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Research Interest

 

MoleculesThe focus of Andrew Thompson’s group is the identification and development of novel ligands acting at ion channels. Using a range of methods including electrophysiology, flow cytometry, confocal microscopy, radioligand binding and in silico modelling & docking, his group investigates ligand binding modes and reconciles them with ligand SAR. These are used to develop new ligands with functional groups such as fluorophores or photo-activatable moieties that allow studies of receptor function, pharmacology and physiology. A brief summary of his main activities are found below and can also be found here.

Single Molecule Imaging (SMI) of Ligand Binding: A main focus of my research is the use of novel fluorescently labelled ligands to directly observe their binding to single receptors using time-lapse TIRF imaging. Understanding how drugs bind to their receptors is the most fundamental purpose of pharmacological research that enables the effective development of novel therapeutics. Currently, these measurements are made using methods that yield ensemble averages (e.g. radioligand binding, SPR, X-ray crystallography). Techniques such as single-channel patch clamp go some way towards monitoring the properties of single molecules, but are composite measurements of both ligand binding and a functional response, and can only be used for receptors that are functionally coupled to ion channels. A more widely applicable and direct method of monitoring drug binding is the use of SMI. In this approach, purified receptors are reconstituted into artificial membranes and the binding of fluorescent ligands is monitored by time-lapse microscopy. The results are direct observations of single receptors bound with one or more of their ligands, and the resultant movies of these events can be analysed to determine key components of the interactions, such as the number, position, and rate constants of individual molecules.

Electrophysiology:   The observations from SMI are combined with electrophysiology to answer questions about how the binding of ligands activates or inhibits a target receptor. My lab routinely uses two-electrode voltage clamp to validate newly identified compounds and probe the effects of receptor mutations on the binding affinities of these ligands. Results from this mutagenesis can confirm the orientations of ligands in the binding site and be used to identify regions of the ligands that are amenable to the attachment of large functional groups such as fluorophores.

Therapeutics: One means of identifying new ligands is fragment-based drug-discovery (FBDD), an efficient and rational approach that is becoming widely adopted by industry and academia. Screening carefully designed small compounds (fragments) in FBDD is considered effective because, 1) a diverse chemical space can be efficiently described by a smaller number of compounds with lower MW and complexity, 2) lower complexity raises the chance of identifying protein-ligand interactions, 3) small fragment libraries (~1500 compounds) typically result in a higher hit rate than large drug libraries, 4) hit fragments are easier to subsequently optimise by further synthesis, despite their low affinity, and 5) FBDD is design-intensive rather than resource-intensive, making it cost-effective for the end user. Using FBDD I have identified many new and novel ligands acting at 5-HT3A, P2X1 and P2X4 receptors. Modifications of these allow me to optimise potency and receptor-selectivity, and develop new molecular probes. The attachment of fluorophores allows the ligands to be monitored using the SMI methods described above, as well as being useful for techniques such as flow cytometry, fluorescent polarisation, confocal microscopy and live imaging.