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Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18

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Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18 biomolecules Article Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18 Alexander Neumann 1,2 , Viktor Engel 1, Andhika B. Mahardhika 1,2 , Clara T. Schoeder 1,2, Vigneshwaran Namasivayam 1 , Katarzyna Kie´c-Kononowicz 3 and Christa E. Müller 1,2,* 1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical & Medicinal Chemistry, University of Bonn, 53121 Bonn, Germany; [email protected] (A.N.); [email protected] (V.E.); [email protected] (A.B.M.); [email protected] (C.T.S.); [email protected] (V.N.) 2 Research Training Group 1873, University of Bonn, 53127 Bonn, Germany 3 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland; [email protected] * Correspondence: [email protected]; Tel.: +49-228-73-2301; Fax: +49-228-73-2567 Received: 10 March 2020; Accepted: 23 April 2020; Published: 29 April 2020 Abstract: GPR18 is an orphan G protein-coupled receptor (GPCR) expressed in cells of the immune system. It is activated by the cannabinoid receptor (CB) agonist D9-tetrahydrocannabinol (THC). Several further lipids have been proposed to act as GPR18 agonists, but these results still require unambiguous confirmation. In the present study, we constructed a homology model of the human GPR18 based on an ensemble of three GPCR crystal structures to investigate the binding modes of the agonist THC and the recently reported antagonists which feature an imidazothiazinone core to which a (substituted) phenyl ring is connected via a lipophilic linker. Docking and molecular dynamics simulation studies were performed. As a result, a hydrophobic binding pocket is predicted to accommodate the imidazothiazinone core, while the terminal phenyl ring projects towards an aromatic pocket. Hydrophobic interaction of Cys251 with substituents on the phenyl ring could explain the high potency of the most potent derivatives. Molecular dynamics simulation studies suggest that the binding of imidazothiazinone antagonists stabilizes transmembrane regions TM1, TM6 and TM7 of the receptor through a salt bridge between Asp118 and Lys133. The agonist THC is presumed to bind differently to GPR18 than to the distantly related CB receptors. This study provides insights into the binding mode of GPR18 agonists and antagonists which will facilitate future drug design for this promising potential drug target. Keywords: cannabinoid; docking studies; GPCR; GPR18; MD simulation; orphan GPCRs 1. Introduction G protein-coupled receptors (GPCR) represent the largest family of membrane proteins in eukaryotes. They are structurally characterized by seven transmembrane (TM) regions connected by three extracellular (ECL1-3) and three intracellular loops (ICL1-3), an extracellular N-terminal and an intracellular C-terminal domain. Upon binding of the cognate agonist (e.g., biogenic amine neurotransmitter, nucleotide, lipid, amino acid, peptide, glycoprotein) conformational changes are induced. These result in coupling with G proteins, and thereby transducing information from the extracellular to the intracellular compartment and inducing or inhibiting downstream signaling pathways [1,2]. Despite persistent efforts, nearly 100 GPCRs remain orphan, with their endogenous ligands unidentified or unconfirmed [3]. The functionalities and roles of orphan GPCRs under Biomolecules 2020, 10, 686; doi:10.3390/biom10050686 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 686 2 of 19 GPCRs remain orphan, with their endogenous ligands unidentified or unconfirmed [3]. The func- (patho)physiologicaltionalities and roles of conditions orphan GPCRs are inunder most (patho)physiological cases poorly understood. conditions The are identification in most cases poorly of the endogenousunderstood. ligandsThe identification would be helpful of the endogenous for target validation ligands would studies be and helpful the design for target of novel validation therapeutic stud- drugsies and for the orphan design GPCRs. of novel therapeutic drugs for orphan GPCRs. δ GPR18 isis suchsuch an an orphan orphan GPCR GPCR of therapeuticof therapeutic interest, interest, phylogenetically phylogenetically belonging belonging to the to-branch the δ- ofbranch class of A, class rhodopsin-like A, rhodopsin-like GPCRs. GPCRs. GPR18 GPR18 was was first first described described in in 1997 1997 and and reported reported to to bebe highly expressed in didifferentfferent tissuestissues andand cellcell lineslines ofof thethe immuneimmune system,system, includingincluding spleen,spleen, thymus,thymus, and leukocytesleukocytes [4]. [4]. The The role role of of GPR18 GPR18 is isstill still uncl unclearear and and controversially controversially debated. debated. GPR18 GPR18 has been has beenpro- proposedposed by independent by independent groups groups to be to involved be involved in immunological in immunological [5–8] [ 5and–8] andneurodegenerative neurodegenerative pro- processescesses including including Alzheimer’s Alzheimer’s disease disease and and multiple multiple sclerosis sclerosis [9–13]. [9–13 Based]. Based on the on theobservation observation that that the theactivation activation of GPR18 of GPR18 lowers lowers the theintraocular intraocular pressure pressure in mice, in mice, GPR18 GPR18 agonists agonists have have been been proposed proposed for forthe thetreatment treatment of glaucoma of glaucoma [14,15]. [14 ,Antagonists15]. Antagonists targeting targeting GPR18 GPR18 may be may effective be effective as anticancer as anticancer drugs drugs[16–18], [16 since–18], the since receptor the receptor was found was found to be toabun bedantly abundantly overexpressed overexpressed in melanoma in melanoma metastases metastases and andreported reported to contribute to contribute to tumor to tumor cell surviv cell survivalal through through inhibition inhibition of apoptosis of apoptosis [17]. [ 17]. In recentrecent years,years, severalseveral studies aimed at the deorphanization of GPR18 have been published. Due to the the lack lack of of selective selective agonists, agonists, the the moder moderatelyately potent potent cannabinoid cannabinoid (CB) (CB) receptor receptor agonist agonist ∆9- 9 Dtetrahydrocannabinol-tetrahydrocannabinol (THC, (THC, 1) has1) been has beenused usedin pharmacological in pharmacological studies studies to activate to activate human GPR18, human GPR18, which led to the suggestion to classify GPR18 as a cannabinoid receptor subtype besides CB which led to the suggestion to classify GPR18 as a cannabinoid receptor subtype besides CB1 and CB12 and[19–22]. CB2 N[19-Arachidonoylglycine–22]. N-Arachidonoylglycine (NAGly, 2) (NAGly, and resolvin2) and D2 resolvin (RvD2, 3 D2) were (RvD2, proposed3) were as proposedendogenous as endogenousagonists of GPR18 agonists [23,24]. of GPR18 However, [23,24 independent]. However, confirmation independent for confirmation both lipids is for still both lacking, lipids as is other still lacking,groups, asincluding other groups, ours, have including not been ours, able have to not confir beenm able their to activation confirm their of GPR18 activation [25,26]. of GPR18 We recently [25,26]. Wedescribed recently the described first GPR18 the first antagonists GPR18 antagonists based on basedan imidazothiazinone on an imidazothiazinone core structure core structure [21,27]. [ 21These,27]. β Thesewere discovered were discovered by screening by screening a compound a compound library at librarythe human at the receptor human in receptora β-arrestin in arecruitment-arrestin recruitmentassay using assayenzyme using complementation enzyme complementation technology and technology THC as an and agonist. THC as Based an agonist. on the screening Based on theresults, screening a library results, of imidazothiazinones a library of imidazothiazinones was synthesized was and synthesized their structure–activity and their structure–activity relationships relationships(SARs) were investigated. (SARs) were PSB-CB-27 investigated. (4) PSB-CB-27and PSB-CB-5 (4) and(5; for PSB-CB-5 structures, (5; forsee structures,Figure 1) were see Figurereported1) wereas the reported first potent as the and first selective potent GPR18 and selective antagonists GPR18 [21]. antagonists [21]. Figure 1. Structures of proposedproposed GPR18 agonists (1–3) andand antagonistsantagonists ((4–66).). In thethe presentpresent study,study, wewe constructedconstructed aa homologyhomology modelmodel ofof thethe humanhuman GPR18GPR18 to elucidateelucidate the binding modemode ofof the the only only confirmed confirmed agonist agonist so so far, fa ther, the natural natural product product THC, THC, and and of selected of selected antagonists antag- by docking and molecular dynamics (MD) simulation studies. Insights into the binding interactions of Biomolecules 2020, 10, 686 3 of 19 agonists and antagonists will provide a basis for the rational design of more potent ligands and may eventually contribute to the deorphanization of GPR18. 2. Material and Methods 2.1. Homology Modeling The crystal structures of the murine µ-opioid receptor in complex with the agonist BU72 (PDB-ID: 5C1M), the human P2Y1 receptor in complex with the allosteric antagonist BPTU (PDB-ID: 4XNV) and the zebrafish lysophosphatidic acid receptor LPA6 in complex with 1-oleoyl-R-glycerol (PDB-ID: 5XSZ) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein
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