Structural basis of selective cannabinoid CB2 receptor activation.

Abstract

Cannabinoid CB₂ receptor (CB₂R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB₂R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB₂R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB₂R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB₂R activation by selective agonists and highlights the role of lipophilicity in CB₂R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

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Subject terms: Electron microscopy, Small molecules, Receptor pharmacology

Web Summary

Cannabinoid CB₂ receptor (CB₂R) agonists are investigated as therapeutic agents in the clinic. Here, authors report the discovery of LEI-102, a CB₂R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate selective CB₂R activation.

Article notes

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Received 2022 Oct 17; Accepted 2023 Mar 2; Collection date 2023.

Author notes

Introduction

Preparations of the plant Cannabis sativa have been used for centuries in the treatment of various diseases, including cancer and neuropathic pain¹. The synthetic version of its psychoactive constituent, Δ⁹-tetrahydrocannabinol (THC, Fig. 1), is in FDA approved drugs Marinol® or Syndros® (dronabinol). The extracted version of THC is one of the active constituents of oromucosal spray Sativex® (nabiximols). These drugs are primarily used for the treatment of chemotherapy-induced nausea, enhancement of appetite in cachexic AIDS-patients, and to alleviate the spasticity and pain associated with multiple sclerosis^2–6. However, THC-based therapies are associated with clinically undesired psychotropic and cardiovascular adverse effects and challenging pharmacokinetic properties due to their high lipophilicity that may limit their therapeutic efficacy^7–10.

Chemical structures.Shown are the main constituent of Cannabis sativa Δ9-tetrahydrocannabinol (THC), and the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as non-selective CBR agonist CP55,940 and CB2R agonists HU308, APD371, LEI-101, and LEI-102. — Fig. 1Chemical structures.Shown are the main constituent of Cannabis sativa Δ⁹-tetrahydrocannabinol (THC), and the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as non-selective CBR agonist CP55,940 and CB₂R agonists HU308, APD371, LEI-101, and LEI-102.

THC exerts its therapeutic effects mostly via the G protein-coupled receptors (GPCRs) cannabinoid CB₁ and CB₂ receptors (CB₁R and CB₂R), which have 68% sequence identity in their seven transmembrane (TM) domains¹¹. Both receptors are activated by the endogenous signaling lipids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) (Fig. 1), the two main endocannabinoids. The CB₁R, which is the most abundantly expressed GPCR in the central nervous system (CNS) is responsible for the psychotropic side effects of THC^12–14. It plays a role in memory, learning, neurogenesis, neuronal migration, and synaptogenesis. Furthermore, its presence in many organ tissues belies more non-neurological functions¹⁵. The CB₂R is mainly found on the cells of the immune system and is upregulated under pathophysiological conditions^16,17. Its activation in general is associated with anti-inflammatory responses in tissue injury of the liver, heart, kidney, colon, and brain as determined in various preclinical models^18–22. Based on preclinical studies, it is thought that selective CB₂R agonists may retain and exceed certain therapeutic properties of THC without inducing psychotropic side effects²³.

Various academic and industrial groups have developed selective CB₂R ligands²⁴. HU308 (Fig. 1) was the first selective CB₂R agonist to be reported that displayed anti-inflammatory and analgesic properties in mouse models without inducing CNS-side effects¹⁸. However, poor physico-chemical properties (e.g. low solubility, high lipophilicity) of HU308, which has a calculated logarithm of octanol-water partition coefficient (cLogP) of 8.0²⁵, and its analogs prevented the successful clinical translation of this class of cannabinoid-based drugs.

A next generation of CB₂R ligands was developed with improved drug-like properties. For instance, Olorinab® (APD371, Fig. 1) is the most polar CB₂R agonist reported to date with a cLogP of −0.4²⁶. A phase 2a small-scale safety and tolerability trial in 14 patients with chronic abdominal pain associated with Crohn’s disease showed mild-to-moderate adverse events and an improvement in abdominal pain scores²⁷. We have previously disclosed pyridinylbenzylimidazolidine-2,4-dione derivatives as selective CB₂R agonists and studied their affinity, target binding kinetics and potency as a function of their lipophilicity, which resulted in the discovery of the orally available and peripherally restricted selective CB₂R agonist LEI-101 (Fig. 1)^28–30. It is intriguing that the CB₂R binding pocket tolerates a wide array of ligands with very different scaffolds and hydrophobicity. For example, HU308 has a 2-billion-fold higher lipophilicity than APD371. Despite the tremendous progress in the field of CB₂R drug discovery, we still do not have any molecular understanding on how these CB₂R agonists selectively activate CB₂R over CB₁R.

Recently, three-dimensional structures of the CB₁R and CB₂R have been elucidated in both the active and inactive states by crystallography or cryo-electron microscopy (cryo-EM) and the binding modes of diverse ligands and their activation mechanism were reported^31–35. Remarkably, those structures revealed that CB₁R and CB₂R possess a highly similar, lipophilic orthosteric agonist binding pocket, which makes it challenging to explain the selective activation of CB₂R. To date, no structural studies with selective CB₂R agonists have been reported that could aid in understanding the molecular basis of CB₂R selectivity.

Here, we present the discovery of LEI-102, a potent and selective CB₂R agonist with good physico-chemical and biological properties. LEI-102 is used in conjunction with CB₂R selective agonists APD371 and HU308, and non-selective agonist CP55,940 to investigate the activation mechanism of CB₂R. For this study, we combine ligand-target binding kinetics, site-directed mutagenesis, and cryo-EM methods. We find that CB₂R has a distinct activation mechanism compared to CB₁R. Additionally, we find that the physico-chemical properties of the ligands influence their entry pathway into the receptor. Highly lipophilic ligands, such as HU308 and the endocannabinoids, may reach the binding pocket through the membrane, whereas more polar ligands, such as LEI-102, APD371 and CP55,940, enter the receptor via an alternative route. Furthermore, we show that the favorable physico-chemical properties of LEI-102 and CB₂R selectivity underscore its promising in vivo efficacy via oral administration in a chemotherapy-induced nephropathy model without inducing CNS-mediated side effects. Together, these studies enhance our insights into how certain physico-chemical properties of ligands translate to in vivo activity and changes their engagement to GPCRs.

Results

LEI-102 as a high affinity and potent CB₂R-selective agonist

To obtain a selective CB₂R agonist with beneficial physico-chemical properties, LEI-102, a pyridinylbenzylimidazolidine-2,4-dione derivative, was designed and synthesized (Supplementary Fig. 1). LEI-102 combined an isobutyl substituent on the imidazolidine with an aminotetrahydropyran to replace the cyclopropyl and thiomorpholine 1,1-dioxide in LEI-101, respectively³⁰. LEI-102 had a cLogP of 2.1 as calculated by ChemDraw 19.0 (Supplementary Table 1). The inhibitory constant (pK_i), potency (pEC₅₀) and intrinsic activity (E_max) of LEI-102 were determined in [³H]RO6957022 displacement assays on stably expressing CB₂R membranes and [³⁵S]GTPγS G protein activation assays using HEK293T membranes transiently expressing recombinant hCB₂R or hCB₁R, respectively (Supplementary Table 2). APD371, HU308, CP55,940 and the endocannabinoids AEA and 2-AG were also explored. LEI-102 had a high binding affinity for CB₂R (pK_i = 8.0 ± 0.1) and was more potent than the selective CB₂R agonists APD371 and HU308. LEI-102 did not bind CB₁R, thereby showing at least 1000-fold selectivity (Supplementary Table 3). In G protein activation assays, LEI-102 activated the receptor as a partial agonist (E_max 76 ± 1%) with a pEC₅₀ value of 6.9 ± 0.2 (Supplementary Table 2).

Distinct target binding kinetic profiles of CB₂R agonists

To quantify the ligand-target binding kinetic parameters of the agonists in more detail, we performed displacement and competition association assays with [³H]RO6957022 on membranes stably expressing hCB₂R (Supplementary Table 2). The equilibrium K_i and kinetic K_D values were well correlated, validating the competition association assay. First, we determined the dissociation rate constants (k_off) of all agonists and converted these into a residence time (RT). LEI-102 had a RT of 16 min, which was around half that of APD371 (45 min) and CP55,940 (32 min), whereas HU308 had the longest RT at the receptor of 71 min (Supplementary Table 2). Endocannabinoids 2-AG and AEA had the shortest RT, both approximately 7 min. Of note, we found that the association rate constants (k_on) varied greatly between the different agonists, ranking from fast to slow engagement CP55,940 > LEI-102 > 2-AG > APD371 > HU308 = AEA. The calculated engagement time (ET) to CB₂R at 1 µM of each agonist further emphasized that CP55,940 arrived at CB₂R within one second, whereas APD371, LEI-102, and 2-AG needed between 16 and 40 s to reach the CB₂R binding site. Interestingly, HU308 and AEA took 143 and 152 s to bind CB₂R, respectively. In view of the distinct target-binding kinetic profiles of the four synthetic CB₂R agonists, we decided to elucidate their binding poses in CB₂R using cryo-EM method.

Overall similar structural comparison of CB₂R-G_i in complex with different agonists

To obtain the stable complex sample of CB₂R-G_i bound with LEI-102, APD371, HU308, or CP55,940, a similar procedure was used as for our previous AM12033-CB₂R-G_i complex preparation (PDB: 6KPF). Single particle analysis of the cryo-EM samples yielded a normal global map for CB₂R-LEI-102-G_i-scFv16, CB₂R-APD371-G_i-scFv16, CB₂R-HU308-G_i-scFv16, and CB₂R-CP55,940-G_i-scFv16, complex, at 2.9, 3.0, 3.0, and 2.9 Å, respectively (Fig. 2, Supplementary Table 4, and Supplementary Figs. 2–5). The ligand, receptor and G protein in the isolated complex were clearly visible in the cryo-EM maps (Fig. 2 and Supplementary Fig. 6). The overall structures of the four complexes were comparable, with root mean square deviation (RMSD) of the Cα atoms of the receptors around 0.35 Å. The ligand binding interfaces of the four CB₂R and G_i complexes were similar to each other, and to those of the previous AM12033-CB₂R-G_i or WIN55212-2-CB₂R-G_i complex structures.

Cryo-EM structures of CB2R-Gi complexes.Cryo-EM density maps of a LEI-102 (Dark green), b APD371 (Sky blue), c HU308 (Olive), and d CP55,940 (Teal) bound CB2R in complex with Gαi (Slate), Gβ (Salmon), Gγ (Pale green), scFv16 (Violet purple). e–l Overall structures of CB2R-Gi complexes and enlarged view of orthosteric pocket of f LEI-102, h APD371, j HU308, and l CP55,940 using the same color codes as (a–d), with agonists shown as cornflower blue (LEI-102), orange (APD371), dark salmon (HU308) and purple (CP55,940) sticks, respectively. — Fig. 2Cryo-EM structures of CB₂R-G_i complexes.Cryo-EM density maps of a LEI-102 (Dark green), b APD371 (Sky blue), c HU308 (Olive), and d CP55,940 (Teal) bound CB₂R in complex with Gα_i (Slate), Gβ (Salmon), Gγ (Pale green), scFv16 (Violet purple). e–l Overall structures of CB₂R-G_i complexes and enlarged view of orthosteric pocket of f LEI-102, h APD371, j HU308, and l CP55,940 using the same color codes as (a–d), with agonists shown as cornflower blue (LEI-102), orange (APD371), dark salmon (HU308) and purple (CP55,940) sticks, respectively.

The binding mode of LEI-102 in CB₂R

A clear electron density in the orthosteric ligand binding pocket in the LEI-102-CB₂R-G_i complex resulted in the unambiguously defined binding pose of LEI-102 (Supplementary Fig. 6a). LEI-102 predominantly interacted with the residues in the binding pocket via hydrophobic interactions (Fig. 3a and Supplementary Fig. 7a). The isobutyl substituent of LEI-102 showed interactions with residues S90^2.60 (Ballesteros-Weinstein numbering in superscript), F106^3.25, K109^3.28, and I110^3.29 in CB₂R. The imidazolidine-2,4-dione formed a π-π interaction with F94^2.64 and showed further hydrophobic interactions with F106^3.25 and P184^ECL2. The benzyl formed an aromatic interaction with F183^ECL2, and hydrophobic interactions with F87^2.57 and S285^7.39. The phenyl ring in the core of LEI-102 formed a cation–π interaction with F183^ECL2 and T-shaped π-π interaction with F281^7.35. The pyridine had hydrophobic contacts with F117^3.36 and W258^6.48. The aminotetrahydropyran sidechain protruded into the long channel and formed hydrophobic interactions with residues I110^3.29, T114^3.33, I186^ECL2, Y190^5.39, L191^5.40, W194^5.43, and M265^6.55. Additionally, a hydrogen bond was formed with T114^3.33 (Supplementary Fig. 7a).

Key interactions between CB2R and agonists.Key residues involved in a LEI-102, b APD371, c HU308, and d CP55,940 binding in CB2R-Gi complex structures. The amino acids involved in interactions are shown as sticks, hydrogen bonds are highlighted with yellow dashed lines. Same color codes as in Fig. 2. — Fig. 3Key interactions between CB₂R and agonists.Key residues involved in a LEI-102, b APD371, c HU308, and d CP55,940 binding in CB₂R-G_i complex structures. The amino acids involved in interactions are shown as sticks, hydrogen bonds are highlighted with yellow dashed lines. Same color codes as in Fig. 2.

The binding mode of APD371 in CB₂R

APD371 mainly formed hydrophobic and aromatic interactions with residues from ECL2, TM2, TM3, TM5, TM6, and TM7 (Fig. 3b and Supplementary Fig. 7b). The carbonyl group of APD371 formed a putative hydrogen bond with S285^7.39 and a hydrophobic interaction with F87^2.57. The pyrazole and pyrazine cores of APD371 formed aromatic interactions with F183^ECL2. Furthermore, the pyrazine core formed hydrophobic contacts with T114^3.33, I186^ECL2, L191^5.40, and W194^5.43. The (S)-1-hydroxy-3,3-dimethylbutyl head formed hydrophobic contacts with residues M26^N-terminus, S90^2.60, F94^2.64, F106^3.25, I110^3.29 and V113^3.32. The cyclopropyl group formed hydrophobic contacts with F117^3.36, W194^5.43, W258^6.48, and V261^6.51.

The binding mode of HU308 in CB₂R

The interactions between HU308 and CB₂R were hydrophobic, including residues from ECL2, TM2, TM3, TM5, TM6, and TM7 (Fig. 3c and Supplementary Fig. 7c). The phenyl of 2,6-dimethoxyphenyl core formed hydrophobic interactions with F87^2.57, F183^ECL2, and S285^7.39, the C2-methoxy formed hydrophobic contacts with A282^7.36 and S285^7.39, and the C6-methoxy formed hydrophobic contacts with I110^3.29, V113^3.32, and T114^3.33, respectively. The dimethylheptyl chain of HU308 extended into the long channel and formed hydrophobic interactions with residues from ECL2 (F183^ECL2), TM3 (T114^3.33, F117^3.36) and TM5 (W194^5.43). The 1,1-dimethyl formed hydrophobic interactions with residues F87^2.57, F117^3.36, F281^7.35, and S285^7.39. The bicyclic head of HU308 formed hydrophobic interactions with M26^N-terminus, F106^3.25, I110^3.29, S90^2.60, F94^2.64, P184^ECL2, and the 2-methanol formed a hydrophobic interaction with F94^2.64 (Supplementary Fig. 7c).

The binding mode of CP55,940 in CB₂R

CP55,940 adopted an L-shape conformation in the orthosteric binding pocket (Fig. 3d and Supplementary Fig. 6d). The cyclohexanol group formed hydrophobic interactions with F94^2.64, L182^ECL2, F183^ECL2, and P184^ECL2. The hydroxyl group established a hydrogen bond with L182^ECL2 and the hydroxypropyl formed hydrophobic contacts with F87^2.57, S90^2.60, F91^2.61, I110^3.29, and V113^3.32. The phenol core formed hydrophobic interactions with F87^2.57, F183^ECL2, F281^7.35, and S285^7.39, and its hydroxyl additionally formed a hydrogen bond with S285^7.39. The dimethyl formed hydrophobic interactions with F183^ECL2, F281^7.35, M265^6.55, F87^2.57, F117^3.36, and C288^7.42. The dimethylheptyl alkyl chain of CP55,940 extended into the long channel and formed hydrophobic interactions with residues I110^3.29, F183^ECL2, I186^ECL2, W194^5.43, T114^3.33 and F117^3.36 (Supplementary Fig. 7d).

LEI-102 and APD371 require H95^2.65 for G protein activation in CB₂R

To study the mechanism of CB₂R activation, five residues in the binding pocket were further characterized based on the complex structures (Fig. 3). Six CB₂R mutants were created, i.e. four residues (S285^7.39, H95^2.65, I110^3.29, and F117^3.36) were replaced by alanine, as these are conserved between CB₂R and CB₁R, and two others (I110^3.29, V261^6.51) were substituted by the hCB₁R reciprocal residue leucine. All mutants were sufficiently expressed at the cell surface as determined with an ELISA (Supplementary Fig. 8, Supplementary Table 5). To characterize the binding mechanisms of LEI-102, APD371, HU308, and CP55,940, their responses were investigated by [³H]CP55,940 displacement and [³⁵S]GTPγS binding assays. Of note, in the [³H]CP55,940 displacement assay, only the CB₂R-I110^3.29L mutant showed a sufficient binding window (data not shown). This prevented the affinity determination of the four agonists on other mutant receptors. Five mutant receptors, except CB₂R-F117^3.36A, were still active in the [³⁵S]GTPγS functional assay, thereby allowing us to study the receptor activation mechanism (Fig. 4a–d and Supplementary Table 6). All four synthetic agonists were unable to activate CB₂R-F117^3.36A, which indicated an important role of this residue in the activation of CB₂R.

Characterization of G protein activation of wild type (WT) and mutant CB2R by synthetic agonists and endocannabinoids.Dose-response curves for G protein activation of WT and mutants that are located in the CB2R binding pocket by a LEI-102, b APD371, c HU308, and d CP55,940. e–j Dose response curves for G protein activation of WT and mutants that are proposed to be involved in ligand entry of CB2R via either the ECL2 or membrane access by e LEI-102, f APD371, g HU308, h CP55,940, i AEA and j 2-AG. a–j The maximum activation level of WT CB2R was set to 100% while the basal levels were set to 0%. Data are presented as mean ± SEM of at least three individual experiments performed in duplicate (specific n values are given in Supplementary Table 6). Source data are provided as a Source Data file. — Fig. 4Characterization of G protein activation of wild type (WT) and mutant CB₂R by synthetic agonists and endocannabinoids.Dose-response curves for G protein activation of WT and mutants that are located in the CB₂R binding pocket by a LEI-102, b APD371, c HU308, and d CP55,940. e–j Dose response curves for G protein activation of WT and mutants that are proposed to be involved in ligand entry of CB₂R via either the ECL2 or membrane access by e LEI-102, f APD371, g HU308, h CP55,940, i AEA and j 2-AG. a–j The maximum activation level of WT CB₂R was set to 100% while the basal levels were set to 0%. Data are presented as mean ± SEM of at least three individual experiments performed in duplicate (specific n values are given in Supplementary Table 6). Source data are provided as a Source Data file.

The potency of LEI-102 was significantly increased at the CB₂R-I110^3.29L mutant to a pEC₅₀ value of 7.8 ± 0.1 in the G protein activation assay, while the binding affinity remained similar to wild type (WT) receptor (Fig. 4a and Supplementary Tables 6 and 7). Three mutations CB₂R-I110^3.29A, CB₂R-S285^7.39A and CB₂R-V261^6.51L had no significant effect on the potency of LEI-102 in the functional assay. In contrast, the potency on mutant receptor CB₂R-H95^2.65A was significantly reduced for LEI-102. No gain in binding affinity for the swap mutant in CB₁R-L359^6.51V was found with LEI-102 (Supplementary Tables 3 and 8).

APD371 acted as a full CB₂R agonist with a pEC₅₀ value of 7.9 ± 0.1 and a higher maximal activation compared to that of CP55,940 in the functional assay (Supplementary Table 2). Mutant receptor CB₂R-I110^3.29L did not affect the G protein response of APD371 (Fig. 4b and Supplementary Table 6), while the binding affinity was significantly reduced to a pK_i of 7.1 ± 0.0 (Supplementary Table 7). APD371 potency was not affected by mutant receptors CB₂R-I110^3.29A or CB₂R-S285^7.39A. The responses of APD371 for CB₂R-H95^2.65A and CB₂R-V261^6.51L were significantly impacted with 158-fold and 10-fold drop in potency, respectively (Supplementary Table 6).

Thus, we uncovered a crucial role for CB₂R-H95^2.65 in G protein activation of CB₂R by LEI-102 and APD371. Furthermore, LEI-102 activation was increased for the CB₂R-I110^3.29L mutant, while APD371 activation relied on CB₂R-V261^6.51.

An important role for S285^7.39 and V261^6.51 in CB₂R activation by HU308 and CP55,940

The potency and affinity of HU308 on CB₂R were not affected by the CB₂R-I110^3.29L swap mutant (Fig. 4c and Supplementary Tables 6, 7). In addition, activation of mutant receptors CB₂R-I110^3.29A and CB₂R-H95^2.65A by HU308 was not affected with pEC₅₀ values of 6.4 ± 0.5 and 6.6 ± 0.6, respectively. The maximum activation level of mutant receptor CB₂R-S285^7.39A was unaffected compared to WT receptor, but a significant 15-fold loss in potency was observed. Lastly, CB₂R-V261^6.51L had a significant loss of potency, i.e. more than 120-fold lower (Fig. 4c and Supplementary Table 6).

Similar to HU308, the potency of CP55,940 on CB₂R was not affected by the CB₂R-I110^3.29 mutations compared to WT in the G protein activation assay, nor was its binding affinity for CB₂R-I110^3.29L (Fig. 4d and Supplementary Tables 6, 7). In response to CP55,940, mutant receptors CB₂R-S285^7.39A and CB₂R-V261^6.51L were significantly affected with decreased pEC₅₀ values of 6.7 ± 0.1 and <5, respectively. Moreover, the potency of CP55,940 was significantly affected on the CB₂R-H95^2.65A with a 40-fold decrease compared to WT receptor (Fig. 4d and Supplementary Table 6). No gain in potency or affinity was observed for the swap mutant CB₁R-L359^6.51V for either HU308 or CP55,940 (Supplementary Tables 3 and 8).

Taken together, this showed that CB₂R-S285^7.39 and CB₂R-V261^6.51 were crucial for HU308 and CP55,940 to activate the G protein at CB₂R, where CP55,940 additionally required an interaction with CB₂R-H95^2.65.

HU308 and endocannabinoids gain access via membrane entry

Our detailed ligand-target binding kinetic analysis revealed that the highly lipophilic HU308 and anandamide had a very slow on-rate compared to the other ligands. Since it has previously been postulated that ligands of lipid receptors may gain access to the binding pocket via a membrane channel, we examined two potential ligand entry pathways at CB₂R, i.e. either via ECL2 or via a membrane channel in TM1 and TM7. To this end, four additional mutant receptors were created. Three residues in the ECL2 of CB₂R, which were different from CB₁R, were mutated towards the reciprocal CB₁R residues, i.e. CB₂R-L185^ECL2H, CB₂R-L182^ECL2I, and CB₂R-E181^ECL2D. In the fourth mutant receptor, four residues in TM1 and TM7 that align the potential membrane channel in CB₂R were mutated to the reciprocal CB₁R residues and combined as a quadruple mutant, i.e. CB₂R-K279^7.33T, CB₂R-K33^1.32Q, CB₂R-V36^1.35I and CB₂R-C40^1.39S (termed “CB₂R-Quadruple^TM1,7”). Next, we tested all four synthetic agonists and the two endocannabinoids on these four CB₂R mutant receptors in [³H]CP55,940 and [³⁵S]GTPγS assays. Only CB₂R-L185^ECL2H and CB₂R-Quadruple^TM1,7 were evaluated in the [³H]CP55,940 displacement assays due to insufficient binding window for the other two mutant receptors (data not shown). The binding affinities of the agonists were not affected for mutant receptors CB₂R-L185^ECL2H and CB₂R-Quadruple^TM1,7 (Supplementary Table 7). Interestingly, the potencies of LEI-102, APD371, and CP55,940 in the functional assay were not significantly affected for any of the mutant receptors, whereas HU308 and the endocannabinoids were less potent on CB₂R-L182^ECL2I (Fig. 4e–j and Supplementary Table 6). Additionally, the endocannabinoids showed a decreased potency on CB₂R-L181^ECL2D, but not on CB₂R-L185^ECL2H. Of note, HU308 and both endocannabinoids completely lost their ability to activate CB₂R in the CB₂R-Quadruple^TM1,7 mutant, suggesting that this may be an important access point to the receptor binding pocket for these agonists (Fig. 4g, i, j).

LEI-102 attenuates cisplatin-induced nephrotoxicity without CB₁R-mediated side effects

In view of the excellent physico-chemical properties of LEI-102 and its selective CB₂R agonist profile, we investigated the compound in a well-established in vivo model of kidney inflammation and injury induced by cisplatin. In this model, CB₂R activation is associated with protective effects²⁹. Cisplatin (25 mg/kg, i.p.) induced marked elevations of serum creatinine and blood urea nitrogen levels (functional markers of kidney injury) 72 h following cisplatin injection in wild type mice compared with vehicle-treated control animals. LEI-102 showed a dose-dependent attenuation of the functional markers of cisplatin-induced kidney injury both when administered p.o. (orally) or i.p. (Fig. 5a). Renal dysfunction was also accompanied by morphological damage to the kidney tubules determined by histological examination following PAS staining. LEI-102 (10 mg/kg) significantly decreased tubular injury as determined by this staining (Fig. 5b). Marked increases in oxidative and nitrative stress markers (4-HNE and 3-nitrotyrosine) were observed in kidneys of cisplatin-treated mice determined by immunostaining and quantitative ELISA. Furthermore, LEI-102 (10 mg/kg by i.p. or p.o.) decreased lipid peroxidation and protein nitration (Fig. 5c, d). Additionally, the pro-inflammatory cytokines TNFα and IL1β that were elevated due to the cisplatin-induced injury were attenuated in LEI-102 treated mice (Fig. 5e). Importantly, the protective effects of LEI-102 against cisplatin-induced renal dysfunction and tubular damage (Fig. 5f), histopathological injury (Fig. 5g) and markers of oxidative-nitrative stress (Fig. 5h, i) were abolished in CB₂R knockout mice, which had enhanced kidney injury/dysfunction compared to their wild types.

CB2R agonist LEI-102 attenuates cisplatin-induced renal dysfunction, oxidative stress, and inflammation in a CB2R-dependent manner.a Cisplatin-induced renal dysfunction 72 h after administration to mice as evidenced by increased serum levels of blood urea nitrogen (BUN) and creatinine (CREA), which were attenuated by CB2R agonist LEI-102 in a dose-dependent manner when administered either i.p. or p.o. (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). b Periodic Acid-Schiff (PAS) staining in representative kidney sections from cisplatin treatment samples showing protein cast, vacuolation, and desquamation of epithelial cells in the renal tubules which are attenuated with LEI-102. Tubular damage score from kidney sections is shown (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). c The cisplatin-induced nitrative and oxidative stress (nitrotyrosine staining (top row) and HNE staining (bottom row)) in representative kidney sections were also attenuated by LEI-102. This was confirmed by quantitative determination of protein nitration and HNE adducts formation by ELISA (d) (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). e The cisplatin-induced kidney pro-inflammatory cytokine expressions were also attenuated by the CB2R agonist. (*p < 0.001 vs. vehicle group, #p < 0.05 vs. cisplatin group). The protective effects of LEI-102 on cisplatin-induced kidney dysfunction (BUN and CREA) and tubular injury (tubular damage score) (f) (*p < 0.001 vs. vehicle WT or KO group, #p < 0.05 vs. cisplatin WT group), histopathological injury (g), nitrative (h) and oxidative stress (i) were abolished in CB2R knockout mice. All results are means ± SEM of n = 6/group for panels a, b, d, e, f Closed and open symbols are used for male and female mice respectively (4 males and 2 females/group). In panels a, b, d, and e one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons were used, in panel f unpaired two-tailed t-test was used. The analysis was conducted using GraphPad Prism 6 software. p < 0.05 was considered statistically significant (the exact p values are indicated in the supplemental data). — Fig. 5CB₂R agonist LEI-102 attenuates cisplatin-induced renal dysfunction, oxidative stress, and inflammation in a CB₂R-dependent manner.a Cisplatin-induced renal dysfunction 72 h after administration to mice as evidenced by increased serum levels of blood urea nitrogen (BUN) and creatinine (CREA), which were attenuated by CB₂R agonist LEI-102 in a dose-dependent manner when administered either i.p. or p.o. (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). b Periodic Acid-Schiff (PAS) staining in representative kidney sections from cisplatin treatment samples showing protein cast, vacuolation, and desquamation of epithelial cells in the renal tubules which are attenuated with LEI-102. Tubular damage score from kidney sections is shown (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). c The cisplatin-induced nitrative and oxidative stress (nitrotyrosine staining (top row) and HNE staining (bottom row)) in representative kidney sections were also attenuated by LEI-102. This was confirmed by quantitative determination of protein nitration and HNE adducts formation by ELISA (d) (*p < 0.001 vs. vehicle group, #p < 0.001 vs. cisplatin group). e The cisplatin-induced kidney pro-inflammatory cytokine expressions were also attenuated by the CB₂R agonist. (*p < 0.001 vs. vehicle group, #p < 0.05 vs. cisplatin group). The protective effects of LEI-102 on cisplatin-induced kidney dysfunction (BUN and CREA) and tubular injury (tubular damage score) (f) (*p < 0.001 vs. vehicle WT or KO group, #p < 0.05 vs. cisplatin WT group), histopathological injury (g), nitrative (h) and oxidative stress (i) were abolished in CB₂R knockout mice. All results are means ± SEM of n = 6/group for panels a, b, d, e, f Closed and open symbols are used for male and female mice respectively (4 males and 2 females/group). In panels a, b, d, and e one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons were used, in panel f unpaired two-tailed t-test was used. The analysis was conducted using GraphPad Prism 6 software. p < 0.05 was considered statistically significant (the exact p values are indicated in the supplemental data).

To determine whether LEI-102 maintained its selectivity for CB₂R over CB₁R in vivo, LEI-102 was tested in the mouse tetrad assay for CB₁R activity¹⁸. In this assay, four consecutive behavioral tests, related to anti-nociception, hypothermia, catalepsy, and spontaneous activity, were performed 120 min after administration of the agonist. LEI-102 (25 mg/kg, p.o.) did not produce any effects in the tetrad assay as compared with vehicle. There were no effects on nociceptive behavior assessed in tail withdrawal test nor on body temperature (Fig. 6 upper row). No effect was found on locomotor behavior (Fig. 6 lower row) in case of distance traveled, time spent mobile, or running speed of mice. Nor was catalepsy observed following administration of LEI-102. These results indicated that LEI-102 (or one of its metabolites) did not produce CB₁R-mediated CNS-side effects at doses up to 25 mg/kg (p.o.). Hence, the CB₂R agonist LEI-102 maintained its selectivity over CB₁R in vivo.

The CB2R agonist LEI‐102 does not induce cannabimimetic CB1R-mediated effects (Tetrad assay) in vivo.LEI‐102 (25 mg/kg, p.o.) did not affect nociceptive behavior assessed in tail withdrawal test and body temperature, as compared with mice receiving vehicle (upper row). No effects on locomotor behavior were found (lower row). Results are means ± SEM; n = 8 per group. — Fig. 6The CB₂R agonist LEI‐102 does not induce cannabimimetic CB₁R-mediated effects (Tetrad assay) in vivo.LEI‐102 (25 mg/kg, p.o.) did not affect nociceptive behavior assessed in tail withdrawal test and body temperature, as compared with mice receiving vehicle (upper row). No effects on locomotor behavior were found (lower row). Results are means ± SEM; n = 8 per group.

Discussion

So far, several crystal and cryo-EM CBR structures have been resolved in which non-selective agonists adopt a nearly identical binding position in the orthosteric pocket, regardless of the receptor^34–38. In this study, we aimed to generate a better understanding of the binding and activation mechanism of CB₂R-selective agonists. Therefore, we combined ligand-target binding kinetics, site-directed mutagenesis, and cryo-EM studies to investigate the activation mechanism of CB₂R for the introduced CB₂R selective agonist LEI-102 supplemented with agonists APD371, HU308, and CP55,940 on a molecular level. Furthermore, we investigated potential hotspots for CB₂R/CB₁R selectivity by creating swap mutants and discovered a ligand entry pathway for CBR agonists and endocannabinoids.

First, our data revealed a crucial role for CB₂R-F117^3.36 as replacement by alanine resulted in a complete loss of G protein activation by all tested agonists (Fig. 4a–d and Supplementary Table 6). It has been shown that the CB₁R counterpart F200^3.36 plays an important regulatory role in activation as part of the “twin toggle switch” with CB₁R-W356^6.48³⁹. In contrast, CB₂R-W258^6.48 has been described to be solely responsible for activation as a toggle switch without the help of CB₂R-F117^3.36 in structural studies, since the conformation of CB₂R-F117^3.36 in agonist-bound structures is comparable to the conformation in the antagonist-bound CB₂R structure as well as the CB₁R agonist-bound structures^33,36. Our mutation data further supports this hypothesis, as we do not see the same constitutive activity pattern (Supplementary Table 6) as observed by McAllister et al. for the reciprocal CB₁R-F220^3.36 excluding CB₂R-F117^3.36 from a suppressive function³⁹. Together, this data provides evidence for a different, but important, role for F117^3.36 in CB₂R activation.

In CB₁R, water-mediated interactions between CB₁R-H178^2.65, CB₁R-S383^7.39, and bound ligands have previously been shown with in silico modeling^40,41. The importance of CB₁R-S383^7.39 for classical synthetic cannabinoids such as AM11542, AM841, and CP55,940 was further emphasized in CB₁R-S383^7.39A mutants³⁶. This is in line with the observation that removal or methylation of the phenolic OH on classical cannabinoids, such as in L-759656, JWH-133, and HU308, always affords selectivity over CB₁R^18,42. Non-classical agonists, such as WIN55,212-2, do not form a hydrogen bond with CB₁R-S383^7.39 and consequently are not affected by an alanine mutation⁴³. This translates to our results that CP55,940 and HU308 are more affected by the CB₂R-S285^7.39A mutation than LEI-102 and APD371 (Fig. 4a–d and Supplementary Table 3). The decrease in activation is at least 30-fold smaller for CB₂R than CB₁R³⁶. The elucidated cryo-EM structures of our four agonists did not show direct interactions with CB₂R-H95^2.65, though we cannot rule out its role in stabilizing the surrounding residues. The large effect seen on G protein activation of CB₂R-H95^2.65A by LEI-102, APD371, and CP55,940 (Fig. 4a–d and Supplementary Table 6) must therefore stem from an indirect interaction, supporting the polar network hypothesis between CB₂R-H95^2.65 and CB₂R-S285^7.39 in CB₂R.

Residues at position 6.51 have previously been described to be involved in the binding sites of µ, δ, and κ opioid receptors, the dopamine D2 receptor, and adenosine receptors, and could play a role in ligand binding selectivity between different subtypes^44–46. In our studies, introduction of the bulkier CB₁R leucine on this position in CB₂R-V261^6.51L reduced the G protein activation by APD371, HU308, and CP55,940, while LEI-102 could still be accommodated in the binding pocket (Fig. 4a–d and Supplementary Table 6). Furthermore, with the swap mutant CB₁R-L359^5.61V we found a trend in partial recovery of displacement of [³H]CP55,940 by the CB₂R selective agonists LEI-102, HU308, and APD371, although not significant (Supplementary Table 3). This supports a role of this residue in the selectivity of agonists in CB₂R.

The ECL2 has frequently been implicated to be important for GPCR activation and some GPCRs even use their ECL2 as a ligand to auto-activation⁴⁷. There are distinct differences between the conformations of ECL2 in CB₁R and CB₂R. In antagonist-bound CB₁R crystal structures, the ECL2 dips into the binding pocket, interacting with the ligand and inducing the inactive conformation^31,32. The inactive state of CB₂R, however, does not expand like CB₁R and instead the ECL2 acts more as a lid on the binding pocket in active and inactive CB₂R, akin to active CB₁R³³. A key distinction seen in the CB₁R crystal structures with AM6538 and taranabant, is the ionic lock formed by CB₁R-E100^N-terminus (CB₂R-L17) and CB₁R‑H270^ECL2 (CB₂R-L185)^31,32. We observed improved binding of [³H]CP55,940 for LEI-102 and HU308 with the CB₁R-H270^ECL2L mutation, while the non-selective agonists showed no change (Supplementary Table 3). Through the loss of this ionic lock, selectivity over CB₁R is partially lost, showing that the expulsion of ECL2 upon ligand entry may play an important role in selectivity.

In recent years, computational studies have suggested that lipophilic ligands for various GPCRs, such as the opsin receptor, sphingosine-1-phosphate receptor 1 (S1P₁) and cannabinoid receptors, might gain access to the binding pocket through lateral diffusion via a membrane channel between TM1 and TM7^{32,41,48–51}. We experimentally examined this membrane entry pathway by creating a CB₂R quadruple mutant (K33^1.32Q, V36^1.35I, C40^1.39S, and K279^7.33T) for which we observed a significant loss of potency and a corresponding trend in reduced affinity, although not significant, for HU308 and the endocannabinoids (Fig. 4e−j, Supplementary Table 6 and 7). These compounds are more lipophilic than LEI-102 and APD371, making them more suitable to traverse the membrane to enter between TM1 and TM7. Notably, HU308 and anandamide also showed a substantially longer ET in our assays compared to the other agonists (Supplementary Table 2). This might suggest a possible relationship between a slower association and membrane channel entry at the CB₂R. Likewise, for a peptide GPCR a trend in reduced association rate was found with increasing lipophilicity⁵². Nevertheless, this is in contrast with the mechanism at the α₂-adrenoceptor at which lipophilic compounds had a faster association rate⁵³. This shows the diversity in drug-target binding kinetics as receptor-specific properties and thus the importance of investigating these mechanisms for individual receptors⁵⁴.

The discovery of a membrane access channel for endocannabinoids on the CB₂R is also intriguing from a physiological perspective. Endocannabinoids are produced on demand and act as autocrine or paracrine effectors in the immune system regulating the migration of CB₂R-expressing immune cells¹⁷. Our results suggest that endocannabinoids first have to travel through the plasma membrane via lateral diffusion to reach the receptor. This may suggest that the trafficking and cellular uptake of endocannabinoids could be regulated through extracellular or intracellular vesicles that merge with the plasma membrane. Regardless of the exact mechanism of endocannabinoid trafficking, this study provides experimental evidence of a membrane channel located between TM1 and TM7 in CB₂R that is being used by the endocannabinoids to enter the receptor.

The ligands of the CB₂R, such as the phytocannabinoids and endocannabinoids, are typically very lipophilic, which comes at a cost of reduced solubility, increased off-target activity, and poor pharmacokinetic properties^10,25. Thus, balancing lipophilicity of a drug candidate is an important goal in medicinal chemistry. The first generation of experimental drugs targeting the CB₂R mimicked the plant-based cannabinoids. Consequently, they were highly lipophilic and suffered from poor clinical translation¹⁰. New generations of CB₂R agonists have optimized physico-chemical properties. For instance, LEI-102 and APD371 are orders of magnitude more hydrophilic than HU308. Remarkably, they can bind the same binding pocket in CB₂R as HU308. Our data revealed that LEI-102 and APD371 do not enter the receptor via the membrane channel like HU308, but gain access most likely via the extracellular space. LEI-102 and APD371 also form a specific (indirect) polar interaction network with H95^2.65 to activate CB₂R, which is not observed for HU308. This flexibility of the CB₂R binding pocket to be activated by a diverse set of chemotypes allows to select for a chemotype with more drug-like properties. This notion is supported by the oral efficacy of LEI-102 in the chemotherapy-induced nephropathy model and lack of CNS-adverse side effects (Figs. 5 and 6).

Targeting CB₂R with agonists is a promising avenue for the treatment of autoimmune diseases, neuroinflammation, and various forms of tissue injury/inflammation/fibrosis in the liver, heart, brain, and kidney¹⁷. In this study, we show that LEI-102 protects against cisplatin-induced nephropathy in a CB₂R-dependent manner by attenuating kidney inflammation and injury (Fig. 5). We also show that CB₂R knockout mice develop more severe nephropathy compared to their wild types suggesting a protective role of endocannabinoid-CB₂R signaling during kidney injury. These results are consistent with protective effects of CB₂R agonists in various models of kidney injury/diseases and deleterious effect of CB₂R deletion in these models^29,55–63.

In conclusion, we have discovered LEI-102 as a selective CB₂R agonist that is efficacious in attenuating tissue injury in chemotherapy-induced nephropathy model without inducing CNS-mediated side effects. Using LEI-102 and five other CBR agonists, we have shown that the physicochemical properties determine not only pharmacokinetic properties of ligands, but also how they engage with their target. Altogether, we elucidated several important molecular mechanisms for selective engagement and activation of the CB₂R, which may have implications for drug design and lipid signaling at GPCRs in general.

Methods

General materials for functional assays

Monoclonal M2 mouse anti-FLAG primary antibody (#F3165) was purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands), while secondary goat anti-mouse HRP-conjugated antibody (#115-035-003) was bought from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Bicinchoninic acid (BCA) ad BCA protein assay reagent was obtained from Pierce Chemical Company (Rockford, IL, USA). [³H]RO6957022 (specific activity 82.83 Ci mmol⁻¹) was custom synthesized at F. Hoffman-La Roche Ltd (Basel, Switzerland). [³⁵S]GTPγS (specific activity 1250 Ci mmol⁻¹ #NEG030H250UC), [³H]CP55,940 (specific activity 108.5 Ci mmol⁻¹ #NET1051250UC) and GF/C filter plates (#6055690) were purchased from PerkinElmer (Waltham, MA, USA). CP55,940 (#C1112), AM630 (#SML0327) and DL-dithiotreitol (DTT, #646563) were obtained from Sigma-Aldrich, HU308 (#H800010) was from LKT Laboratories (St. Paul, MN, USA), APD371 was provided by F. Hoffmann-La Roche Ltd, anandamide (AEA, #1339), 2-Arachidonylglycerol (2-AG, #1298) and phenylmethylsulfonyl fluoride (PMSF, #4486) were purchased from Tocris Bioscience (Bristol, UK) and GDP (#J61646) was from Thermo Fisher Scientific (Waltham, MA, USA). All buffers and solutions were prepared using Millipore water (deionized using a MilliQ A10 Biocel with a 0.22 µm filter) and analytical grade reagents and solvents. Buffers are prepared at room temperature (RT) and stored at 4 °C, unless stated otherwise.

Cell lines

Spodoptera frugiperda (Sf 9) cells were used for CB₂R-G_i co-expression for cryo-EM studies. Sf 9 cells were grown in ESF 921 medium (Expression systems) at 27 °C and 125 rpm. For transfections, human embryonic kidney 293T (HEK293T; female, ATCC #CRL-3216) cells were grown as monolayers in culture medium i.e. Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich #6546), supplemented with 10% fetal calf serum (Sigma-Aldrich #F7524), 2 mM L-glutamine (Sigma-Aldrich #G8541), 100 IU/mL penicillin and 100 µg/mL streptomycin (Duchefa Biochemie #P0142 and #S0148) under a humidified atmosphere at 37 °C with 5% CO₂. Subculture was done twice a week at 80–90% confluence on 10 cm ø plates by trypsinization. CHO cells stably expressing hCB₂R (CHOK1_hCB₂bgal; PathHunter EA Parental Cell line, female, DiscoverX #93-0706C2) were cultured in Ham’s F12 Nutrient Mixture (Sigma-Aldrich #4888) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, 300 µg/mL hygromycin (Bio-Connect #ANT-HG-5) and 800 µg/mL G418 (Bio-Connect #SC-29065B) in a humidified atmosphere at 37 °C with 5% CO₂. Cells were subcultured twice a week when reaching 80–90% confluence on 10 or 15 cm ø plates by trypsinization.

Synthesis of LEI-102

All reagents and solvents were purchased from commercial sources and were of analytical grade (Sigma-Aldrich, BroadPharm®). Reagents and solvents were not further purified before use. All moisture sensitive reactions were performed under inert atmosphere. Solvents were dried using 4 Å molecular sieves prior to use when anhydrous conditions were required. Water used in reactions was always demineralized. Analytical Thin-layer Chromatography (TLC) was routinely performed to monitor the progression of a reaction and was conducted on Merck Silica gel 60 F254 plates. Reaction compounds on the TLC plates were visualized by UV irradiation (λ₂₅₄) and/or spraying with potassium permanganate solution (K₂CO₃ (40 g), KMnO₄ (6 g), and H₂O (600 mL)), ninhydrin solution (ninhydrin (1.5 g), n-butanol (100 mL) and acetic acid (3.0 mL)) or molybdenum solution ((NH₄)₆MO₇ · 4 H₂O (25 g/L) and (NH₄)₄Ce(SO₄)4 · H₂O (10 g/L) in sulfuric acid (10%)) followed by heating as appropriate. Purification by flash column chromatography was performed using Screening Devices B.V. silica gel 60 (40–63 µm, pore diameter of 60 Å). Solutions were concentrated using a Heidolph laborata W8 4000 efficient rotary evaporator with a Laboport vacuum pump.

Analytical purity was determined with Liquid Chromatography-Mass Spectrometry (LC-MS) using a Finnigan LCQ Advantage MAX apparatus with electrospray ionization (ESI), equipped with a Phenomenex Gemini 3 μm NX-C18 110 Å column (50 × 4.6 mm), measuring absorbance at 254 nm using a Waters 2998 PDA UV detector and the m/z ratio by using an Acquity Single Quad (Q1) detector. Injection was with the Finnigan Surveyor Autosampler Plus and pumped through the column with the Finnigan Surveyor LC pump plus to be analyzed with the Finnigan Surveyor PDA plus detector. Samples were analyzed using eluent gradient 10% → 90% ACN in MilliQ water (+0.1% TFA (v/v)).

For purification by mass guided preparative High-Performance Liquid Chromatography (Prep-HPLC) the Waters AutoPurification HPLC/MS apparatus was used with a Gemini prep column 5 μm 18 C 110 Å (150 × 21.2 mm), Waters 2767 Sample manager, Waters 2545 Binary gradient module, Waters SFO System fluidics organizer, Waters 515 HPLC pump M, Waters 515 HPLC pump L attached to a Waters SQ detector Acquity Ultra performance LC.

¹H, ¹³C, ¹H-COSY and HSQC Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker AV 300 (300/75 MHz), AV 400 (400/100 MHz) or AV 500 (500/125 MHz) spectrometer at ambient temperature using CDCl₃ as solvent. Chemical shifts (δ) are referenced in parts per million (ppm) with tetramethylsilane (TMS) or CDCl₃ resonance as the internal standard peak (CDCl₃/TMS, δ 0.00 for ¹H (TMS), δ 77.16 for ¹³C (CDCl₃)). Multiplicity is reported as s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, p = quintet, m = multiplet. Coupling-constants (J) are reported in Hertz (Hz) (Supplementary Fig. 1)

(6-bromo-3-fluoropyridin-2-yl)methanol (2)

To a solution of 6-bromo-3-fluoro-2-methylpyridine (1, 10.7 g, 56.3 mmol, 1 eq) under an inert atmosphere at 0 °C in DCM (370 mL) was added portion-wise m-CPBA (23.6 g, 70–75%, 100 mmol, 1.8 eq). The reaction mixture was stirred at room temperature (rt) for 4 days. Sat. NaHCO₃ and sat. Na₂S₂O₃ was added (1:1, v/v) and the layers were separated. The aqueous layer was extracted thrice with DCM. The combined organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. To the residue was added TFAA (17 mL, 122 mmol, 2.2 eq) at 0 °C. After 15 min, the temperature was increased to 55 °C for 3 h. The mixture was concentrated under reduced pressure, redissolved in DCM and sat. Na₂CO₃ was added. The layers were separated and the organic layer was washed with sat. NaHCO₃. The solvent was evaporated and the crude was dissolved in THF:MeOH (20:1, v/v) and K₂CO₃ (18.2 g, 132 mmol, 2.3 eq) was added. After 17 h H₂O was added and the layers were separated. The aqueous layer was extracted thrice with EtOAc. The combined organic layers were dried over MgSO₄, filtered, and the solvent evaporated under reduced pressure. The crude was purified with flash column chromatography (10–20% EtOAc in pentane) to yield 5.79 g (19.7 mmol, 35%) of a white solid. ¹H‑NMR (500 MHz, CDCl₃) δ 7.42 (ddt, J = 8.5, 3.5, 0.7 Hz, 1H), 7.29 (t, J = 8.5 Hz, 1H), 4.80 (d, J = 3.3 Hz, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 156.10 (d, J = 256.2 Hz), 148.74 (d, J = 19.1 Hz), 135.01 (d, J = 2.9 Hz), 128.17 (d, J = 4.2 Hz), 126.09 (d, J = 19.8 Hz), 59.07.

(6-bromo-3-fluoropyridin-2-yl)methyl methanesulfonate (3)

To a cooled (0 °C) mixture of (6-bromo-3-fluoropyridin-2-yl)methanol (1.6 g, 7.8 mmol, 1 eq) and Et₃N (2.5 mL, 17.9 mmol, 2.3 eq) in dry THF (40 mL) was added dropwise MsCl (1.0 mL, 12.9 mmol, 1.7 eq). After stirring at rt for 1 h the solution was concentrated under reduced pressure. DCM and H₂O were added and the layers were separated. The aqueous layer was extracted thrice with DCM. The combined organic layers were washed with brine, dried over MgSO₄, filtered, and the solvent evaporated under reduced pressure to yield 1.65 g (5.8 mmol, 75%) of an yellow solid. ¹H‑NMR (500 MHz, CDCl₃) δ 7.52 (dd, J = 8.6, 3.5 Hz, 1H), 7.37 (t, J = 8.5 Hz, 1H), 5.33 (d, J = 2.1 Hz, 2H), 3.13 (s, 3H). ¹³C‑NMR (126 MHz, CDCl₃) δ 157.82 (d, J = 261.3 Hz), 142.15 (d, J = 16.0 Hz), 130.74 (d, J = 4.4 Hz), 127.06 (d, J = 20.4 Hz), 65.50 (d, J = 1.6 Hz), 38.39.

N-((6-bromo-3-fluoropyridin-2-yl)methyl)tetrahydro-2H-pyran-4-amine (4)

(6-Bromo-3-fluoropyridin-2-yl)methyl methanesulfonate (1.49 g, 5.3 mmol, 1 eq), K₂CO₃ (1.6 g, 11.6 mmol, 2.2 eq) and tetrahydro-2H-pyran-4-amine (0.66 mL, 6.7 mmol, 1.3 eq) were suspended in acetonitrile and stirred at 50 °C for 6 h, then an additional 3 days at rt. After dilution with DCM and H₂O the layers were separated. The aqueous layer was extracted thrice with DCM. The combined organic layers were dried over MgSO₄, filtered, and the solution evaporated under reduced pressure. The crude was purified with flash column chromatography (20–100% EtOAc in pentane) to yield 1.01 g (3.5 mmol, 67%) as a yellow oil. ¹H‑NMR (300 MHz, CDCl₃) δ 7.40 (dd, J = 8.6, 3.6 Hz, 1H), 7.35–7.26 (m, 1H), 4.08–3.95 (m, 4H), 3.42 (td, J = 11.6, 2.2 Hz, 2H), 2.74 (tt, J = 10.5, 4.1 Hz 1H), 1.89 (ddd, J = 12.7, 4.5, 2.3 Hz, 2H), 1.52 (dtd, J = 13.1, 11.0, 4.5 Hz, 2H). ¹³C‑NMR (75 MHz, CDCl₃) δ 157.12 (d, J = 255.9 Hz), 149.21 (d, J = 17.0 Hz), 127.83 (d, J = 4.2 Hz), 125.97 (d, J = 21.2 Hz), 66.76, 53.64, 44.90, 33.59.

2-((4-bromobenzyl)amino)acetamide (6)

To a mixture of 4-bromobenzaldehyde (5, 9.2 g (49.7 mmol, 1.1 eq) and 2‑aminoacetamide hydrochloride (5.06 g, 45.8 mmol, 1.0 eq) in MeOH:H₂O (170 mL, 5:1, v/v) was added NaOH (2.06 g, 51.5 mmol, 1.1 eq) and left to stir at rt overnight. NaBH₄ (3.6 g, 95.2 mmol, 2.1 eq) was added and the solution was stirred overnight at rt. The solution was acidified to pH 3 with 2 M HCl, then neutralized with sat. aqueous NaHCO₃. Methanol was evaporated under reduced pressure and the resulting slurry was filtered to yield 11.0 g (45.2 mmol, 91%) of a white solid. ¹H‑NMR (300 MHz, methanol-d₄) δ 7.69–7.59 (m, 2H), 7.47–7.38 (m, 2H), 4.22 (s, 2H), 3.81 (s, 2H).

1-(4-bromobenzyl)imidazolidine-2,4-dione (7)

To a suspension of 2-((4-bromobenzyl)-amino)acetamide (10.0 g, 40.1 mmol, 1,0 eq) in acetonitrile (300 mL) were added CDI (13.86 g, 85.5 mmol, 2.1 eq) and DMAP (10.2 g, 83.5 mmol, 2.1 eq). The mixture was heated to 60 °C under inert atmosphere for 70 h. HCl (1 M, 250 mL) was added and the aqueous layer extracted thrice with EtOAc. The combined organic layers were washed with H₂O and brine, dried over MgSO₄, filtered, and the solvent evaporated under reduced pressure. The crude was purified with flash column chromatography with dry loading over Celite (5-10% acetone in DCM) to yield 3.95 g (14.7 mmol, 37%) of a yellow solid. ¹H‑NMR (300 MHz, CDCl₃) δ 7.83 (bs, 1H), 7.56 – 7.45 (m, 2H), 7.20 – 7.10 (m, 2H), 4.49 (s, 2H), 3.79 (s, 2H). ¹³C‑NMR (75 MHz, CDCl₃) δ 132.41, 129.95, 77.58, 77.16, 76.74, 50.36, 46.01.

1-(4-bromobenzyl)-3-isobutylimidazolidine-2,4-dione (8)

To solution of 1-(4-bromobenzyl)imidazolidine-2,4-dione (2.00 g, 7.4 mmol, 1,0 eq) in anhydrous DMF (18 mL) were subsequently added K₂CO₃ (3.08 g, 22.3 mmol, 3,0 eq) and 1-bromo-2-methylpropane (1.62 mL, 14.9 mmol, 2,0 eq) and the mixture was stirred for 20 h at rt. The mixture was filtered and the filtrate diluted with diethyl ether and washed thrice with water (3 × 50 mL). The combined organic layers were washed with brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. The crude was purified with flash column chromatography (10–40% EtOAc in pentane) to yield 2.12 g (6.52 mmol, 88%) of a white solid. LCMS (LCQ Fleet, 10–90%): t_r = 7.00 min, m/z: 325.17 [M + H]⁺, 327.08 [M + H]⁺ (Br). ¹H-NMR (300 MHz, CDCl₃) δ 7.47 (d, J = 8.3 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H), 4.52 (s, 2H), 3.74 (s, 2H), 3.33 (d, J = 7.4 Hz, 2H), 2.15–2.04 (m, 1H), 0.91 (d, J = 6.8 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 169.57, 156.78, 134.41, 131.79, 129.48, 121.77, 60.01, 48.61, 45.98, 45.71, 28.57, 19.70.

3-isobutyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)imidazolidine-2,4-dione (9)

A mixture of 1-(4-bromobenzyl)-3-isobutylimidazolidine-2,4-dione (0.50 g, 1.54 mmol, 1 eq), KOAc (0.66 g, 6.76 mmol, 4.4 eq) and bis(pinacolato)diboron (0.59 g, 2.31 mmol, 1.5 eq) in DMF (10 mL) was sonicated for 15 min under argon flow. Subsequently, Pd(dppf)Cl₂ (0.07 g, 0.09 mmol, 0.06 eq) was added and the mixture was stirred at 75 °C for 20 h. The mixture was cooled to rt, diluted with EtOAc (100 mL) and water (10 mL) and the layers were separated. The water layer was extracted thrice with EtOAc (3 × 20 mL). The combined organic layers were extracted with sat. aqueous NaHCO₃, water and brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. The raw product was co-evaporated with CHCl₃ and used in the next step without further purification.

1-(4-(5-fluoro-6-(((tetrahydro-2H-pyran-4-yl)amino)methyl)pyridin-2-yl)benzyl)-3-isobutylimidazolidine-2,4-dione (LEI-102)

To a degassed mixture of N-((6-bromo-3-fluoropyridin-2-yl)methyl)tetrahydro-2H-pyran-4-amine (4, 0.29 g, 1.0 mmol, 1,0 eq), 3-isobutyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)imidazolidine-2,4-dione (9, 0.56 g, ~1.5 mmol, crude) and K₂CO₃ (1.29 g, 6.0 mmol, 6,0 eq) in toluene:ethanol (10 mL, 4:1, v/v) was added under argon atmosphere Pd(PPh₃)₄ (0.18 g, 0.10 mmol, 0.1 eq). The resulting mixture was stirred for 18 h at 75 °C, subsequently cooled to rt, and filtered. The filtrate was diluted with EtOAc and washed with water and brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. The crude was purified with flash column chromatography (0‑20% MeOH in EtOAc) to yield 0.24 g of a white solid (0.53 mmol, 53%). Further purification with preparative HPLC resulted in a yield of 0.204 g (0.45 mmol, 45%). LCMS (LCQ Advantage, 10–90%): t_r = 5.32 min, m/z: 455.27 [M + H]⁺, 908.93 [2 M + H]⁺. HRMS (ESI+) m/z: calcd. for C₂₅H₃₂FN₄O₃ [M + H], 455.245; found, 455.245. ¹H NMR (400 MHz, CD₃CN) δ 8.05 (d, J = 8.3 Hz, 2H), 7.86 (dd, J = 8.7, 3.6 Hz, 1H), 7.61 (t, J = 9.0 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 4.53 (s, 2H), 4.45 (s, 2H), 3.93 (dd, J = 11.4, 4.4 Hz, 2H), 3.77 (s, 1H), 3.47 (tt, J = 11.8, 3.8 Hz, 2H), 3.30 (td, J = 11.9, 1.9 Hz, 2H), 3.24 (d, J = 7.3 Hz, 2H), 2.05 (br d, J = 13.3 Hz, 2H), 1.99 (dt, J = 13.2, 6.6 Hz, 1H), 1.83 (qd, J = 12.1, 4.5 Hz, 2H), 0.88 (d, J = 6.7 Hz, 6H). ¹³C NMR (100 MHz, CD₃CN) δ 171.54, 157.25 (d, J = 226.6 Hz), 156.12, 153.03 (d, J = 4.5 Hz), 140.51 (d, J = 16.1 Hz), 138.83, 137.70, 129.13, 128.22, 125.56 (d, J = 18.8 Hz), 122.81 (d, J = 4.3 Hz), 118.38, 66.55, 55.55, 50.30, 46.81 (d, J = 7.9 Hz), 42.95, 30.02, 28.32, 20.32.

Constructs

The N-BRIL fused wild type (WT) human CB₂R construction and co-expression of G protein for cryo-EM study were performed using the similar procedure as described before³⁴. In brief, the WT human CB₂R was modified to contain a fusion protein BRIL to improve the protein expression and thermostability, along with a 10×His-tag and a FLAG-tag at the N-terminal. The CB₂R, Gα_i1 and Gβ₁γ₂ subunits were cloned into the pFastBac vector separately using cloning kits.

Expression and purification of CB₂R-G_i-Scfv16 complexes

Methods of complex expression and purification in the current study have been described previously³⁴. The CB₂R and G_i heterotrimer were co-expressed in Sf 9 insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Cells were infected with three separate virus preparations for CB₂R, Gα_i1 and Gβ₁γ₂ at a ratio of 1:2:2 at a cell density of 2.5 × 10⁶ cells/mL. After 48 h, the cell culture was collected by centrifugation and the cell pellets were stored at −80 °C until use. The cell pellets were thawed and lysed in the hypotonic buffer of 10 mM HEPES (pH 7.5), 10 mM MgCl₂, 20 mM KCl with EDTA-free complete protease inhibitor cocktail tablets (Roche, #5056489001). The CB₂R-G_i complex was formed in membranes by addition of 25 μM agonist (LEI-102, APD371, HU308, and CP55,940, respectively) and 2 units of apyrase (NEB, #M0398S) in the presence 500 µg scFv16. The lysate was incubated for overnight at 4 °C and discard the supernatant by centrifugation at 186,000 × g for 30 min. Subsequently, the solubilization buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.75% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace, #4216588), 0.15% (w/v) cholesterol hemisucinate (CHS, Sigma-Aldrich, #C6512) supplemented with 25 μM agonist and 2 units of apyrase (NEB) were added to solubilize complexes for 2 h at 4 °C. Insoluble material was removed by centrifugation at 186,000 × g for 30 min and the supernatant was immobilized by batch binding to TALON IMAC resin (Clontech, #635507) including 20 mM imidazole over 6 h at 4 °C. Then, the resin was packed and washed with 15 column volumes (CVs) of washing buffer I containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.1% (w/v) LMNG, 0.02% (w/v) CHS, 30 mM imidazole and 20 μM agonist, and 15 CVs of washing buffer II containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) LMNG, 0.006% (w/v) CHS, 50 mM imidazole and 20 μM agonist. After that, the protein was eluted using 3 CVs of elution buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, 250 mM imidazole and 25 μM agonist. Finally, the complex was concentrated using the centrifugal filter with 100 kDa molecular weight cutoff and loaded onto a Superdex200 10/300 GL column (GE Healthcare) with buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% GDN (Anatrace, #GDN101), 0.0001% (w/v) CHS, 100 μM TCEP. The fractions consisting of purified CB₂R-G_i complex were collected and concentrated to 0.8–1.0 mg/mL for electron microscopy experiments.

Cryo-EM grid preparation and data collection

For cryo-EM grids preparation of the CB₂R-G_i complexes, 3 μL of the concentrated protein was loaded to a glow-discharged holey carbon grid (CryoMatrix Amorphous alloy film R1.2/1.3, 300 mesh), and subsequently were plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The chamber of Vitrobot was set to 100% humidity at 4 °C. The sample was blotted for 2.5 s with blot force 2. Cryo-EM images were collected on a Titan Krios microscope operated at 300 kV equipped with a Gatan Quantum energy filter, with a slit width of 20 eV, a Gatan K2 summit direct electron camera (Gatan). Images were taken at a dose rate of 8e⁻/Å²/s with a defocus range of −0.8 to −2.0 μm using SerialEM software⁶⁴ in EFTEM nanoprobe mode, with 50 μm C2 aperture, at a calibrated magnification of 130,000 corresponding to a magnified pixel size of 1.04 Å. The total exposure time was 8.1 s and 45 frames were recorded per micrograph.

Cryo-EM image processing

The cryo-EM data processing was performed with CryoSPARC⁶⁵. For CB₂R-Gα_i-scFv16-APD371/LEI-102/HU308/CP55,940 dataset, a total of 7443, 5282, 7530, and 6473 movies were collected, respectively. For all datasets, patch motion correction was used for beam-induced motion correction. Contrast transfer function (CTF) parameters for each micrograph were determined by patch CTF estimation. Using Blob Picker in CryoSPARC to auto pick particles in the first 500 micrographs of CB₂R-Gα_i-scFv16-APD371 complex dataset and then 258,347 particles were extracted to conduct 2D classification. 9277 particles in good 2D patterns were selected as templates to pick better particles. 5,239,870, 3,398,611, 4,653,294, and 3,595,875 particles extracted, respectively, in a 256 Å box were divided into three hundred two-dimensional (2D) class averages with a maximum alignment resolution of 6 Å. Then, 1,152,146, 762,471, 355,832, and 440,292 particles were selected from good 2D classification after two round 2D classification, individually. Following 2D classification, these particles were subjected for ab initio reconstruction into four classes. After heterogeneous refinement, homogeneous refinement, non-uniform refinement and local refinement of the best-looking dataset in CryoSPARC, the final map has an indicated global resolution of 3.08, 2.98, 2.97, and 2.84 Å at a Fourier shell correlation (FSC) of 0.143, respectively. Local resolution was determined using the Bsoft package with half maps as input maps⁶⁶.

Model building and refinement

For CB₂R-G_i-scFv16 complex, the CB₂R-AM12033 cryo-EM structure and G_i protein in CB₂R were used as the starting model. The model was docked into the EM density map using Chimera⁶⁷, followed by iterative manual adjustment and rebuilding in COOT⁶⁸ and phenix.real_space_refine in Phenix⁶⁹. The model statistics were validated using MolProbity⁷⁰. Structural figures were prepared in Chimera and PyMOL (http://www.pymol.org). The final refinement statistics were provided in Supplementary Table 4. The extent of any model overfitting during refinement was measured by refining the final model against one of the half-maps and by comparing the resulting map versus model FSC curves with the two half-maps and full model.

Generation of mutants

The WT CB₁R and CB₂R genes were subcloned into vector pcDNA3.1 with an N-terminal HA signal peptide and FLAG-tag. Mutations were introduced by QuikChange PCR (as described by supplier).

Transfection

24 h prior to transfection, HEK293T cells were seeded on 10 cm ø plates to reach approximately 50% confluence at the start of transfection. The cells were transfected with 10 µg plasmid DNA of WT hCB₂R or hCB₁R receptor, or mutant receptor using the calcium phosphate precipitation method⁷¹. In short, a DNA-calcium mix was made containing 270 mM CaCl₂ and 10 µg plasmid DNA to which Hank’s Balanced Salt Solution (HBSS; 280 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, and 50 mM HEPES) was added in a 1:1 (v/v) ratio and mixed by aeration to create consistent calcium phosphate precipitates. For transfection, 1 mL DNA-calcium mix was added per 10 cm ø plate, followed by a 48 h incubation under a humidified atmosphere at 37 °C with 5% CO₂.

Enzyme-linked immunosorbent assay (ELISA)

Receptor expression after transfection was measured in an enzyme-linked immunosorbent assay (ELISA). After 24 h of transfection, HEK293T cells were detached with phosphate-buffered saline (PBS)/EDTA and seeded into a sterile 96-well poly-D-lysine coated plate at a density of 100,000 cells per well and kept under a humidified atmosphere at 37 °C with 5% CO₂. After an additional 24 h, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature (rt). Cells were washed twice with Tris-buffered saline (TBS) and were blocked with TBS supplemented with 0.1% TWEEN 20 (TBST) and 2% BSA (w/v)) for 30 min at rt while shaking. Subsequently, the cells were incubated with monoclonal M2 mouse anti-FLAG primary antibody (1:4000) for 2 h at rt while shaking. After removal of the antibody, the cells were washed three times with TBST and incubated with the secondary goat anti-mouse HRP-conjugated antibody (1:10,000) for 1 h at rt while shaking. After a final wash with TBS, the cells were treated with 3,3’,5,5’-Tetramethylbenzidine (TMB, Sigma-Aldrich #T0440) in the dark for maximally 10 min at rt to visualize immunoreactivity. The reaction was quenched with 1 M H₃PO_4, and absorbance was read at 450 nm with a Wallac EnVision 2104 Multilabel reader (PerkinElmer).

Membrane preparation

For membrane preparation, HEK293T cells were harvested 48 h after transfection. Cells were detached by scraping into 3 mL of PBS and subsequently centrifuged at 2000 × g for 5 min. Pellets were resuspended in ice-cold Tris buffer (50 mM Tris-HCl, pH 7.4) and homogenized with an Ultra Turrax homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany). Cytosolic and membrane fractions were separated using a high-speed centrifugation step of 31,000 rpm in a Beckman Optima LE-80K ultracentrifuge with Ti70 Rotor for 20 min at 4 °C. After a second cycle of homogenization and centrifugation, the final pellets were resuspended in 50 mM Tris-HCl pH 7.4, 5 mM MgCl₂ and stored in 100 µL aliquots at −80 °C until use. CHOK1_hCB₂bgal cells were harvested when reaching 90% confluence in 15 cm ø plates after one week subculture at a 1:6 ratio. Membrane preparation followed a similar procedure as described above. Final membrane pellets were resuspended in 50 mM Tris-HCl pH 7.4 and stored in 100 µL aliquots at −80 °C until use. Membrane protein concentrations were determined using a BCA protein determination assay as described by the manufacturer⁷².

[³H]RO6957022 competition association assays

For assessment of kinetic agonist binding at hCB₂R, [³H]RO6957022 competition association assays were executed. These assays were previously described with the main difference of incubation at 25 °C compared to 10 °C for identification of more distinct kinetic differences⁷³. In short, prior to kinetic assessment of agonist binding, the affinity (IC₅₀) of the agonists at the hCB₂R was determined in [³H]RO6957022 displacement assays. CHOK1_hCB₂bgal were thawed, homogenized, and subsequently diluted to 1 µg protein per well. When studying endocannabinoids, membranes were preincubated with 50 µM PMSF for 30 min. Membranes were incubated with ~1.5 nM [³H]RO6957022 and six increasing concentrations of competing agonists in a total volume of 100 µL assay buffer (50 mM Tris-HCl (pH 7.4), 0.1% (w/v) BSA). Incubations were done for 2 h at 10 °C to reach equilibrium. Subsequently, in competition association assays, agonists were incubated at their IC₅₀ concentration in the presence of ~1.5 nM [³H]RO6957022 in a total volume of 100 µL assay buffer at 10 °C. Competition was initiated by addition of membrane homogenates at different time points for 2 h. Nonspecific binding (NSB) was determined with 10 µM AM630 and organic solvent (DMSO or acetonitrile) concentrations were <1% in all samples. Total radioligand binding (TB) did not exceed 10% of the amount added to prevent ligand depletion. Incubations were terminated by rapid vacuum filtration with ice-cold 50 mM Tris-HCl (pH 7.4), 0.1% (w/v) BSA buffer through Whatman GF/C filters using a Filtermate 96-well harvester (PerkinElmer). Filters were dried for at least 30 min at 55 °C, and subsequently 25 µL MicroScint scintillation cocktail (PerkinElmer #6013621) was added per well. Filter-bound radioactivity was measured by scintillation spectrometry using a Microbeta² 2450 counter (PerkinElmer).

[³⁵S]GTPγS binding assays

G protein activation by agonists LEI-102, APD371, HU308, CP55,940 AEA, and 2-AG was measured by binding of radiolabeled [³⁵S]GTPγS to the cannabinoid receptors as previously described²⁵. In short, transient HEK293T membrane homogenates (10 µg/well) were diluted in assay buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 150 mM NaCl, 1 mM EDTA, 0.05% BSA (w/v) and 1 mM DTT, freshly prepared every day) and were pretreated with 10 µg saponin and 1 µM GDP. For endocannabinoid samples, the membranes were additionally pretreated for 30 min with 50 µM PMSF before agonist addition. To determine the G protein activation, the membranes were incubated with 10 µM or six increasing concentrations of agonist (ranging from 0.01 nM to 10 µM) for 30 min at rt. Basal receptor activity was determined in the presence of vehicle only (0.2% DMSO/acetonitrile). [³⁵S]GTPγS (0.3 nM) was added and the mixture was co-incubated for an additional 90 min at 25 °C while shaking at 400 rpm. Filtration was performed, and filter-bound radioactivity was determined as described under [³H]RO6957022 Competition Association Assays except for using ice-cold 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂ buffer.

[³H]CP55,940 homologous and heterologous displacement assays

Agonist affinity (K_i) on WT and mutant receptors was determined in [³H]CP55,940 displacement assays. The amount of transient HEK293T membrane, ranging from 0.75 µg to 10 µg protein per well, was chosen to obtain a specific [³H]CP55,940 binding window of 1200-1500 disintegrations per minute (dpm) except for the CB₂R-Quadruple^TM1,7 mutant, for which a window of ~500 dpm could be obtained using 20 µg protein per well. Membranes were thawed and subsequently homogenized using the Ultra Turrax homogenizer. For the endocannabinoid assays, the membranes were preincubated for 30 min with 50 µM PMSF. Homologous displacement assays were performed with 1.5 nM final concentration [³H]CP55,940 and when necessary supplemented with an additional concentration of 0.55 nM [³H]CP55,940 in the presence of competing CP55,940 (ranging from 0.01 nM to 1 µM) in assay buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 0.1% (w/v) BSA). Heterologous displacement assays were executed for LEI-102, APD371, HU308, AEA, and 2-AG using 1.5 nM final concentration [³H]CP55,940 with one concentration (10 µM) or six increasing concentrations (ranging from 0.1 nM to 10 µM) in assay buffer. For both assays, binding was initiated by addition of membrane homogenates to reach a final volume of 100 µL. NSB was determined using 10 µM CP55,940 and organic solvent (DMSO or acetonitrile) concentrations were <1% in all samples. TB did not exceed 10% of the amount added to prevent ligand depletion. Incubation was done for 2 h at 25 °C to reach equilibrium. Filtration was performed, and filter-bound radioactivity was determined as described under [³H]RO6957022 Competition Association Assays except for using ice-cold 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 0.1% (w/v) BSA buffer.

Cisplatin-induced nephropathy

Ten to twelve-week-old male/female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). CB₂R knockout mice (CB₂R^−/−) and their wild-type littermates (CB₂R^+/+) were developed as described previously and had been backcrossed to a C57BL/6J background⁷⁴. All animals were kept in a temperature-controlled environment (20–22 °C) with a 12 h light–dark cycle and were always allowed free access to food and water. All animal experiments reported in this manuscript complied with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (NIH publication 86–23 revised 1985) and were approved by the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (Bethesda, MD).

The well-established model of cisplatin-induced nephropathy was used⁶³. Mice (CB₂R^−/− and CB₂R^+/+) were sacrificed 72 h after a single injection of cisplatin (cis-diamine platinum (II) dichloride (Sigma#P4394) 25 mg/kg i.p.; freshly dissolved in physiological saline) by cervical dislocation under deep anesthesia with 5% isoflurane, for collection of blood and tissue samples. LEI-102 was given i.p. or by oral gavage (p.o.) at 0.3, 3.0, and 10 mg/kg every day, starting 1.5 h before the cisplatin exposure. The drug was dissolved in a vehicle of DMSO:Tween 80:saline, 1:1:18. After administration of LEI‐102, mice were killed by cervical dislocation under deep anesthesia with 5% isoflurane, for collection of blood and tissue samples at the time described in the figure. The tetrad assay in mice has previously been described in detail²⁹.

Biochemistry, histopathology, immunostaining, real-time PCR

Markers of kidney dysfunction (BUN and CREA), histopathology (PAS staining), immunostaining or ELISA for 3-nitrotyrosine (3-NT; Cell Biolabs #STA-305) and 4-hydroxynonenal (4-HNE; Cell Biolabs#STA-838), and real-time PCR (Primers from Qiagen, SYBER Green Vita Scientific#MEIF01301, High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific#4368813) for inflammatory cytokines were performed as previously described⁶³. Tubular damage scores were determined based on the percentage of tubules showing epithelial necrosis where 0= normal; 1, <10%; 2, 10–25%; 3, 26–75%; 4, >75%. Tubular necrosis was defined as the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane, or intraluminal aggregation of cells and proteins. The morphometric examination was performed in a blinded manner. Ten fields were scored from each mouse kidneys at 200× magnification, and average scores were determined for each mouse. For final quantification graph, average tubular damage scores of six mice/group were plotted.

Quantification and statistical analysis

All experimental data were analyzed using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA). All values obtained are means ± standard error of the mean (SEM) of at least three independent experiments performed in duplicate, unless stated otherwise.

From [³H]RO6957022 competition association assays, the k_on and k_off were determined by non-linear regression analysis, using the “kinetics of competitive binding” model as described by Motulsky and Mahan (Motulsky and Mahan, 1984):

K_{a} = k_{1} \cdot (L) \cdot 10^{- 9} + k_{2}

K_{b} = k_{3} \cdot (I) \cdot 10^{- 9} + k_{4}

S = \sqrt{{(K_{a} - K_{b})}^{2} + 4 \cdot k_{1} \cdot k_{3} \cdot (L) \cdot (I) \cdot 10^{- 18}}

K_{f} = 0.5 \cdot (K_{a} + K_{b} + S)

K_{s} = 0.5 \cdot (K_{a} + K_{b} - S)

Q = \frac{B \max \cdot k_{1} \cdot (L) \cdot 10^{- 9}}{K_{f} - K_{s}}

(Y) = Q \cdot (\frac{k_{4} \cdot (K_{f} - K_{s})}{K_{f} \cdot K_{s}} + \frac{k_{4} - K_{f}}{K_{f}} \cdot e^{(- K_{f} \cdot X)} - \frac{k_{4} - K_{s}}{K_{s}} \cdot e^{(- K_{S} \cdot X)})

Where [L] is the radioligand concentration per experiment (~1.5 nM), I is the IC₅₀ concentration of agonist (nM), X is the time (s), and Y is the specific binding of the radioligand (dpm). K_a and K_b are the observed association rate constants (k_obs) of the radioligand and the agonist of interest, respectively. k₁ and k₃ are the association rate constants (k_on in M⁻¹s⁻¹) of [³H]RO6957022 (determined per experiment) and the agonist of interest, respectively. Similarly, k₂ and k₄ are the dissociation rate constants (k_off in s⁻¹) of [³H]RO6957022 (experimentally determined at 4.3 × 10⁻⁴ s⁻¹, data not shown) and the agonist of interest, respectively. The engagement time (ET in seconds) of the agonists of interest was determined at 1 µM of agonist using the equation ET = 1/(k_on · 1 × 10⁻⁶). The residence time (RT in min) was calculated using the equation RT = 1/(60 · k_off)⁷⁵. The association and dissociation rate constants were used to calculate the kinetic K_D using: K_D = k_off/k_on.

[³⁵S]GTPγS agonist responses on hCB₂R constructs were baseline-corrected for the individual mutant’s basal activity. The responses were normalized to the basal activity of the construct (0%) and top of the CP55,940 (for WT responses only) or WT curve (for mutants, 100%). The potency (pEC₅₀) and efficacy (E_max) values were obtained by non-linear regression to a sigmoidal concentration-effect curve with a Hill slope of 1 by using the “log(agonist) vs response (three parameters)” model. [³⁵S]GTPγS data from hCB₁R constructs were expressed as fold over the mutant’s basal activity to also quantify the effects of CB₂R selective agonists.

Displacement assays were baseline-corrected with NSB and normalized to this value (0%) and TB (100%). The equilibrium dissociation constants (K_D) of [³H]CP55,940 on different mutants were calculated from homologous displacements by non-linear regression analysis, using the “one-site homologous” model. The half-maximal inhibitory concentrations (pIC₅₀) of the agonists in [³H]CP55,940 and [³H]RO6957022 assays were obtained by non-linear regression analysis of the homologous and heterologous displacement curves and further converted into inhibitory constant pK_i using the Cheng-Prusoff equation⁷⁶. In which the experimentally determined K_D for each construct was used for [³H]CP55,940 assays or 0.78 nM for [³H]RO6957022 assays (data not shown).

Differences in pEC₅₀, E_max, pK_D and pK_i values for each mutant compared to WT were analyzed using a one-way Welch’s ANOVA with Dunnett’s T3 multiple comparisons test or an unpaired Student’s t-test with Welch’s correction. Significant differences are displayed as *p < 0.05; **p < 0.01, ***p < 0.001 and ****p < 0.0001. For the animal experiments all the values are represented as mean ± SEM. Statistical analysis of the data was performed by analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons or t-test if appropriate. The analysis was conducted using GraphPad Prism 9 software. p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

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Acknowledgements

This work was supported by the National Science Fund for Distinguished Young Scholars 32022038 (T.H.), the National Natural Science Foundation of China grant 91953202 (Z.-J.L.), the CAS Strategic Priority Research Program XDB37030104 (Z.-J.L.), the National Natural Science Foundation of China grant 31870744 (T.H.), the National Key Research and Development Program of China grant 2018YFA0507000 (T.H.), the Shanghai Rising-Star Program 20QA1406500 (T.H.); the Dutch Research Council (NWO, Vidi #16573) (L.H.H) and (NWO, Navistroke #15851) (M.v.d.S.). and Intramural Research Program of National Institutes of Health/NIAAA (P.P.).

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Source data

Author contributions

Conceptualization, T.H., Z.J.L., J.B., L.V.d.P., A.P.A.J., M.v.d.S., L.H.H and P.P.; Conducting Experiments, X.T.L., H.C., J.B., L.V.d.P., C.v.d.H, S.S.K, L.J.W., Y.N. Y., R.J.B.H.N.v.d.B, A.P.A.J., M.M., P.M., J.P.; Writing-Original Draft, X.T.L., H.C., J.B., L.V.d.P., A.P.A.J., and P.P.; Writing Review & Editing, M.v.d.S., L.H.H., A.L., P.P. T.H., and Z.J.L.; Visualization, X.T.L., H.C., J.B., A.P.A.J., P.M., L.J.W.; Supervision, M.v.d.S., L.H.H, P.P., T.H., and Z.J.L.

Peer review

Peer review information

Nature Communications thanks Matthew Belousoff, Frank Park and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Data availability

The data that support this study are available from the corresponding authors upon request. The atomic coordinates for CB₂R-LEI-102-G_i-scFv16, CB₂R-APD371-G_i-scFv16, CB₂R-HU308-G_i-scFv16, and CB₂R-CP55,940-G_i-scFv16 have been deposited in the Protein Data Bank with the accession codes 8GUT, 8GUQ, 8GUS, and 8GUR. The EM maps for CB₂R-LEI-102-G_i-scFv16, CB₂R-APD371-G_i-scFv16, CB₂R-HU308-G_i-scFv16 and CB₂R-CP55,940-G_i-scFv16 have been deposited in EMDB with the codes EMD-34279, EMD-34276, EMD-34278, and EMD-34277, respectively. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Footnote Group

Contributor Information

Zhi-Jie Liu, Email: liuzhj@shanghaitech.edu.cn.

Pal Pacher, Email: pacher@mail.nih.gov.

Mario van der Stelt, Email: m.van.der.stelt@chem.leidenuniv.nl.

Laura H. Heitman, Email: l.h.heitman@lacdr.leidenuniv.nl

Tian Hua, Email: huatian@shanghaitech.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-023-37112-9.

References

Untitled section

References

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Studies

Conditions

Cannabinoids and active compounds

Related research

Abstract

Untitled section

Web Summary

Article notes

Untitled section

Author notes

Introduction

Results

LEI-102 as a high affinity and potent CB2R-selective agonist

Distinct target binding kinetic profiles of CB2R agonists

Overall similar structural comparison of CB2R-Gi in complex with different agonists

The binding mode of LEI-102 in CB2R

The binding mode of APD371 in CB2R

The binding mode of HU308 in CB2R

The binding mode of CP55,940 in CB2R

LEI-102 and APD371 require H952.65 for G protein activation in CB2R

An important role for S2857.39 and V2616.51 in CB2R activation by HU308 and CP55,940

HU308 and endocannabinoids gain access via membrane entry

LEI-102 attenuates cisplatin-induced nephrotoxicity without CB1R-mediated side effects

Discussion

Methods

General materials for functional assays

Cell lines

Synthesis of LEI-102

(6-bromo-3-fluoropyridin-2-yl)methanol (2)

(6-bromo-3-fluoropyridin-2-yl)methyl methanesulfonate (3)

N-((6-bromo-3-fluoropyridin-2-yl)methyl)tetrahydro-2H-pyran-4-amine (4)

2-((4-bromobenzyl)amino)acetamide (6)

1-(4-bromobenzyl)imidazolidine-2,4-dione (7)

1-(4-bromobenzyl)-3-isobutylimidazolidine-2,4-dione (8)

3-isobutyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)imidazolidine-2,4-dione (9)

1-(4-(5-fluoro-6-(((tetrahydro-2H-pyran-4-yl)amino)methyl)pyridin-2-yl)benzyl)-3-isobutylimidazolidine-2,4-dione (LEI-102)

Constructs

Expression and purification of CB2R-Gi-Scfv16 complexes

Cryo-EM grid preparation and data collection

Cryo-EM image processing

Model building and refinement

Generation of mutants

Transfection

Enzyme-linked immunosorbent assay (ELISA)

Membrane preparation

[3H]RO6957022 competition association assays

[35S]GTPγS binding assays

[3H]CP55,940 homologous and heterologous displacement assays

Cisplatin-induced nephropathy

Biochemistry, histopathology, immunostaining, real-time PCR

Quantification and statistical analysis

Reporting summary

Supplementary information

Untitled section

Acknowledgements

Untitled section

Source data

Author contributions

Peer review

Peer review information

Data availability

Competing interests

Footnotes

Footnote Group

Contributor Information

Supplementary information

References

Untitled section

References

Associated Data

Supplementary Materials

Data Availability Statement

Additional materials

LEI-102 as a high affinity and potent CB₂R-selective agonist

Distinct target binding kinetic profiles of CB₂R agonists

Overall similar structural comparison of CB₂R-G_i in complex with different agonists

The binding mode of LEI-102 in CB₂R

The binding mode of APD371 in CB₂R

The binding mode of HU308 in CB₂R

The binding mode of CP55,940 in CB₂R

LEI-102 and APD371 require H95^2.65 for G protein activation in CB₂R

An important role for S285^7.39 and V261^6.51 in CB₂R activation by HU308 and CP55,940

LEI-102 attenuates cisplatin-induced nephrotoxicity without CB₁R-mediated side effects

Expression and purification of CB₂R-G_i-Scfv16 complexes

[³H]RO6957022 competition association assays

[³⁵S]GTPγS binding assays

[³H]CP55,940 homologous and heterologous displacement assays