Biology subjects

Holliday, N. D.

Publications and source records attributed to Holliday, N. D..

4 recordsLinked to original sources

bioRxiv2023

A time-resolved Forster resonance energy transfer assay to investigate 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 Forster 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. HighlightsO_LIABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitors C_LIO_LIWe introduce a time-resolved Forster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2 C_LIO_LIWe confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor C_LI

biochemistry↗

bioRxiv2022

Development of fluorescent peptide G Protein Coupled Receptor activation biosensors for NanoBRET characterisation of intracellular allosteric modulators

G protein coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at this class of receptors offer further potential for exploitation. In particular GPCR intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g. G protein) and so inhibit agonist responses non-competitively. This potentially offers a tailored mode of action and greater selectivity between conserved receptor subtypes compared to classical orthosteric ligands. However, while specific examples of the IAM class of ligands are well described (particularly for chemokine receptors), a more general methodology for assessing compound interactions at the GPCR IAM site is lacking. Here fluorescent labelled peptides based on the G peptide C terminus are developed as novel binding and activation biosensors for the GPCR IAM binding site. In TR-FRET binding studies, unlabelled peptides derived from the GS subunit C-terminus were first characterised for their ability to positively modulate agonist affinity at the {beta}2-adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labelled tracer was synthesized based on the 19 amino acid C terminal GS peptide (TMR-GS19cha18, where cha=cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-GS19cha18 was recruited to Gs coupled {beta}2-adrenoceptor and EP2 receptors in an agonist dependent manner (correlated with ligand efficacy), but not to the Gi coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-GS19cha18 enabled direct assessment of the affinity of unlabelled ligands for {beta}2-adrenoceptor IAM site. Thus the NanoBRET platform using fluorescent-labelled G protein peptide mimetics offers novel potential for medium-throughput affinity screens to identify new IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.

pharmacology and toxicology↗

bioRxiv2020

Measurement of non-purified GPCR thermostability using the homogenous ThermoBRET assay

Measurements of membrane protein thermostability allows indirect detection of ligand binding. Current thermostability assays require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established target proteins. Alternative methods detect protein aggregation which requires sufficiently high level of protein expression. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the {beta}2-adrenoceptor ({beta}2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non-purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 C vs 59 C). ThermoBRET allows determination of GPCR thermostability, which is useful for protein purification optimisation and as part of drug discovery screening strategies.

biochemistry↗

bioRxiv2019

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and impacts on diseases such as cancer, Alzheimers and inflammatory diseases. ADAM10 is a molecular scissor that proteolytically cleaves the extracellular region from over 100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. ADAM10 was recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function is critically dependent on a TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex.

cell biology↗