Covalent Isothiocyanate Inhibitors of Macrophage Migration Inhibitory Factor as Potential Colorectal Cancer Treatments.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity.

Development of Putative Bivalent Dicovalent Ligands for the Adenosine A1 Receptor.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (AR) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each AR of an AR homodimer.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors).

Characterisation of AMB-FUBINACA metabolism and CB-mediated activity of its acid metabolite.

Purpose: AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it.

Developing the Cannabinoid Receptor 2 (CB2) pharmacopoeia: past, present, and future.

Cannabinoid Receptor 2 (CB2) is a G protein-coupled receptor (GPCR) with considerable, though as yet unrealised, therapeutic potential. Promising preclinical data supports the applicability of CB2 activation in autoimmune and inflammatory diseases, pain, neurodegeneration, and osteoporosis. A diverse pharmacopoeia of cannabinoid ligands is available, which has led to considerable advancements in the understanding of CB2 function and extensive preclinical evaluation.

Covalent cannabinoid receptor ligands - structural insight and selectivity challenges.

X-ray crystallography and cryogenic electronic microscopy have provided significant advancement in the knowledge of GPCR structure and have allowed the rational design of GPCR ligands. The class A GPCRs cannabinoid receptor type 1 and type 2 are implicated in many pathophysiological processes and thus rational design of drug and tool compounds is of great interest. Recent structural insight into cannabinoid receptors has already led to a greater understanding of ligand binding sites and receptor residues that likely contribute to ligand selectivity.

Development of Covalent, Clickable Probes for Adenosine A and A Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A receptor (AR) and adenosine A receptor (AR) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship.

Human liver-derived MAIT cells differ from blood MAIT cells in their metabolism and response to TCR-independent activation.

Mucosal associated invariant T (MAIT) cells are anti-microbial innate-like T cells that are abundant in blood and liver. MAIT cells express a semi-invariant T-cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)-MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection.

Neutrophils suppress mucosal-associated invariant T cells in humans.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a nonredundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied.

Type I interferons are important co-stimulatory signals during T cell receptor mediated human MAIT cell activation.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as TLR agonists, that can modulate TCR-mediated activation.

TCR- or Cytokine-Activated CD8 Mucosal-Associated Invariant T Cells Are Rapid Polyfunctional Effectors That Can Coordinate Immune Responses.

Mucosal-associated invariant T (MAIT) cells can be activated via either their T cell receptor (TCR), which recognizes MR1-bound pyrimidines derived from microbial riboflavin biosynthesis, or via cytokines. These two modes of activation may act in concert or independently, depending upon the stimulus. It is unknown, however, how MAIT cell responses differ with the mode of activation.

Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor.

Cannabinoid type 2 receptor (CBR) is an attractive target for the treatment of pain and inflammatory disorders. Availability of a selective CBR fluorescent ligand to study CBR expression and localization in healthy and disease conditions would greatly contribute to improving our understanding of this receptor. Herein, we report a series of chromenopyrazole-based CBR fluorescent ligands.

Development of selective, fluorescent cannabinoid type 2 receptor ligands based on a 1,8-naphthyridin-2-(1)-one-3-carboxamide scaffold.

Cannabinoid type 2 (CB) receptor has been implicated in several diseases and conditions, however no CB receptor selective drugs have made it to market. The aim of this study was to develop fluorescent ligands as CB receptor tools, to enable an increased understanding of CB receptor expression and signalling and thereby accelerate drug discovery. Fluorescent ligands have been successfully developed for other receptors, however none with adequate subtype selectivity or imaging properties have been reported for CB receptor.

Cannabinoid Receptor 2 Signalling Bias Elicited by 2,4,6-Trisubstituted 1,3,5-Triazines.

Cannabinoid receptor 2 (CB) is predominantly distributed in immune tissues and cells and is a promising therapeutic target for modulating inflammation. In this study we designed and synthesised a series of 2,4,6-trisubstituted 1,3,5-triazines with piperazinylalkyl or 1,2-diethoxyethane (PEG2) chains as CB agonists, all of which were predicted to be considerably more polar than typical cannabinoid ligands. In this series, we found that triazines containing an adamantanyl group were conducive to CB binding whereas those with a cyclopentyl group were not.

Synthesis of novel (benzimidazolyl)isoquinolinols and evaluation as adenosine A1 receptor tools.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in eukaryotes. The adenosine A receptor (AAR) is a class A GPCR that is of interest as a therapeutic target particularly in the treatment of cardiovascular disease and neuropathic pain. Increased knowledge of the role AAR plays in mediating these pathophysiological processes will help realise the therapeutic potential of this receptor.

Alkyl indole-based cannabinoid type 2 receptor tools: Exploration of linker and fluorophore attachment.

Cannabinoid type 2 (CB) receptor continues to emerge as a promising drug target for many diseases and conditions. New tools for studying CB receptor are required to further inform how this receptor functions in healthy and diseased states. The alkyl indole scaffold is a well-recognised ligand for cannabinoid receptors, and in this study the indole C5-7 positions were explored for linker and fluorophore attachment.

Development of novel fluorescent histamine H-receptor antagonists to study ligand-binding kinetics in living cells.

The histamine H-receptor (HR) is an important mediator of allergy and inflammation. HR antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor HR ligand binding kinetics in living cells.

Chemical Tools for Studying Lipid-Binding Class A G Protein-Coupled Receptors.

Cannabinoid, free fatty acid, lysophosphatidic acid, sphingosine 1-phosphate, prostanoid, leukotriene, bile acid, and platelet-activating factor receptor families are class A G protein-coupled receptors with endogenous lipid ligands. Pharmacological tools are crucial for studying these receptors and addressing the many unanswered questions surrounding expression of these receptors in normal and diseased tissues. An inherent challenge for developing tools for these lipid receptors is balancing the often lipophilic requirements of the receptor-binding pharmacophore with favorable physicochemical properties to optimize highly specific binding.

Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist.

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.

Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers.

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance.

The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs.

The past decade has witnessed fluorescently tagged drug molecules gaining significant attraction in their use as pharmacological tools with which to visualize and interrogate receptor targets at the single-cell level. Additionally, one can generate detailed pharmacological information, such as affinity measurements, down to almost single-molecule detection limits. The now accepted utilization of fluorescence-based readouts in high-throughput/high-content screening provides further evidence that fluorescent molecules offer a safer and more adaptable substitute to radioligands in molecular pharmacology and drug discovery.

Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A(1) and -A(3) receptors.

Highly potent and selective fluorescent antagonists of the human adenosine A₃ receptor based on the 1,2,4-triazolo[4,3-a]quinoxalin-1-one scaffold.

The adenosine-A(3) receptor (A(3)AR) is a G protein-coupled receptor that shows promise as a therapeutic target for cancer, glaucoma, and various autoimmune inflammatory disorders, and as such, there is a need for molecular probes to study this receptor. Here, we report a series of fluorescent ligands containing different linkers and fluorophores based around a 1,2,4-triazolo[4,3-a]quinoxalin-1-one antagonist. One of these conjugates (19) displayed high affinity for the A(3)AR (pK(D) = 9.