Identification, optimization and characterization of pharmacological tools for the cannabinoidactivated orphan G protein-coupled receptor GPR18 and related receptors

Cannabinoid receptors comprise two classical subtypes, CB1 and CB2, which belong to the large family of G protein-coupled receptors (GPCRs). They are the major targets of cannabinoids such as tetrahydrocannabinol (THC), the main psychoactive ingredient of Cannabis sativa. However, cannabinoids also interact with additional GPCRs, namely the orphan receptors GPR18 and GPR55. These receptors have been described to be involved in inflammatory diseases, but due to the lack of well-characterized pharmacological tool compounds their validation as drug targets has remained elusive. Here, we aimed to identify new ligands, agonists and antagonists, for these orphan cannabinoid-interacting GPCRs to support their (patho-)physiological characterization. In a β-arrestin recruitment assay, we identified agonists and antagonists for both, GPR18 and GPR55, evaluated their suitability as lead structures and analyzed structure-activity relationships. For GPR18, we identified the first non-lipid-derived synthetic agonist known to date, PSB-KK-107, with an EC50 value of 0.556 µM. Subsequently, potency and selectivity was optimized based on structure-activity relationship studies resulting in the optimized GPR18 agonist PSB-KK-1415 with an EC50 value of 0.0191 µM. This compound class was found to be selective versus GPR55, CB1 and CB2 receptors. In a further study, chromen-4-one derived GPR55 agonists were studied and pharmacologically characterized. The obtained compounds will serve as valuable tool compounds for studying GPR55. Furthermore, synthetic cannabinoids were investigated, which had been reported as constituents of incenses (called “spice”). The pharmacological characterization of these highly potent CB receptor agonists will help to classify their toxicological potential and guide decisions on their legal restriction

Computational investigations on the binding mode of ligands for the cannabinoid-activated G protein-coupled receptor GPR18

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 A9-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

Ligand-binding and -scavenging of the chemerin receptor GPR1

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other. Graphic abstractDeutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Universität Leipzig (1039)Peer Reviewe

Discovery of Tricyclic Xanthines as Agonists of the Cannabinoid-Activated Orphan G-Protein-Coupled Receptor GPR18

[Image: see text] GPR18 is a rhodopsin-like orphan G-protein-coupled receptor (GPCR) that is activated by the natural cannabinoid (CB) Δ(9)-tetrahydrocannabinol (THC). It is highly expressed in immune cells and represents a promising new drug target. However, THC is much more potent in activating CB receptors than GPR18, and several other proposed lipidic agonists for GPR18 have not been independently confirmed. Herein we describe the first non-lipid-like agonists for GPR18 based on a tricyclic xanthine-derived scaffold, along with initial structure–activity relationships. PSB-KD107 (5) and PSB-KD477 (16) displayed significantly higher potency and efficacy than THC, determined in a GPR18-dependent β-arrestin recruitment assay, and were found to be selective versus the CB-sensitive receptors CB(1), CB(2), and GPR55. Structure–activity relationships were steep, and indole substitution was crucial for biological activity. These first selective agonists, which are structurally distinct from the lipidic agonist(s), will allow target validation studies and may eventually contribute to the deorphanization of GPR18

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABA(A) receptors.

Biphenylic compounds related to the natural products magnolol and 4'-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA(A) receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5'-hexylbiphenyl-2,2'-diol (45) and the honokiol analogs 4'-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4'-methoxybiphenyl-2-ol (62) and 5-hexyl-4'-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA(A) receptor agonists

Computational Structure Prediction for Antibody-Antigen Complexes From Hydrogen-Deuterium Exchange Mass Spectrometry: Challenges and Outlook

Although computational structure prediction has had great successes in recent years, it regularly fails to predict the interactions of large protein complexes with residue-level accuracy, or even the correct orientation of the protein partners. The performance of computational docking can be notably enhanced by incorporating experimental data from structural biology techniques. A rapid method to probe protein-protein interactions is hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS has been increasingly used for epitope-mapping of antibodies (Abs) to their respective antigens (Ags) in the past few years. In this paper, we review the current state of HDX-MS in studying protein interactions, specifically Ab-Ag interactions, and how it has been used to inform computational structure prediction calculations. Particularly, we address the limitations of HDX-MS in epitope mapping and techniques and protocols applied to overcome these barriers. Furthermore, we explore computational methods that leverage HDX-MS to aid structure prediction, including the computational simulation of HDX-MS data and the combination of HDX-MS and protein docking. We point out challenges in interpreting and incorporating HDX-MS data into Ab-Ag complex docking and highlight the opportunities they provide to build towards a more optimized hybrid method, allowing for more reliable, high throughput epitope identification

Unraveling the Molecular Basis of Substrate Specificity and Halogen Activation in Vanadium-Dependent Haloperoxidases

Vanadium-dependent haloperoxidases (VHPOs) are biotechnologically valuable and operationally versatile biocatalysts that do not require complex electron shuttling systems. These enzymes share remarkable active-site structural similarities yet display broadly variable reactivity and selectivity. The factors dictating substrate and halogen specificity and, thus, a general understanding of VHPO reaction control still need to be discovered. This work\u27s strategic single-point mutation in the cyanobacterial bromoperoxidase AmVHPO facilitates a selectivity switch to allow aryl chlorination. This mutation induces loop formation absent in the wild-type enzyme, and that interacts with the neighboring protein monomer, creating a tunnel to the active sites. Structural analysis of the substrate-R425S-mutant complex reveals a substrate-binding site at the interface of two adjacent units. There, residues Glu139 and Phe401 interact with arenes, extending the substrate residence time close to the vanadate cofactor and stabilizing intermediates. Our findings validate the long-debated existence of direct substrate binding and provide detailed VHPO mechanistic understanding. This work will thus pave the way for a broader application of VHPOs in diverse chemical processes