Current Medicinal Chemistry 1908 researchers havesolvedstructure the ofmembrane the formation ogy because of theirability tomediate the flow of in- 1. INTRODUCTION Alegre-RS 90619-900,Brazil Bioinformatics,Pontifical Catholic University ofRioGrande(PUCRS),Av. doSul Ipiranga,Porto 6681, (PUCRS), Av. Ipiranga, 6681, Porto Alegre-RS 90619-900, Brazil; Cellular andMolecular Biology, School ofSciences,Pontifical Catholic University ofRio Grande doSul E-mails: [email protected], [email protected]. Alegre-RS, Brazil; Tel/Fax: ++55-51-3353-4529, Pontifical Catholic University of Grande Rio doSul-PUCRS, Porto *Address correspondence to this author at the School of Sciences, Keywords: a Silvana Russo Grande doSul(PUCRS),Av. Ipiranga, 6681, PortoAlegre-RS 90619-900,Brazil; 10.2174/092986732566618041716524 DOI: 2018 03, April Accepted: Revised: February 21, 2018 2017 18, December Received: Y R T O I S E H L I C T R A Laboratory of Computational Systems Biology, ofSciences, School Pontifical Catholic University ofRio Membrane proteinsessential are structures inbiol- REVIEW ARTICLE Focusing on the Inverse Agonists Interactions Agonists Inverse onthe Focusing 1 – the of Understanding the in Advances across them intothe cell core [1]. R Cannabinoid r

a,b,c andWalter FilgueiradeAzevedo Jr.

7 actionsthein development inverse ofnovel agonists. lecular hydrogen bonds highlighting theimportance ofthe exploration ofintermolecular inter- otal role played byresidues Phe 170and Leu 359 in their interactions and the strong intermo- Abstract: Methods tures. agonists boundstruc- inverse the on emphasis with studies, structural recent the on focusing Objective of new drugs. development the in role its and ligands its with complexes CB1 investigate to needed be will the central nervous system, whosecrystallographic structure has recently been solved. Studies Conclusion 178 ()and Thr Ser 383(). and 197 otenabant and taranabant, we observed intermolecular hydrogen bonds involving residues His Leuin all 359 complex structuresinvestigated the in present study.thecomplexes For with teractions involve hydrophobic residues, with the participation ofthe residues Phe 170and responsible forthe specificity ofthe inverse agonists forCB1. Most ofthe intermolecular in- Results plex structuresinverse ofCB1withagonists. protein- interactions. also We describe the molecular docking method to obtain com- tallographic structure and docking simulations. We usethis structural information to depict eceptor, drug de drug eceptor,

: : Analysis of the crystallographicAnalysis ofthe structure docking and results revealedthe residues : We startWe a with literature review,and then we describe recentstudies CB 1crys- on 1875-53 Background Our goal hereis to reviewthe studiesCB1, on starting with generalaspects and : Analysis ofthe structuresinvolving inverse agonists CBand 1revealed the piv-

1 5.0.0 ©2019Bentham Science Publishers 3X/19 $58.00+.00 sign, docking, inverse agonist,membrane protein, GPCR. : Current Medicinal Chemistry, 1(CB1) isa membrane protein prevalent in ecently, llular loopsandintrace three extracellular loops[2]. C-terminal, an extracellular N-terminal, three of seven transmembranealpha helices,an int topologywith the group, acommon composedshares proteins. CB1 isan integralmembrane proteinthat Receptor group, (GPCR) new challenges and opportunities for research. drug protein Cannabinoid Receptor 1(CB1) thatbrought has CB1 is a member of the Class A G AProtein-C Class the of amember is CB1 a,b,c,* c Postgraduate Specialization Course in Send Orders for Reprints to [email protected] to Reprints for Orders Send 2019,

26, 1908-1919 b which consistsof Graduate Program in

nearly800 llular racellular oupled oupled Factor: Impact 3.469 eISSN: 1875-533X Chemistry Medicinal Current Timely In-depth International in Medicinal Journal for Chemistry SCIENCE BENTHAM Reviews The ISSN: 0929-8673

Advances in the Understanding of the Cannabinoid Receptor 1 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 1909

Being the most abundant metabotropic membrane with behavioral actions, mainly in the cortex, receptor in the brain, CB1 has a vital role in cell basal ganglia, hippocampus, amygdala, substantia signaling [3]. This protein participates in the control of nigra, and cerebellum. Its low expression in brainstem several processes, including brain development, respiratory centers is accounted for Cannabis low learning, memory, motor behavior, regulation of respiratory toxicity [11]. CB1 is located on presynaptic appetite, body temperature, pain perception, terminals, in intracellular compartments (mitochondria, inflammation, besides being involved in psychiatric endosomes, lysosomes) [4,12], and is also found in and neurological disorders [4]. peripheral tissues like fat tissue, cardiovascular system, Until recently, there was no crystallographic data skeletal muscle, lung, small intestine, uterus, testis for the structure of CB1, so the newly determined CB1 [11,12]. crystal structures provide three-dimensional informa- Cannabinoid Receptor 2 (CB2) is present in micro- tion, which is useful for drug design and development glia, peripheral tissues immune-related like spleen and [5]. If the research of new drugs is focused on a recep- lymph nodes and immune cells [11]. The orphaned re- tor structure using its complex with an antagonist, the ceptors GPR18, GPR55, and GPR119 are known to be result may develop antagonists instead of agonists. activated by endo-, phyto-, and/or synthetic cannabi- Thus, it seems plausible that the receptor structure of noids, which suggest their possible role in the endo- the inverse agonist-bound form is necessary for drug cannabinoid system [3, 13]. design of inverse agonists. Hua et al. [6] have reported The CRs have two crucial lipophilic ligands derived two crystal structures of CB1, one in which the recep- from arachidonic acid, N-arachidonoylethanolamine or tor is bound with the agonist AM11542 (Protein Data (AEA), and 2-arachidonoylglycerol (2- Bank (PDB): 5XRA), and in the other, we have a com- AG), known as the endocannabinoids (EC). It is now plex with the agonist AM841 (PDB: 5XR8). Before generally accepted that AEA is a partial agonist, and 2- that, Hua et al. [7] reported the crystal structures of AG is a full agonist, at both CB1 and CB2 receptors [3, CB1 complexed with the inverse agonist AM6538 9, 14]. An essential characteristic of these ligands is (PDB: 5TGZ) and Shao et al. [8] reported the crystal that their precursors are present in lipid membranes, structure of CB1 complexed with the inverse agonist and they are liberated and released rapidly upon de- MK-0364 (taranabant) (PDB: 5U09). It is well-known mand into the extracellular space [15]. EC act in CB1 that CB1 inverse agonists will have a crucial role in the and CB2 receptors and additional GPCRs such as ion treatment of diseases like [5-8]. channels, ion channel receptors and nuclear receptors Our focus in this review is on the intermolecular in- [9]. Structural data could bring further insights into the teractions of CB1 with its inverse agonists using the mode of binding of these ligands against CB1, but so structure contained in the PDB under code 5TGZ, and far, there are no crystallographic structures for the we explain the structural basis for the interaction of complexes of CB1 with these lipophilic ligands. CB1 with inverse agonists using its available crystallo- On the other hand, the recently determined struc- graphic information. Also, we describe the application tures of CB1 open the possibility of using these lipo- of molecular docking simulations to determine the philic ligands against the CB1 structure for docking mode of binding of inverse agonists against the struc- simulations, but due to the presence of 17 torsional an- ture of CB1. gles in the structure of AEA, this system is going to be a challenging one to simulate using protein-ligand 2. THE docking programs. The Endocannabinoid System (ECS) was catego- The EC are hydrolyzed intracellularly mainly by rized after the cloning of CB1 in the early nineties and two . Fatty acid amide hydrolase (FAAH) (EC the discovery of the endogenous ligand anandamide 3.5.1.99) is an integral membrane that hydro- (AEA) right after that [9]. ECS has multiple compo- lyzes the AEA and related amidated signaling lipids nents, consisting of the cannabinoid receptors (CR), into arachidonic acid(AA). FAAH is a serine hydrolase their ligands, and the enzymes that metabolize them. also known as hydrolase or anandamide ami- Some authors also include in ECS, the naturally found dohydrolase. (MAGL) (EC [10], usually called phytocannabinoids. 3.1.1.23) controls the degradation of 2-AG into AA and We describe these components in the following. glycerol, which transforms 85% of all 2-AG [16, 17]. We find CB1 in the central nervous system (CNS), MAGL is also a member of the serine hydrolase en- widely distributed in a way that it is said to correlate zyme family. 1910 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 Russo and de Azevedo Jr.

Especially interesting is the observation that AEA 4. SIGNALLING PATHWAYS can participate on both CB1 and transient receptor po- EC are retrograde synaptic messengers, suppressing tential vanilloid type 1 ion channels (TRPV1) interac- transmitter release in a transient or long-lasting man- tions, usually controlled by degradation via the enzyme ner, at both excitatory and inhibitory synapses, inhibit- FAAH [18]. A previously published work hypothesized ing synaptic transmission [21]. ECS retrograde signal- that such multifaceted modulation of neuronal excit- ing occurs via the spillover of neurotransmitter which ability might clarify the mixed hyperalgesic and anal- initiates the synthesis of EC by the postsynaptic neuron gesic actions described for molecules targeting can- membrane, which are then released and transported to nabinoid CB1 receptors and TRPV1 [18]. Structural activate CB1 expressed on the presynaptic terminal studies focused on the interaction between AEA, CB1, [22]. A signaling cascade initiates with activated CB1 and TRPV1 may guide the development of a novel decoupling associated G proteins, where Gαi/o targets generation of drugs for the treatment of pain. adenyl cyclase. As a consequence, we have a reduction The capacity of the plant Cannabis to alter human of cyclic AMP production, and the interaction of Gβγ consciousness was discovered at least 12,000 years protein subunits with ion channels such as N- and P/Q- ago. Cannabis constituents, the phytocannabinoids, are type Ca2+ channels inhibiting calcium currents and a family of lipophilic compounds structurally related to activating the Kir3 class of potassium channels [3]. Δ9- (THC), isolated by Gaoni and Ligand-activated CB1, with altered shape after G- Mechoulam [19] as the primary psychoactive ingredi- protein binding, and presenting different ent in Cannabis. THC interacts with CB1 receptors in phosphorylation patterns in multiple sites of the car- areas of the cortex involved in the control of emotional boxy-terminus or cytoplasmic loops will bind to a β- behavior [20]. The structural information of THC-CB1 arrestin molecule. This CB1-β-arrestin binding will end interaction has the potential to improve our understand- and prevent further G-protein activation and signaling, ing of the analgesic effects of THC and its ability to start receptor desensitization and internalization into control emotional and eating behavior [6]. endosomes besides initiating β-arrestin signaling cas- cades. After internalization, CB1 can return to the 3. AGONISTS AND ANTAGONISTS plasma membrane or go through degradation by the We may classify the CB1 agonists into one of four lysosomes [23]. CB1 activate several cellular path- groups named classical, nonclassical, aminoalkylindole ways, such as the mitogen-activated protein and [3]. The conventional group consists of kinases/extracellular signal-regulated kinase (MAPK/ (–)-Δ9-tetrahydrocannabinol (THC), the principal psy- ERK), c-Jun N-terminal kinases (JNK), phosphatidyli- choactive constituent of Cannabis, and (–)-11-hydroxy- nositol 3-kinases (PI3K), and mammalian target of ra- Δ8-tetrahydrocannabinol-dimethylheptyl (HU-210), a pamycin (mTOR) pathways [24]. synthetic analog of (–)-Δ8-tetrahydrocannabinol. The CB1 is widely expressed in several levels at the nonclassical group contains bicyclic and tricyclic ana- plasma membrane of axonal shafts and presynaptic logs of THC that lack a pyran ring, a member of this terminals [21], and in different neuronal types. In group being CP55940. The aminoalkylindole group GABAergic neurons, CB1 can control food intake, best-known agonist is R-(+)-WIN55212. The eicosa- running-related behaviors, drug addiction, and learning noid group includes the EC, AEA, and 2-AG. and memory processes [4]. CB1 in glutamatergic neu- The CB1-selective agonists are all synthetic analogs rons controls neuroprotection, olfactory processes, fear of AEA: R-(+)-, arachidonyl-2’- memories, social behaviors, and anxiety [4]. The func- chloroethylamide and arachidonylcyclopropylamide. tional roles of CB1 are relevant to motor regulation, Main CB2-selective agonists include the classical can- control of appetite, and memory processing. It helps to nabinoid, JWH-133, the nonclassical cannabinoid, HU- regulate metabolic processes related to energy storage 308, and the aminoalkylindoles, JWH-015 and and expenditure, being relevant to the study of obesity AM1241. Antagonists that display significant CB1- [17]. CB1 has recently been found in intracellular selectivity include (SR141716A), AM251, compartments, being predominantly intracellular [25]. AM281, LY320135, and taranabant. CB1-selective Activation of mitochondrial CB1 decreases respiration competitive antagonists include NESS O327 and and cause memory impairment, whereas blockade in- AM4113, both of which are structural analogs of ri- creases energy production [26]. Structural studies fo- monabant. The EC , which acts as a CB1 cused on the complexes of CB1 with different ligands , also behaves as a non-competitive might shed light on the binding mode of these mole- negative allosteric modulator of CB1 [3, 4]. cules. Advances in the Understanding of the Cannabinoid Receptor 1 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 1911

5. DIFFICULTIES IN GCPR DRUG RESEARCH CB1 also shows biased signaling, meaning that con- If the screening of novel compounds is directed by formational changes caused by different ligands will using a receptor structure from its complex with an an- initiate several kinds of signaling cascades and subse- tagonist or inverse agonist, one may end up with an- quent cellular responses [31]. This involves G-proteins tagonists or inverse agonists rather than with agonists. or β-arrestin pathway activation. It is expected that the It seems plausible that the receptor structure of the in- study of biased signaling will take to the development verse agonist-bound form is necessary for drug design of drugs with the potential to reach a specific pathway of inverse agonists, and it is essential to understand the [32]. ligand-initiated GPCRs conformational change to de- 6. MATERIALS AND METHODS velop new drugs targeting these receptors. Understand- ing of binding efficacy in GPCRs is challenging due to Application of X-ray diffraction crystallography their conformational complexity. was able to solve the structure of CB1 in 2016 [7]. So far, there are four structures deposited in the PDB [33- Evidence indicates that GPCRs may show agonist- 35] with crystallographic resolution ranging from 2.6 to independent constitutive activity, meaning that CB1 2.95 Å (PDB access codes: 5TGZ [7], 5U09 [8], 5XR8 shows some grade of activation even in the absence of [6], 5XRA [6]) (search carried out on November 27th, an agonist [9]. Together with this concept, we also 2017). To facilitate the crystallization of CB1, crystal- know that inverse-agonists are compounds that inhibit lographers deleted residues from Val 306 to Pro 332 this constitutive activity and destabilize a receptor con- and substituted them by flavodoxin (residues 1002- formation in the presence, and in the absence, of en- 1148). The flavodoxin works as a fusion protein and dogenous agonists. A hyperactive ECS seems to be does not participate in the transmembrane helix bundle involved in the etiology of several diseases [27, 28]. [7]. Fig. (1) shows the complete structure 5TGZ with The inverse agonist AM6538 bound to CB1 in the flavodoxin insertion. structure 5TGZ is derived from AM251, a rimonabant analog. Rimonabant (SR141716A), a drug used to treat The available crystallographic structures of CB1 obesity related disorders, is an inverse agonist at CB1 made possible the use of these structures to carry out only when higher concentrations are reached and molecular docking and molecular dynamics simula- potentially involving an allosteric site mechanism [29]. tions [7]. Our focus here is on the description of the This drug has been removed from the market because it structure with special attention to the inverse agonist has produced adverse effects including nausea and interactions with CB1. We also describe the inverse suicide risk that limited its clinical use [30]. agonist interactions with CB1 obtained through mo- lecular docking simulations.

AB GGGRGENFMDIECFMVLNPSQQLAIAVLSLTLGTF TVLENLLVLCVILHSRSLRCRPSYHFIGSLAVADL LGSVIFVYSFIDFHVFHRKDSRNVFLFKLGGVTAS FTASVGSLFLAAIDRYISIHRPLAYKRIVTRPKAV VAFCLMWTIAIVIAVLPLLGWNCEKLQSVCSDIFP HIDKTYLMFWIGVVSVLLLFIVYAYMYILWKAHSH AVAKALIVYGSTTGNTEYTAETIARELADAGYEVD SRDAASVEAGGLFEGFDLVLLGCSTWGDDSLELQD DFIPLFDSLEETGAQGRKVACFGCGDSSWEYGCGA VDAIEEKLKNLGAEIVQDGLRIDGDPRAARDDIVG WAHSVRGAIPDQARMDIELAKTLVLILVVLIICWC PLLAIMVYDVFGKMNKLIKTVFAFCSMLCLLNSTV NPIIYALRSKDLRHAFRSMFPS

Flavodoxin

Fig. (1). A) Ribbon diagram of CB1 showing the flavodoxin insertion on the bottom of the structure. We used the program Visual Molecular Dynamics (VMD) [36] to generate this figure. B) The sequence of the structure 5TGZ highlighting the fla- vodoxin residues. 1912 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 Russo and de Azevedo Jr.

A previously published study about the prediction 7. RESULTS AND DISCUSSION of ligand binding to CB1 used the program Glide 6.9 7.1. Crystal Structure of CB1 [37-39] to carry out docking simulations. Here we per- formed molecular docking simulations of the inverse A search in the PDB looking for protein sequences agonists against the structure of CB1 (PDB access that are similar to CB1 with sequence identity higher code: 5TGZ) using the program Molegro Virtual than 25 % returned nine protein structures (search car- Docker (MVD) [40-43]. We used the program SAn- ried out on November 27th, 2017). In this search, we DReS [44-47] to analyze the docking results. We vali- used the CB1 sequence without the fusion protein. Be- dated the docking protocol using the crystallographic sides the CB1 structures, there are also structures of position of the ligand AM6538 (ligand code: ZDG) in Adenosine A2A Receptor (A2A) (PDB access codes: the structure 5TGZ. We assigned atomic charges for 3PWH, 3REY, 3RFM [50], 3UZA, 3UZC [51]). The protein and ligands using the default-charging scheme A2A shows only 25 % of identity with CB1 sequence, available in the MVD program [40]. but both proteins share the same overall structure. We employed the Iterated Simplex algorithm with In addition to the structure of A2A, CB1 presents adaptive sampling strategy based Ant Colony Optimi- the same folding observed in the rhodopsin (rho) struc- zation [40, 41] as a search engine and the MolDock ture [52] (PDB access code: 1F88). Fig. (3) shows the scoring function to calculate pose energy for all dock- sequence alignment of these proteins, which presents ing simulations. This scoring function represents the the canonical GPCR 7 transmembrane-helical bundle interaction energy between the pose and the protein fold. CB1 shows an overall structure, which is similar [40]. SAnDReS [44] determined root-mean-square de- to rho and A2A, with a canonical GPCR 7 transmem- viation (RMSD) between poses and the crystallo- brane-helical bundle fold with a short helix VIII packed graphic position for the ligand AM6538. parallel to the membrane bilayer. We downloaded the structures of the inverse ago- Fig. (4) shows the structure of CB1 complexed with nists rimonabant (ZINC01540228), otenabant the antagonist AM6538 (PDB access code: 5TGZ) [7]. (ZINC03948997), and taranabant (ZINC28701758) In CB1 structure, the transmembrane helices vary in from the ZINC database [48, 49] and the atomic length from 25 to 35 residues and do not line up per- charges for them were assigned as described for the pendicular to the phospholipid bilayer, as observed for AM6538 structure. We applied the previously de- the rho structure. Analysis of CB1 structure indicates scribed docking protocol to determine the binding of that the transmembrane helical bundle involves the these inverse agonists into the structure of CB1. Fig. following regions: helix I (Pro 112-His 143), helix II (2) shows the structures of the antagonists/inverse ago- (Ser 152-Val 179), helix III (Arg 186-Arg 220), helix nists studied in the present work. IV (Arg 230- Gly 254), helix V (Lys 273-His 304),

H3C helix VI (Asp 333-Ile 362), and helix VII (Lys 373-Arg A B O 400). CH3 N N Cl H There is an additional amphipathic helix at the C- N CH3 N terminal involving residues Lys 402-Met 411, named N O Cl Cl N C here as helix VIII. These helices show an assembly that HN Cl N Cl exhibits variation on their tilt angles and bends. CB1 Cl structure shows kinks in helix IV (Pro 231 and Pro H C H N C 3 2 + D 251) and helix VII (Pro 358) which are due to the pres- NH2 O ence of proline residues. Also, the helix III bends at a CH3 pair of glycine residues, Gly 194-Gly 195, allowing the N O

N HN main chain oxygen of Thr 197 to make a strong hydro- N CH3 N H3C gen bond with the side chain at residue Thr 201. One N N O Cl N proline residue neighboring to the antagonist-binding site in helix VII (Pro 358) serves to bend the helix VII, F Cl which brings residues Leu 359 and Met 363 close to F F the antagonist-binding site. Fig. (2). Structures of the inverse agonists A) AM6538, B) In summary, the kinks observed in the transmem- rimonabant (ZINC01540228), C) otenabant (ZINC0394- brane helical bundle help enable them to pack more 8997), and D) taranabant (ZINC28701758). closely and to make a binding pocket with a volume of Advances in the Understanding of the Cannabinoid Receptor 1 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 1913

Helix I Rho MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAE----PWQFSMLAAYMFLLIMLGFPINF CB1 ------GENFMDIECFMVLNPSQQLAIAVLSLTLGTFTVLE---NL A2A ------MPIMGSSVYITV----ELAIAVLA------ILG---NV :.::. ::::* :**. Helix II Rho LTLYVTVQHKKLRTPLNY-ILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGC-NLEG CB1 LVLCVILHSRSLRCRPSYHFIGSLAVADLLG--SVIFVYSFIDFHVFHRKDSRNVFLFKL A2A LVCWAVWLNSNLQNVTNY-FVVSLAAADILV--GVLAIPFAITISTGFCAACHGC-LFIA *. ...* .* :: .**.::.: ** : . . : Helix III Helix IV Rho FFATLGGEIALWSLVVLAIERYVVVCKPMSNFRF-GENHAIMGVAFTWVMALACAAPPLV CB1 GGVTASFTASVGSLFLTAIDRYISIHRPLAYKRIVTRPKAVVAFCLMWTIAIVIAVLPLL A2A CFVLVLAQSSIFSLLAIAIDRYIAIAIPLRYNGLVTGTRAAGIIAICWVLSFAIGLTPML .::.** **:: ** ::* : ...:* *...::: *:: Helix V Rho GWSRY------IPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFC CB1 GWN------CEKLQSVC--SDIFPHIDKTY--LMFWIGVTSVLLLFIVYAYM A2A GWNNCGQPKEGKNHSQGCGEGQVACLFEDVVP---MNY--MVYFNFFACVLVPLLLMLGV **. **. * . :: : ...::: :: Helix VI Rho Y------GQLVFTVKEAAAQQQESA--TTQKAEKEVTR------MVIIMVIAAFL CB1 YILWKAHSHAVERMIQRGTQKSIIH--TSEDGKVQVTRPDQARMDIRLAKTLVLILVVLI A2A Y------LRIFAAARRQLKQMESQPLPGERARSTLQK------EVHAAKSAAIIAGLFA * ::..:.:.::: Helix VII Helix VIII Rho ICWLPYAGVAFYIFTHQGSDFGPIFMTIPAF-FAKTSAVYNPVIYIMMNKQFRNCMVTTL CB1 ICWGPLLAIMVYDVFGKMNKLIKTVFAFCSM-LCLLNSTVNPIIYALRSKDLRHAFRSMF A2A LCWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLAHTNSVVNPFIYAYRIREFRQTFRKII :** * ::. . : :::..:..** ** .::*::.:

Rho ------CCGKNPLGDDEASTTVSKTETSQVAPA------CB1 PSCEGTAQPLDNSMGDSDCLHKHANNAASVHRAAESCIKSTVKIAKVTMSVSTDTSAEAL A2A ------RSHVLRQQEPFKAAAAH------.:.. : Fig. (3). Sequence alignment of rho, CB1, and A2A generated using MUltiple Sequence Comparison by Log-Expectation (Muscle) [53, 54]. The character “*” indicates conserved residues, the symbol “:” indicates similar residues, and the symbol “.” has been used here to define similar partial residues.

A ECL3 N-terminal B VII ECLI III ECL2 IV I II ECL2 II

VI I IV V V III ECL3 ECL1 VIII VII

C-terminal VIII C

VI ICL1

ICL2 ICL3

Fig. (4). Crystallographic structure of human CB1 in complex with AM6538 (PDB access code: 5TGZ) [7]. A. Ribbon diagram of CB1 showing the GPCR 7 transmembrane-helical bundle fold. We numbered the eight helices in Roman numerals with AM6538 near the center. B. The view from the exo-cytoplasmic side shows the access to the antagonist. C. Cavity identified in CB1 structure. This cavity was determined with the program MVD [40] using a probe with a radius of 1.2 Å and grid resolution of 0.5 Å. We used the program VMD [36] to generate Fig. (4A) and Fig. (4B).

424.3 Å3 that binds to the antagonist/inverse agonist. the helices reveals one water-mediated hydrogen bond Fig. (5) shows the antagonist-binding pocket and the involving Arg 214 (helix III) and Gln 334 (helix VI) residues involved in the kinking of helical structures. and a hydrogen bond involving Tyr 224 (short helix An elaborate water hydrogen-bonding network links between helix III and helix IV) and Asp 213 (helix III). the helices in the rho structure [52]. Analysis of water- The lower number of water-mediated hydrogen bonds mediated hydrogen bonds and hydrogen bonds between in the crystallographic structure of CB1 is due to the 1914 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 Russo and de Azevedo Jr. reduced number of water molecules identified in CB1 results for docking rimonabant, otenabant, and tarana- structure (7 water molecules) against the 27 water bant against CB1 structure. molecules identified in rho structure. Fig. (5) shows the hydrogen bonds observed between helices in the CB1 7.3. Structural Basis for CB1-ligand Interactions structure. To analyze CB1-ligand interactions, we applied the In addition to the transmembrane helical bundle, program LigPlot+ [55, 56] to the structure of CB1 there are three extracellular loops (ECL1–ECL3) and (PDB code: 5TGZ) and the docked structures obtained three intracellular loops (ICL1–ICL3) connecting these for the following ligands: rimonabant, otenabant, and helical regions. ECL1 is in the N-terminal and involves taranabant. Since the specificity and affinity between a 14 residues (Gly 99-Asn 112). ECL1 forms a V-shaped protein target and its related ligand depend on intermo- loop, which inserts into the antagonist-binding pocket lecular hydrogen bonds, ionic interactions, and van der and functions as a lid, limiting entry into the pocket Waals contacts as well as on shape complementarity of from the extracellular side (Fig. (4A) and Fig. (4B)). the contact surfaces of both partners [57-61], it is nec- ECL2 contains 18 residues (Trp 255-Asp 272) folding essary to have a standard to evaluate protein-ligand into a complex arrangement that protrudes four resi- interactions. dues (Phe 268, Pro 269, His 270, and Ile 271) into The program LigPlot+ allows establishing structural CB1-binding pocket. ECL3 contains ten residues (Met criteria to evaluate CB1-ligand interactions. It brings 363 – Asn 372) between helix VI and helix VII. consistency in the analysis of protein-ligand interac- tions since it uses the same strong structural evidence Helix V to assign a given interaction for a pair of atoms. We employed LigPlot+ to analyze the binding of a series of Helix III Arg 214 small molecules to the CB1 structure. Fig. (8) shows the protein-ligand interactions for the crystal (PDB ac- Helix VI cess code: 5TGZ) and docked structures obtained using Asp 213 the program MVD. Water Analysis of the crystallographic structure of CB1 in Tyr 224 complex with AM6538 reveals an intricate network of Gln 334 van der Waals contacts as previously reported [7]. There are no intermolecular hydrogen bonds in the CB1 structure, and all contacts involve van der Waals Fig. (5). Water-mediated hydrogen bond and hydrogen-bond interactions. Detailed analysis of the CB1-AM6538 linking helices in CB1 structure. Fig. (5) generated using the structure shows mainly hydrophobic contacts with program VMD [36]. ECL2 and the N-terminus. There are also hydrophobic interactions with all CB1 helices except for helix IV. 7.2. Docking Simulations Analysis of the structure of AM6538 shows a pyrazole ring core with three functional groups (Fig. (7A)). To validate the MVD docking protocol, we carried out docking simulation of the ligand AM6538 (ligand Following the previously published description of code: ZDG) against the atomic coordinates of CB1 the AM6538 structure [7], we named the 2,4- (PDB access code: 5TGZ). Fig. (6) shows the docking dichlorophenyl ring as “arm 1”, the 4-aliphatic moiety results. The coordinates of the center for the docking substituted phenyl ring as “arm 2”, and the piperidine- sphere were x = 43.44 Å; y = 27.17 Å; and z=318.99 Å 1-ylcarbamoyl as “arm 3” (Fig. (7A)). The pyrazole with a radius of 14 Å (Fig. (6A)). The docking RMSD ring core lies between helices II and VII, where we ob- was 0.8 Å, close to the one previously published for the serve hydrophobic contacts with the side chains of Phe structure 5TGZ [7] (Fig. (6B)). 170 (helix II) and Ser 383 (helix VII). Most of the contacts found for the Arm 1 involves residues from Analysis of the docking results indicates a correla- helices II (Gly 166 and Phe 170), III (Val 196), and VI tion of 0.63 between protein scoring function and dock- (Cys 386 and Leu 387). Arm 1 forms π-π interactions ing RMSD (Fig. (6C)). Application of this docking with the residue Phe 170, and with the main-chain at- protocol to the structures of the others inverse agonists oms of the residue Gly166. There are additional van (rimonabant, otenabant, and taranabant) showed the der Waals contacts involving residue Phe 268. binding mode of these ligands. Fig. (7) displays the Advances in the Understanding of the Cannabinoid Receptor 1 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 1915

B A B

Prctein Score vs Docking RMSD C 0 Prctein Score vs Docking RMSD C 0

-50 -50

-100-100

-150-150

-200-200 Prctein Score (kcel/mol) Prctein Score (kcel/mol)

-250-250

-300-300 02468024681010 12 Docking RMSD (A) Fig. (6). A) Ribbon diagram of CB1 structure with docking sphere used in all docking simulations. The structure shows the pose and the crystallographic position for the ligand AM6538. B) Superposition of the lowest energy pose and the crystallo- graphic position of the AM6538 structure. C) Scattering plot for Protein Scoring against Docking RMSD. We used the program MVD [40] to generate Fig. (6A) and Fig. (6B). The program SAnDReS [44] generated the Fig. (6C). Arm 2 A Arm 2 B

Arm 3 Arm 3

Arm 1 Arm 1

C Arm 2 D Arm 2

Arm 3 Arm 3

Arm 1 Arm 1 Fig. (7). Docking results obtained using the program MVD [40]. A) All four inverse agonist structures together. B) Structures of AM6538 and rimonabant (ZINC01540228). C) Structures of AM6538 and otenabant (ZINC03948997). D) Structures of AM6538 and taranabant (ZINC28701758). Considering intermolecular interactions of the arm 2 the residue Phe 170 plays a pivotal role in the intermo- of AM6538 structure, we observe π-π interactions with lecular interaction between AM6538 and CB1. Phe 170 Phe102 and Phe268. Also, we identify hydrophobic interacts with the pyrazole ring as well as rings in arm contacts with helices III (Leu 193 and Val 196) and VI 1 and arm 3. (Leu 359). Finally, for the arm 3, we observe no π-π Analysis of the complex CB1-rimonabant interac- interactions for this region of the structure. There are tions reveals a similar network of van der Waals con- hydrophobic contacts involving the following residues: tacts observed for the crystallographic structure of the Met 103, Ile105, Phe170, Phe174, Ala 380, Ser 383 complex CB1-AM6538. We have no intermolecular and Met 384. In summary, intermolecular interactions hydrogen bonds between rimonabant and CB1. The of AM6538 and CB1 involves van der Waals contacts pyrazole ring core of rimonabant occupies approxi- solely. Analysis of the structure using LigPlot+ [55, 56] mately the same site of the pyrazole ring of AM6538 shows no intermolecular hydrogen bonds. Furthermore, (Fig. (7)). 1916 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 Russo and de Azevedo Jr.

Thr197 The docked structure of rimonabant also shows A Val196 CAC similar positioning for the equivalent structures of the Cys386 CAA

CAB Gly166 Leu193 Phe268 arms. Among the 16 residues involved in van der

CAC

CAN Leu359 Waals contacts with AM6538, three residues are not CAX CAN CAW CAN CAU Phe102 Leu387 CAN CAI conserved in the docked structure of rimonabant; they CAT CAX CAO

CAS CAN CAI NAV are Phe 174, Thr 197, and Ala 380. Also, we observe ANO CAA ZDG Pbe170 CUS Met103 CBC CAI the interaction of His 178 in the complex of rimonabant

NHD

NIT Ser383 and CB1. The residue His 178 shows no interactions in CDG CBK Ile105 CHK Phe174 CTJ the structure of CB1-AM6538. These slight differences CTO observed in the network of van der Waals contacts be- Ala380 Met384 tween these two complexes are probably due to the longer chain in the Arm 2 of the AM6538 and the dif-

His178 ferent positioning of the Arm 3 in the structure of ri-

B C7 Phe170 C9 C9 monabant (Fig. (7B)).

C6 C30 Ile105 N1 For the docked structure of CB1-otenabant, there is N1 Met384 Met103 C3 RIM one intermolecular hydrogen bond involving residue C4

N1 C1 Gly166 C11 N1 C2 His 178. This histidine residue shows van der Waals C3 C16 Ser383

C15 C19 C14 C11 interactions in the complex CB1-rimonabant, but there Leu387 C12 Phe268 C22 C18 C14

C31C30 C12 C15 is no hydrogen bond interaction with rimonabant. Tak- C13 Leu359 ing the structure of CB1-AM6538 as a reference, we Val196 Cys386 did not observe interactions involving the residues Thr Leu193 197, Phe 268, and Cys 386. There are four additional Phe102 contacts involving the ligand otenabant, besides the His

His178 N C 178, we observe van der Waals interactions involving O

C4 C C14 Ile19 C4 the residues Ile 119, Ala 120, and Phe 379. These dif- CH

CG NI DE Ala120 ferences in the pattern of intermolecular interactions 125 Phe174 C1 C2 C3 N1C9 are probably due to the bulky moiety of the otenabant Ala380

C7 C3 C2

C4 C4 structure that replaces the pyrazole ring core found in Ile105 Met384 N1C5

C1C2 the structures of AM6538 and rimonabant. C14 C4 C3 C4 Phe379 C10N1 C12 C13 Met103 C19 Finally, for the structure of the complex CB1- Phe170 N1 C13 OTE C14 C14 C18 C24 C31 taranabant, we observe four intermolecular hydrogen C15 C25 C23 Gly166 Phe102 C26 Leu387 bonds involving residues Thr 197 and Ser 383, two in- Val196 C12 Ser383 teractions for each residue. Analysis of the previous

Leu193 structures showed no participation of these residues in Leu359 intermolecular hydrogen bonds. The structure of the

Met384 complex CB1-taranabant shows most of the van der D Ile105 C11 Waals contacts observed for the structure CB1- Phe102 C9

C6 C3 AM6538, with two exceptions, the residues Gly 166 C4 C5 C6

C1 C0 Met103 C10 C4 and Ala 380. The inverse agonist taranabant shows ad- C1 C Ser383 2.75 C12 O

C1 C13 Phe174 C2 ditional hydrophobic interactions involving residues N2 C11 C36 Phe170 C14 C30 C13 C TAR 3.20 Trp 356 and Phe 379. We suggest that these differences O2 C19 C17 C24 C22 Phe379 C23 N1 might be due the substitution of the pyrrole ring present X1 Cys386 O C24 C36 Thr197 C in the structures of AM6538 and rimonabant by a Phe268 CO2 C25 Trp356 F1 CA F2 C22 flexible moiety, with six torsional angles, that we set 2.98 C4 N P2 CO2 Leu387 3.31 free to rotate in the docking simulation of taranabant Leu359

Leu193 Val196 against the structure of CB1. Fig. (8). Protein-ligand interactions for the structures of the CONCLUSION antagonists/inverse agonists A) AM6538, B) rimonabant (ZINC01540228), C) otenabant (ZINC03948997), and D) Taken together, we may say that the analysis of the taranabant (ZINC28701758). Fig. (8) generated using Lig- crystallographic structure and the docked poses involv- Plot+ [55, 56]. ing CB1 and inverse agonists revealed some interesting Advances in the Understanding of the Cannabinoid Receptor 1 Current Medicinal Chemistry, 2019, Vol. 26, No. 10 1917 features that establish the structural basis for interac- PDB = Protein Data Bank tions of inverse agonists with CB1. The pivotal role Rho = Rhodopsin played by residues Phe 170 and Leu 359 in the interac- tions of complexes and the identification of strong in- RMSD = Root-mean-square Deviation termolecular hydrogen bonds (involving otenabant and 2-AG = 2-arachidonoylglycerol taranabant) highlight the importance of the exploration VMD = Visual Molecular Dynamics of intermolecular interactions in the design and devel- opment of novel inverse agonists. Identification of the TRPV1 = Transient Receptor Potential Vanilloid key residues responsible for the specificity of the Type 1 ligand is especially interesting if we consider the de- velopment of drugs that do not cross the blood-brain CONSENT FOR PUBLICATION barrier, for instance, those intended for treating obe- Not applicable. sity-driven metabolic diseases [7, 62-65]. Therefore, the description of the key residues responsible for drug FUNDING specificity helps us to focus on that part of the inverse This work was supported by grants from Conselho agonists which we may alter to modify lipophilicity. Nacional de Desenvolvimento Científico e Tecnológico For instance, adding hydrogen bond donors and accep- (CNPq, Brazil) (Process Numbers: 472590/2012-0 and tors to the structure of the arm 2 in the taranabant may 308883/2014-4). SR acknowledges support from improve specificity and decrease lipophilicity. On the CAPES. WFA is a senior researcher for CNPq (Brazil). other hand, the addition of a methyl group on the struc- ture of the arm 3 of taranabant has the potential to in- CONFLICT OF INTEREST crease lipophilicity. The authors declare no conflict of interest, financial LIST OF ABBREVIATIONS or otherwise.

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