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.

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.

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.

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.

Identification of a novel toxicophore in anti-cancer chemotherapeutics that targets mitochondrial respiratory complex I.

Disruption of mitochondrial function selectively targets tumour cells that are dependent on oxidative phosphorylation. However, due to their high energy demands, cardiac cells are disproportionately targeted by mitochondrial toxins resulting in a loss of cardiac function. An analysis of the effects of mubritinib on cardiac cells showed that this drug did not inhibit HER2 as reported, but directly inhibits mitochondrial respiratory complex I, reducing cardiac-cell beat rate, with prolonged exposure resulting in cell death. We used a library of chemical variants of mubritinib and showed that modifying the 1H-1,2,3-triazole altered complex I inhibition, identifying the heterocyclic 1,3-nitrogen motif as the toxicophore. The same toxicophore is present in a second anti-cancer therapeutic carboxyamidotriazole (CAI) and we demonstrate that CAI also functions through complex I inhibition, mediated by the toxicophore. Complex I inhibition is directly linked to anti-cancer cell activity, with toxicophore modification ablating the desired effects of these compounds on cancer cell proliferation and apoptosis.

The pharmaceutical industry needs to make safe and effective drugs. At the same time this industry is under pressure to keep the costs of developing these drugs at an acceptable level. Drugs work by interacting with and typically blocking a specific target, such as a protein in a particular type of cell. Sometimes, however, drugs also bind other unexpected targets. These "off-target" effects can be the reason for a drug's toxicity, and it is important ­ both for the benefit of patients and the money that can be saved when developing drugs ­ to identify how drugs cause toxic side effects. The earlier researchers detect off-target effects, the better. Recent data has suggested that an anti-cancer drug called mubritinib has off-target effects on the compartments within cells that provide the cell with most of their energy, the mitochondria. This drug's intended target is a protein called HER2, which is found in large amounts on the surfaces of some breast cancer cells. Yet if mubritinib has this off-target effect on mitochondria, it may be harmful to other cells including heart cells because the heart is an organ that needs a large amount of energy from its mitochondria. Stephenson et al. have now performed experiments to show that mubritinib does not actually interact with HER2 at all, but only targets mitochondria. The effect of mubritinib as an anti-cancer drug is therefore only due to its activity against mitochondria. Digging deeper into the chemistry revealed the small parts of its chemical structure that was responsible for mubritinib's toxicity against heart cells, the so-called toxic substructure. Another anti-cancer drug called carboxyamidotriazole also has the same toxic substructure. Carboxyamidotriazole is supposed to stop cells from taking up calcium ions, but a final set of experiments demonstrated that this drug also only acts by inhibiting mitochondria. Often there is not enough information about many drugs' substructures, meaning off-target effects and toxicities cannot be predicted. The pharmaceutical industry will now be able to benefit from this new knowledge about the toxic substructures within some drugs. This research may also help patients who take mubritinib or carboxyamidotriazole, because their doctors will have to check for side effects on the heart more carefully.

Visualizing Ligand Binding to a GPCR In Vivo Using NanoBRET.

The therapeutic action of a drug depends on its ability to engage with its molecular target in vivo. However, current drug discovery strategies quantify drug levels within organs rather than determining the binding of drugs directly to their specific molecular targets in vivo. This is a particular problem for assessing the therapeutic potential of drugs that target malignant tumors where access and binding may be impaired by disrupted vasculature and local hypoxia. Here we have used triple-negative human breast cancer cells expressing ß2-adrenoceptors tagged with the bioluminescence protein NanoLuc to provide a bioluminescence resonance energy transfer approach to directly quantify ligand binding to a G protein-coupled receptor in vivo using a mouse model of breast cancer.

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.