Communication Chemistry—A European Journal doi.org/10.1002/chem.202003389 & Medicinal Chemistry Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB2 Receptor** Paula Morales+,[a] Gemma Navarro+,[b] Marc Gjmez-Autet+,[c] Laura Redondo,[a] Javier Fern#ndez-Ruiz,[d] Laura P8rez-Benito,[c, f] Arnau Cordom&,[c] Leonardo Pardo,*[c] Rafael Franco,*[b, e] and Nadine Jagerovic*[a] the ~ 360 non-olfactory GPCRs).[2] One of the reasons is that Abstract: Single chemical entities with potential to simul- the orthosteric binding site for a particular endogenous ligand taneously interact with two binding sites are emerging is often highly conserved across a GPCR subfamily, thus strategies in medicinal chemistry. We have designed, syn- making it difficult to achieve high selectivity for specific recep- thesized and functionally characterized the first bitopic li- tor subtypes.[3] Novel approaches to overcome this problem in- gands for the CB2 receptor. These compounds selectively clude the discovery of bitopic ligands that bind the orthosteric target CB2 versus CB1 receptors. Their binding mode was site as well as a less conserved site within the same receptor studied by molecular dynamic simulations and site-direct- unit.[4–6] This type of complementary cavity is often located at ed mutagenesis. the entrance of the orthosteric binding site, as identified in ligand binding pathway simulations, which have been named extracellular vestibule[7] or entrance,[8] or secondary[9] or meta- G protein-coupled receptors (GPCRs) regulate a vast amount of stable[10] binding site, or exosite.[11] In this work, we will name cellular processes,[1] thus, they form one of the most important this cavity as receptor vestibule or exosite. Bitopic ligands that pharmaceutical drug-target class (475 drugs in the market that target the orthosteric site and the receptor vestibule improve represent ~ 34 % of all drugs approved by the US Food and selectivity,[11, 12] off-rates and signaling bias,[4, 13, 14] maintaining Drug Administration).[2] However, these drugs target only 108 bioavailability and brain penetration properties in mice.[15] unique GPCRs that are < 15 % of the ~ 800 genes (or ~ 30 % of Other type of comparable ligands are designed to simultane- ously bind two orthosteric sites of a (homo/hetero) GPCR dimer.[16] These type of ligands have been recently reviewed.[17] [a] Dr. P. Morales,+ L. Redondo, Dr. N. Jagerovic Medicinal Chemistry Institute, Spanish Research Council, Madrid (Spain) While most GPCRs recognize polar ligands, GPCRs for lipid E-mail: nadine@iqm.csic.es mediators are activated by hormone-like signaling molecules [b] Dr. G. Navarro,+ Prof. Dr. R. Franco derived from lipid species, which possess long hydrophobic Department of Biochemistry and Physiology, CIBERNED moieties. This subfamily is mostly composed of the sphingo- Faculty of Pharmacy and Food Sciences, Universitat de Barcelona sine-1-phosphate (S1P), lysophosphatidic acid (LPA) and canna- Barcelona (Spain) [c] M. Gjmez-Autet,+ Dr. L. P8rez-Benito, Dr. A. Cordom&, Prof. Dr. L. Pardo binoid (CB1R and CB2R) receptors.[18] In the crystal structures of Laboratory of Computational Medicine, Biostatistics Unit these receptors[19] the extracellular N-terminus and extracellular Faculty of Medicine, Universitat Autjnoma de Barcelona, Barcelona (Spain) loop 2 folds over the ligand binding pocket blocking the E-mail: leonardo.pardo@uab.es access to the orthosteric binding cavity from the extracellular [d] Prof. Dr. J. Fern#ndez-Ruiz environment. These structures together with binding pathway Department of Biochemistry and Molecular Biology, CIBERNED, IRYCIS Faculty of Medicine, Universidad Complutense de Madrid, Madrid (Spain) simulations suggest that binding of lipid-like ligands to a lipid [e] Prof. Dr. R. Franco GPCR occurs through a narrow channel between transmem- Department of Biochemistry and Molecular Biology, CIBERNED brane helices (TMs) 1 and 7 that connects the orthosteric bind- School of Chemistry, Universitat de Barcelona, Barcelona (Spain) ing site to the lipid bilayer.[20] Notably, the access to the ligand E-mail: rfranco@ub.edu pocket of the MT1 melatonin receptor that binds polar ligands [f] Dr. L. P8rez-Benito (serotonin-derived compounds) is also via the lipid bilayer.[21] Present address: Computational Chemistry Janssen Research & Development, Janssen Pharmaceutica N.V. (Belgium) Thus, the design of bitopic ligands for lipid GPCRs is more [+] These authors contributed equally to this work. challenging than for other GPCRs that fully expose the binding [**] A previous version of this manuscript has been deposited on a preprint site to the extracellular environment, due to the narrow chan- server https://doi.org/10.26434/chemrxiv.12583604.v1. nel linking the binding site and the lipid bilayer. Supporting information and the ORCID identification number(s) for the In the present communication, we have designed bitopic li- author(s) of this article can be found under: gands for the cannabinoid CB2R. We have selected CB2R, in- https://doi.org/10.1002/chem.202003389. stead of CB1R, due to its lack of adverse psychotropic effects T 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access along with its wide therapeutic application in pathologies such article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the as cancer, neuroinflammation and pain.[22] Bivalent ligands original work is properly cited. have already been reported for CB1R[23–25] and CB2R.[26] These li- Chem. Eur. J. 2020, 26, 15839 – 15842 15839 T 2020 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202003389 gands were reported before the release of crystal structures, thus, their binding characteristics remain unclear.[27, 28] Here, we have used the recently released structure of CB2R in its active[29] Gi-bound conformation to identify the binding mode of the designed ligands. The design of bitopic ligands requires the selection of a moiety able to bind the orthosteric site (pharmacophore). In this regard, we have selected chromenopyrazole derivatives A and B (Figure 1), which have been previously identified as CB2R orthosteric agonists.[30] Then, a second pharmacophore unit needs to be developed for the vestibule or exosite. This is chal- lenging because this additional cavity has not been properly characterized yet for most GPCRs. We have taken advantage of the simulated binding process of a lipid inhibitor to the S1P1 receptor.[20] The process consists of the diffusion of the ligand through the bilayer leaflet to contact the vestibule at the top of TM 7 (the rate-limiting step), subsequently moving from this lipid-facing vestibule to the orthosteric binding cavity through the channel between TMs 1 and 7. We propose that lipid GPCRs are capable to recognize orthosteric ligands at the vesti- bule of the receptor. Thus, we have also selected the chrome- nopyrazole moiety as the second pharmacophore so that the Scheme 1. Synthesis of 9-alkoxychromenopyrazoles 6–17 and bivalent chro- menopyrazoles 18–29. Reagents and conditions: (i) 3,3-dimethylacrylic acid, designed bitopic ligands are symmetrical (it contains two methanesulfonic acid, P2O5, 8 h, 70 8C, 81 %; (ii) a) NaH, THF, MW, 25 min, copies of the same pharmacophore). In addition, an appropri- 45 8C; b) ethyl formate, MW, 25 min, 45 8C, 76 %; (iii) corresponding hydra- ate length spacer to cover the distance between both pharma- zine, EtOH, 1–4 h, 40 8C, 28–74 %; (iv) a) NaH, anhydrous THF, 10 min, b) 1- cophores is required. Importantly, this approach has been re- bromoalkane, reflux, 2–12 h, 32–75 %; (v) a) Cs2CO3, anhydrous THF, 10 min, b) 1,(n + 2)-dibromoalkane, reflux, 8–72 h, 6–59 %. cently supported in the model of the bitopic ligand CTL01-05- B-A05, a symmetrical agomelatine molecule linked by an ethoxyethane spacer, which binds both the orthosteric binding site and the exosite of the MT1 melatonin receptor.[21] ing chromenopyrazoles with 0.5 equivalents of the desired di- Chromenopyrazole A, in its most stable conformation (Fig- bromoalkanes. Different bases were tested, and finally cesium ure S1), was docked into the orthosteric and vestibule sites of carbonate was selected and used under inert atmosphere. CB2R, and its stability was assessed by molecular dynamic (MD) Thus, the desired bivalent compounds (18–29) were achieved simulations (Figure S2). Results showed that pharmacophore in low to moderate yields. units remain highly stable at the orthosteric site and moder- In vitro binding affinities of the alleged bitopic ligands 18– ately stable at the exosite. Visual inspection of the models 29 (Table 1) and the corresponding monovalent counterparts shows that the @CH3 group of the methoxy moieties of both 6–17 (Table S1) were obtained from [3H]CP-55,940 competi- pharmacophores are suitable attachment points to link the tion-binding assays using membrane fractions of the human spacer moiety. Linker lengths from six to sixteen methylene CB1R and CB2R, respectively, expressed in HEK-293T cells. None units were chosen for the bivalent molecules. of the monovalent chromenopyrazoles exhibits affinity towards Bivalent chromenopyrazoles and their monovalent ana- any of the cannabinoid receptors. Addition of the second phar- logues were synthesized, starting from chromenopyrazoles 4 macophore makes bivalent ligands capable of binding CB2R in and 5 (Scheme 1).[31] Preparation of 9-alkoxychromenopyrazoles a selective manner. Optimal spacer length for N1- and N2-ethyl (6–17) was achieved in high yields by deprotonation of the hy- derivatives is from 10 (n = 8) to 14 (n = 12) methylene units. droxyl group with sodium hydride, followed by rapid addition Compounds with CB2R affinity constants in the low micro- of an excess of the appropriate 1-bromoalkane. Bivalent com- molar range (22, 24–27) were selected for functional evalua- pounds 18–29 were achieved by alkylation of the correspond- tion by measuring their effect on forskolin-induced cAMP levels in HEK-293 cells expressing hCB2R (Figure 2 A). Dose-re- sponse experiments demonstrated that these compounds are able to inhibit cAMP accumulation as efficiently as CP55,940 but with a slight drop of potency (27: pEC50 = 7.6 vs. CP55,940: pEC50 = 8.2) (Table 2), while their monovalent counterparts are inactive (Figure S3). None of the compounds displayed an effect in non-transfected HEK-293T cells, confirming that the re- sults are fully mediated by CB2R (Figure S4). Figure 1. Structures of chromenopyrazoles A and B.[30] These compounds are isomers differing in the position of the N-ethyl at the pyrazole (N1- or N2- In order to assess the binding mode of homobivalent chro- ethyl). menopyrazoles 22, 25 and 27 (10, 12 and 14 methylene units), Chem. Eur. J. 2020, 26, 15839 – 15842 www.chemeurj.org 15840 T 2020 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202003389 Table 1. Binding affinities of bivalent chromenopyrazoles (18–29) for hCB1R and hCB2R. Compd R1 N[a] CB1R Ki [mm][b] CB2R Ki [mm][b] 18 N1-Et 4 > 40 28.1 : 1.6 19 N2-Et 4 > 40 12.4 : 2.0 20 N1-Et 6 > 40 2.2 : 0.7 21 N2-Et 6 nd nd 22 N1-Et 8 > 40 0.9 : 0.2 23 N2-Et 8 > 40 5.8 : 1.5 24 N1-Et 10 > 40 0.4 : 0.14 25 N2-Et 10 > 40 0.3 : 0.1 26 N1-Et 12 > 40 0.8 : 0.1 27 N2-Et 12 > 40 0.3 : 0.1 28 N1-Et 14 > 40 > 40 29 N2-Et 14 > 40 2.12 : 0.21 A30 N1-Et – 5.0 : 0.7 0.16 : 0.03 B30 N2-Et – 2.9 : 0.5 0.09 : 0.02 WIN[c] – – 0.04 : 0.01 0.003 : 0.002 [a] n refers to Scheme 1. Total number of methylenes in the spacer is n + 2. [b] Values obtained from competition curves using [3H]CP55,940 as radioligand for hCB1R and hCB2R and are expressed as the mean : SEM of at least three experiments. nd: not determined. [c] WIN55,212,2. Figure 3. A) General view of the binding mode of bitopic ligand 22 into the orthosteric site and the vestibule of the CB2R-Gi complex (depicted as cylin- ders for CB2R and grey surfaces for Gi). B) Mesh surface of Val361.35 and Ala2827.36 (spheres in orange) that formed the channel between TMs 1 and 7. C, D) Detailed views of the binding mode of 22 (C) and 25 (D) into the re- ceptor vestibule obtained during the MD simulations (the longer spacer of 27 permits higher flexibility at the vestibule and some of these interactions Figure 2. Decrease of forskolin-induced cAMP (normalized to 100 %), in HEK- are not observed, Figure S6). TMs 1 and 7 are shown in orange and blue, re- 293T cells, upon stimulation of wild type CB2R (A) and Val361.35Met (B) and spectively; Val361.35 and Ala2827.36, which were mutated to Met, are shown in Ala2827.36Met (C) mutant receptors with the CP55,940 agonist and ligands orange; and the pharmacophore units and spacer of bitopic ligands are 22 and 27. shown in green and yellow, respectively. methylene spacer expands toward the lipid-facing vestibule in- Table 2. Functional properties of compounds 22, 24–27 and the refer- teracting with the hydrophobic side chains of Phe912.61, ence compound CP55,940 at wild type and mutant CB2R. Ala2827.36, Met2867.40, and Val361.35. In the vestibule, the chro- Wild type Val361.35Met Ala2827.36Met menopyrazole moiety forms aromatic–aromatic interactions pEC50 Emax[a] pEC50 Emax[a] pEC50 Emax[a] with Phe2837.37. In the simulations Phe2837.37 adopts the trans 22 7.1 : 0.2 42 : 5.4 7.7 : 0.1 75 : 1.5 7.2 : 0.2 77 : 2.5 conformation that opens the channel between TMs 1 and 7 24 7.2 : 0.2 45 : 6.3 nd nd and permits the ligand to reach the membrane, in contrast to 25 7.2 : 0.2 40 : 5.9 nd nd the gauche + conformation observed in the crystal structure of 26 6.5 : 0.2 52 : 4.1 nd nd 27 7.6 : 0.1 44 : 3.7 7.1 : 0.2 68 : 3.5 7.3 : 0.2 84 : 1.4 CB2R[29, 32] that closes the channel. The heptyl chain is accom- CP[b] 8.2 : 0.1 48 : 1.7 8.1 : 0.1 50 : 2.3 7.2 : 0.1 48 : 3.43 modated in a hydrophobic cavity of TMs 1 and 7, facing the membrane, which is formed by Leu391.38, Cys401.39, Met2867.40, [a] Emax (%), the maximum inhibition of forskolin-stimulated cAMP levels (normalized to 100 %), values were calculated using nonlinear regression Leu431.39, Ile2907.44, Met2937.47 and Leu461.45. In the case of the analysis. Data are expressed as the mean : SEM of at least three inde- N1-ethyl derivative the lone pair in the N2 atom forms a hydro- pendent experiments performed in triplicates. [b] CP is CP55,940. nd: not gen bond with Gln321.31. A detailed description of these inter- determined. actions is shown in Figure S6. Figure 3 B shows the mesh surface formed by Val361.35 and Ala2827.36. Clearly, these TMs side chains, which are located we docked their most favorable conformation (Figure S1) into midway between the orthosteric site and the receptor vesti- CB2R in its active state in such a manner that both pharmaco- bule, delimit the channel between TMs 1 and 7. Thus, in order phoric units bind into the orthosteric and vestibule sites (Fig- to validate the proposed binding mode of bitopic ligands, we ure 3 A). Unbiased MD simulations show that these chain mutated the side chains of Val361.35 (Figure 2 B) and Ala2827.36 lengths can simultaneously bind both sites (Figure 3, S5). The (Figure 2 C) to the much larger Met side chain. As expected, Chem. Eur. J. 2020, 26, 15839 – 15842 www.chemeurj.org 15841 T 2020 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202003389 these mutations do not influence the function of the orthoster- [11] M. Masureel, Y. Zou, L. P. Picard, E. van der Westhuizen, J. P. Mahoney, ic agonist CP55,490, but clearly impair signaling of bitopic li- J. P. G. L. M. Rodrigues, T. J. Mildorf, R. O. Dror, D. E. Shaw, M. Bouvier, E. Pardon, J. Steyaert, R. K. Sunahara, W. I. Weis, C. Zhang, B. K. Kobilka, gands 22 and 27 (Table 2) by occupying the volume of the Nat. Chem. Biol. 2018, 14, 1059 – 1066. channel. Notably, these results contrast with other MD simula- [12] R. A. Medina, H. V#zquez-Villa, J. C. Gjmez-Tamayo, B. Benhamffl, M. tions, suggesting the entrance of the ligands from the extracel- Mart&n-Fontecha, T. De La Fuente, G. Caltabiano, P. B. Hedlund, L. Pardo, lular environment.[33, 34] M. L. Ljpez-Rodr&guez, J. Med. Chem. 2014, 57, 6879 – 6884. [13] C. Valant, J. R. Lane, P. M. Sexton, A. Christopoulos, Annu. Rev. Pharma- In summary, we present herein the discovery of CB2R homo- col. Toxicol. 2012, 52, 153 – 178. bivalent bitopic ligands. These compounds were designed as [14] B. I. Gaiser, M. Danielsen, E. Marcher-Rørsted, K. Røpke Jørgensen, T. M. symmetrical bivalent compounds using the previously reported Wrjbel, M. Frykman, H. Johansson, H. Br-uner-Osborne, D. E. Gloriam, chromenopyrazole scaffold as potential pharmacophore for J. M. Mathiesen, D. Sejer Pedersen, J. Med. Chem. 2019, 62, 7806 – 7839. [15] L. Tan, Q. Zhou, W. Yan, J. Sun, A. P. Kozikowski, S. Zhao, X. Huang, J. both units linked by a methylene spacer. Binding and function- Cheng, J. Med. Chem. 2020, 63, 4579 – 4602. al cAMP studies revealed their ability to selectively activate [16] D. Pulido, V. Casadj-Anguera, L. P8rez-Benito, E. Moreno, A. Cordom&, L. CB2R versus CB1R. The longer Ile1.35 in CB1R than Val1.35 in CB2R Ljpez, A. Cort8s, S. Ferr8, L. Pardo, V. Casadj, M. Royo, J. Med. Chem. seems responsible for the observed selectivity (Figure S7). MD 2018, 61, 9335 – 9346. [17] A. H. Newman, F. O. Battiti, A. Bonifazi, J. Med. Chem. 2020, 63, 1770 – simulations and site-directed mutagenesis studies show that 1779. these bitopic ligands bind into the orthosteric site and in a [18] G. P#ndy-Szekeres, C. Munk, T. M. Tsonkov, S. Mordalski, K. Harpsøe, A. S. vestibule/exosite located at the ligands entry/egress channel Hauser, A. J. Bojarski, D. E. Gloriam, Nucleic Acids Res. 2018, 46, D440 – that connects the orthosteric site with the lipid bilayer mem- D446. [19] M. Audet, R. C. Stevens, Protein Sci. 2019, 28, 292 – 304. brane. Whether these bitopic ligands at CB2R show beneficial [20] N. Stanley, L. Pardo, G. De Fabritiis, Sci. Rep. 2016, 6, 22639. therapeutic application needs further investigation such as [21] B. Stauch, L. C. Johansson, J. D. McCorvy, N. Patel, G. W. Han, X. P. structure–activity relationship studies to improve potency. Huang, C. Gati, A. Batyuk, S. T. Slocum, A. Ishchenko, W. Brehm, T. A. White, N. Michaelian, C. Madsen, L. Zhu, T. D. Grant, J. M. Grandner, A. Shiriaeva, R. H. J. Olsen, A. R. Tribo, S. Yous, R. C. Stevens, U. Weierstall, V. Katritch, B. L. Roth, W. Liu, V. Cherezov, Nature 2019, 569, 284 – 288. Acknowledgements [22] A. Dhopeshwarkar, K. Mackie, Mol. Pharmacol. 2014, 86, 430 – 437. [23] Y. Zhang, A. Gilliam, R. Maitra, M. I. Damaj, J. M. Tajuba, H. H. Seltzman, This work was supported by the MICIU/FEDER (RTI2018- B. F. Thomas, J. Med. Chem. 2010, 53, 7048 – 7060. 095544-B-I00, PID2019-109240RB-I00, SAF2017-84117-R), and [24] C. Fern#ndez-Fern#ndez, J. Decara, F. J. Bermffldez-Silva, E. S#nchez, P. Morales, M. Glmez-CaÇas, M. Glmez-Ru&z, L. F. Callado, P. Goya, F. Rodr&- by the CSIC (PIE-201580E033). P.M. is grateful to the CM pro- guez De Fonseca, M. I. Mart&n, J. Fern#ndez-Ru&z, J. J. Meana, N. Jagerov- gram “Atraccijn de Talento Investigador” number 2018-T2/ ic, Arch. Pharm. 2013, 346, 171 – 179. BMD-1081. [25] G. Huang, D. Pemp, P. Stadtmeller, M. Nimczick, J. Heilmann, M. Decker, Bioorg. Med. Chem. Lett. 2014, 24, 4209 – 4214. [26] M. Nimczick, D. Pemp, F. H. Darras, X. Chen, J. Heilmann, M. Decker, Bioorg. Med. Chem. 2014, 22, 3938 – 3946. Conflict of interest [27] M. Glass, K. Govindpani, D. P. Furkert, D. P. Hurst, P. H. Reggio, J. U. Fla- nagan, Trends Pharmacol. Sci. 2016, 37, 353 – 363. The authors declare no conflict of interest. [28] L. P8rez-Benito, A. Henry, M. T. Matsoukas, L. Lopez, D. Pulido, M. Royo, A. Cordom&, G. Tresadern, L. Pardo, Bioinformatics 2018, 34, 3857 – 3863. [29] C. Xing, Y. Zhuang, C. Xing, Y. Zhuang, T. Xu, Z. Feng, X. E. Zhou, M. Keywords: bitopic ligands · CB2 cannabinoid · G protein- Chen, L. Wang, Cell 2020, 180, 645 – 654. coupled receptors · molecular dynamics · site-directed [30] P. Morales, M. Gjmez-CaÇas, G. Navarro, D. P. Hurst, F. J. Carrillo-Salinas, L. Lagartera, R. Pazos, P. Goya, P. H. Reggio, C. Guaza, R. Franco, J. mutagenesis Fern#ndez-Ruiz, N. Jagerovic, J. Med. Chem. 2016, 59, 6753 – 6771. [31] J. Cumella, L. Hern#ndez-Folgado, R. Girjn, E. S#nchez, P. Morales, D. P. [1] D. Wootten, A. Christopoulos, M. Marti-Solano, M. M. Babu, P. M. Sexton, Hurst, M. Gjmez-CaÇas, M. Gjmez-Ruiz, D. C. G. A. Pinto, P. Goya, P. H. Nat. Rev. Mol. Cell Biol. 2018, 19, 638 – 653. Reggio, M. I. Martin, J. Fern#ndez-Ruiz, A. M. S. Silva, N. Jagerovic, Chem- [2] A. S. Hauser, M. M. Attwood, M. Rask-Andersen, H. B. Schiçth, D. E. Glori- MedChem 2012, 7, 452 – 463. am, Nat. Rev. Drug Discovery 2017, 16, 829 – 842. [32] X. Li, T. Hua, K. Vemuri, J. H. Ho, Y. Wu, L. Wu, P. Popov, O. Benchama, N. [3] D. J. Foster, P. J. Conn, Neuron 2017, 94, 431 – 446. Zvonok, K. Locke, L. Qu, G. W. Han, M. R. Iyer, R. Cinar, N. J. Coffey, J. [4] J. R. Lane, P. M. Sexton, A. Christopoulos, Trends Pharmacol. Sci. 2013, Wang, M. Wu, V. Katritch, S. Zhao, G. Kunos, L. M. Bohn, A. Makriyannis, 34, 59 – 66. R. C. Stevens, Z. J. Liu, Cell 2019, 176, 459 – 467. [5] K. Mohr, J. Schmitz, R. Schrage, C. Tr-nkle, U. Holzgrabe, Angew. Chem. [33] T. Hua, K. Vemuri, M. Pu, L. Qu, G. W. Han, Y. Wu, S. Zhao, W. Shui, S. Li, Int. Ed. 2013, 52, 508 – 516; Angew. Chem. 2013, 125, 530 – 538. A. Korde, R. B. Laprairie, E. L. Stahl, J. H. Ho, N. Zvonok, H. Zhou, I. Kufar- [6] S. J. Bradley, A. B. Tobin, Annu. Rev. Pharmacol. Toxicol. 2016, 56, 535 – eva, B. Wu, Q. Zhao, M. A. Hanson, L. M. Bohn, A. Makriyannis, R. C. Ste- 559. vens, Z. J. Liu, Cell 2016, 167, 750 – 762. [7] R. O. Dror, A. C. Pan, D. H. Arlow, D. W. Borhani, P. Maragakis, Y. Shan, H. [34] N. Saleh, O. Hucke, G. Kramer, E. Schmidt, F. Montel, R. Lipinski, B. Xu, D. E. Shaw, Proc. Natl. Acad. Sci. USA 2011, 108, 13118 – 13123. Ferger, T. Clark, P. W. Hildebrand, C. S. Tautermann, Angew. Chem. Int. Ed. [8] C. Wang, Y. Jiang, J. Ma, H. Wu, D. Wacker, V. Katritch, G. W. Han, W. Liu, 2018, 57, 2580 – 2585; Angew. Chem. 2018, 130, 2610 – 2615. X. P. Huang, E. Vardy, J. D. McCorvy, X. Gao, X. E. Zhou, K. Melcher, C. Zhang, F. Bai, H. Yang, L. Yang, H. Jiang, B. L. Roth, V. Cherezov, R. C. Ste- vens, H. E. Xu, Science 2013, 340, 610 – 614. Manuscript received: July 19, 2020 [9] A. Gonz#lez, T. Perez-Acle, L. Pardo, X. Deupi, PLoS One 2011, 6, e23815. Revised manuscript received: August 12, 2020 [10] P. Fronik, B. I. Gaiser, D. Sejer Pedersen, J. Med. Chem. 2017, 60, 4126 – Accepted manuscript online: August 13, 2020 4134. Version of record online: November 9, 2020 Chem. Eur. J. 2020, 26, 15839 – 15842 www.chemeurj.org 15842 T 2020 The Authors. Published by Wiley-VCH GmbH