo[c]chromen-1-ol;

AM4073,

(6aR,9R,10aR)-9-(isothiocyanatomethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-h exahydro-6H-benzo[c]chromen-1-ol;

AM4099,

(6aR,9R,10aR)-3-(8-isothiocyanato-2-methyloctan-2-yl)-9-(isothiocyanatomethyl)-6,6-dimethyl6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol; (-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol;


CP55,940,

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[3H]CP55,940, 5-(1,1-dimethy)-[2,3,4,4-3H]-heptyl-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]p henol; NCS, isothiocyanate; LAPS, ligand-assisted protein structure; hCB2R*, active-state human cannabinoid receptor 2; SAR, structure-activity relationship; HEK, human embryonic kidney; WT, wild type; ECL, extracellular loop; ICL, intracellular loop; CI, confidence interval; PBS, phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; TR-FRET, time-resolved fluorescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; hCB1R, human cannabinoid receptor 1; MM/GBSA, molecular mechanics, generalized Born model and solvent accessibility

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Table 1.

Ligand-binding Parameters for WT hCB2R and Cys Mutants

hCB2R variant

Bmax (95 % CI; pmol/mg)a

WT

11.3

Kd (95 % CI; nM)a

(10.9-11.8)

2.1 (1.8-2.3)

(1.3-1.6)

7.2 (5.4-9.0)

C1.39(40)S

1.5

C2.59(89)S

11.5

(11.0-12.0)

1.1 (1.0-1.3)

C6.47(257)S

15.2

(14.6-15.9)

3.8 (3.3-4.2)

C7.38(284)S

7.4

(7.1-7.7)

1.0 (0.8-1.1)

C7.42(288)S

1.7

(1.6-1.7)

0.7 (0.6-0.8)

C7.38(284)7.42(288)S

0.7

(0.6-0.7)

0.5 (0.4-0.6)

a

Cell-surface expression levels (as Bmax values) and binding affinities (as Kd

values) were derived from saturation binding assays with [3H]CP55,940 radioligand and hCB2R membrane preparations from stably transfected HEK293 cells.

Data are the means of at least three independent experiments

conducted in triplicate, with 95% confidence intervals (CIs) shown in parentheses.


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Table 2. Inhibition of Forskolin-stimulated cAMP Accumulation in WT hCB2R-expressing HEK293 Cells by Test Agonists Agonist CP55,940

AM4073

AM4099

a

The

IC50 (95% CI, nM)a

Efficacy (95% CI, %)a

1.02

59.59

(0.39-2.67)

(51.63-72.71)

9.31

39.95

(2.60-33.43)

(31.21-53.51)

22.88

74.05

(13.61-33.83)

(63.88-86.61)

concentration-dependence

of

each

agonist

on

forskolin-stimulated cyclic AMP accumulation in HEK293 cells expressing WT hCB2R was determined. Data are the means of two independent experiments performed in triplicate, with 95% confidence intervals (CIs) in parentheses.



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FIGURE LEGENDS Figure 1. Chemical structures of classical cannabinoid agonists. (A) The prototypical classical cannabinoid agonist, (−)-trans-∆9-tetrahydrocannabinol (THC), with its four principal pharmacophores indicated:

C1-phenolic hydroxyl group, C3-side chain, C6-sourthern group,

and C9-northern head group. (B) Synthetic classical cannabinoid ligands relevant to this study.

Figure 2. Sequence representation of hCB2R.

The full-length hCB2R sequence is presented

with the five transmembrane-helix cysteine residues subjected to mutation in this study, C1.39(40), C2.59(89), C6.47(257), C7.38(284) and C7.42(288), highlighted in red. The respective location of each of the two TMH hCB2R cysteine residues that are the main focus of this study-- TMH2 C2.59(89) and TMH6 6.47(257)-- are explicitly indicated with arrows to the serpentine plot. TMH, transmembrane helix; ICL, intracellular loop; ECL, extracellular loop; H, helix.

Figure 3.

AM4073 (red) and AM4099 (green) act as agonists to inhibit in a

concentration-dependent manner forskolin-stimulated cyclic AMP accumulation in HEK293 cells expressing hCB2R, as does CP55,940 standard (black).

Data are the means ± SEM of

three independent experiments performed in triplicate.

Figure 4.

Engagement of AM4073 and AM4099 by hCB2R requires TMH2 cysteine residue

C2.59(89). Membranes prepared from HEK293 cells expressing WT hCB2R were preincubated


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37

with either (A) 33 nM AM4073 (red) or (B) 126 nM AM4099 (green) (i.e., 10-fold each respective Ki*) for 1 h at 37 °C and then extensively washed to remove free (non-membrane associated) ligand. The washed membranes were then subjected to a saturation binding assay using [3H]CP55,940 as the radioligand.

AM4073 and AM4099 reduced WT hCB2R maximal

[3H]CP55,940 specific binding (i.e., Bmax) by at least 60% (arrows). The differences in Bmax values of WT and cysteine-mutant hCB2Rs with or without pre-incubation with either (C) AM4073 or (D) AM4099.

WT hCB2R and all cysteine mutants

except C2.59(89)S evidenced an ~60% reduction in maximal [3H]CP55,940 specific binding following the preincubation, implicating C2.59(89) as an important residue in the irreversible binding of both AM4073 and AM4099. Data shown represent the means ± SEM of three independent experiments performed in triplicate.

Figure 5.

Illustration of modeled AM4073 binding pose in the WT hCB2R* active-state

complex from the (A) side and (B) extracellular view.

The headgroup orientation allows

AM4073 (green) to interact with hCB2R C2.59(89) (red) by its head thiol group (yellow). The AM4073 phenyl ring evidences π–π stacking with F2.57(87). The hydroxyl group from the phenol ring forms a hydrogen bond with the hydroxyl group of S3.35(116) (d = 2.24 Å; O – H-O angle = 158°). The aliphatic C3 side chain of AM4073 is disposed parallel to the transmembrane helices of the hCB2R hydrophobic binding pocket. (For clarity, loops and TMHs 4, 5, and 6 are not depicted.)



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C9-northern group

Figure 1.


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C6.47(257)

C2.59(89)

Figure 2.

Figure 3.



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Figure 4.

.

Figure 5.


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C2.59(89) 1.15

C6.47(257) )1.15 1.19

TOC FIGURE.


Human Cannabinoid Receptor 2 Ligand-Interaction Motif

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