Andrea J. Vernall

Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay

Summary 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-A1 and -A3 receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.

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. One such drug‐target family that has received considerable attention are the GPCRs; this review therefore summarizes the most recent developments in the area of fluorescent ligand design for this important drug target. We assess recently reported fluorescent conjugates by adopting a receptor‐family‐based approach, highlighting some of the strengths and weaknesses of the individual molecules and their subsequent use. This review adds further strength to the arguments that fluorescent ligand design and synthesis requires careful planning and execution; providing examples illustrating that selection of the correct fluorescent dye, linker length/composition and geographic attachment point to the drug scaffold can all influence the ultimate selectivity and potency of the final conjugate when compared with its unlabelled precursor. When optimized appropriately, the resultant fluorescent conjugates have been successfully employed in an array of assay formats, including flow cytometry, fluorescence microscopy, FRET and scanning confocal microscopy. It is clear that fluorescently labelled GPCR ligands remain a developing and dynamic research arena.

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) = 9.36 ± 0.12) and is >650-fold selective over other adenosine receptor subtypes. Confocal microscopy revealed clear, displaceable membrane labeling of CHO-A(3) cells with 19, with no detectable labeling of CHO-A(1) cells under identical conditions. This fluorescent ligand was also able to specifically label the A(3)AR in HEK293T cells containing a mixed adenosine receptor population. The subtype specificity, along with its excellent imaging properties, make 19 an ideal tool for studying A(3)AR distribution and organization, particularly in the presence of other adenosine receptor subtypes.

A Single α‐Helical Turn Stabilized by Replacement of an Internal Hydrogen Bond with a Covalent Ethylene Bridge

Walking the carbon bridge to helix stabilization: α-Helix mimetics have been synthesized that contain an ethylene linkage in place of an (i,i+4) hydrogen bond (see structure). The smallest unit possible, a modified pentapeptide, shows the characteristic features of a helix (see CD spectrum). Incorporation of this carbon bridge into a peptide represents the first example of replacement of an internal backbone hydrogen bond.

Ring-deactivated hydroxyalkylpyrrole-based inhibitors of α-chymotrypsin: synthesis and mechanism of action

13C NMR and mass spectrometry studies have been used to demonstrate that the inhibition of alpha-chymotrypsin by N-sulfonylhydroxymethylpyrrole inhibitors (10) is non-covalent. Hydroxyalkylpyrroles in which an electron-withdrawing group (acyl substituent) is introduced at the alternative C2 position have been synthesised and also shown to inactivate alpha-chymotrypsin. SAR studies on this class suggests that the incorporation of phenylalanine at C2 is favoured, however, there is little gain in introducing a hydrophobic substituent at C5.

Cross-metathesis coupling of sugars and fatty acids to lysine and cysteine

Attachment of an olefin tether to the side chain of either lysine or cysteine allows cross metathesis (CM) conjugation with olefin-containing sugar and fatty acid analogues.

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. In this study, we review the radioligands, fluorescent ligands, covalent ligands, and antibodies that have been used to study these lipid-binding receptors. For each tool type, the characteristics and design rationale along with in vitro and in vivo applications are detailed.

Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells

The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R 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 H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H1R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H1R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications.

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 A1 receptor (A1AR) 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 A1AR plays in mediating these pathophysiological processes will help realise the therapeutic potential of this receptor. There is a lack of enabling tools such as selective fluorescent probes to study A1AR, therefore we designed a series of (benzimidazolyl)isoquinolinols conjugated to a fluorescent dye (31–35, 42–43). An improved procedure for the synthesis of isoquinolinols from tetrahydroisoquinolinols via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and atmospheric oxygen is reported. This synthetic method offers advantages over previous metal-based methods for the preparation of isoquinolinols and isoquinolines, which are important scaffolds found in many biologically active compounds and natural products. We report the first synthesis of the (benzimidazolyl)isoquinolinol compound class, however the fluorescent conjugates were not successful as A1AR fluorescent ligands.

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

Cannabinoid type 2 (CB2) receptor has been implicated in several diseases and conditions, however no CB2 receptor selective drugs have made it to market. The aim of this study was to develop fluorescent ligands as CB2 receptor tools, to enable an increased understanding of CB2 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 CB2 receptor. A series of 1,8-naphthyridin-2-(1H)-one-3-carboxamides with linkers and fluorophores appended in the N1 and C3-positions were developed. Molecular modelling indicated the C3 cis-cyclohexanol-linked compounds directed the linker out of the CB2 receptor between transmembrane helices 1 and 7. Herein we report fluorescent ligand 32 (hCB2 pK i = 6.33 ± 0.02) as one of the highest affinity, selective CB2 receptor fluorescent ligands reported. Despite 32 displaying poor specific labelling of CB2 receptor, the naphthyridine scaffold with this linker remains highly promising for future development of CB2 receptor tools.