A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2.

The human ATP-binding cassette (ABC) transporter, ABCG2, is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H1 Receptor.

The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Subtype selective fluorescent ligands based on ICI 118,551 to study the human ß2-adrenoceptor in CRISPR/Cas9 genome-edited HEK293T cells at low expression levels.

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective ß2 -adrenoceptor (ß2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the ß2 AR (pKD >7.0) with good selectivity over the ß1 AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; ß2 AR pKD 7.48), 9c (ICI 118,551-ßAla-ßAla-BODIPY-FL-X; ß2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; ß2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; ß2 AR pKD 7.42). 9a (ICI 118,551-ßAla-ßAla-BODIPY-X-630/650) had the highest affinity at recombinant ß2 ARs (pKD 7.57), but also exhibited significant binding affinity to the ß1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular ß2 ARs in HEK293 T cell expressing exogenous ß2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular ß2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T ß2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.

The use of fluorescence correlation spectroscopy to monitor cell surface ß2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying ß2 -adrenoceptor (ß2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged ß2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged ß2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface ß2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface ß2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting ß2AR agonists.

The synthesis and biological evaluation of multifunctionalised derivatives of noscapine as cytotoxic agents.

Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, is a well-known antitussive drug that has a relatively safe in vitro toxicity profile. Noscapine is also known to possess weak anticancer efficacy, and since its discovery, efforts have been made to design derivatives with improved potency. Herein, the synthesis of a series of noscapine analogues, which have been modified in the 6', 9', 1 and 7-positions, is described. In a previous study, replacement of the naturally occurring N-methyl group in the 6'-position with an N-ethylaminocarbonyl was shown to promote cell-cycle arrest and cytotoxicity against three cancer cell lines. Here, this modification has been combined with other structural changes that have previously been shown to improve anticancer activity, namely halo substitution in the 9'-position, regioselective O-demethylation to reveal a free phenol in the 7-position, and reduction of the lactone to the corresponding cyclic ether in the 1-position. The incorporation of new aryl substituents in the 9'-position was also investigated. The study identified interesting new compounds able to induce G2/M cell-cycle arrest and that possess cytotoxic activity against the human prostate carcinoma cell line PC3, the human breast adenocarcinoma cell line MCF-7, and the human pancreatic epithelioid carcinoma cell line PANC-1. In particular, the ethyl urea cyclic ether noscapinoids and a compound containing a 6'-ethylaminocarbonyl along with 9'-chloro, 7-hydroxy and lactone moieties exhibited the most promising biological activities, with EC50 values in the low micromolar range against all three cancer cell lines, and these derivatives warrant further investigation.