publications cover art editorials books methods contact

Flex.jpg

FlexStation III: The FlexStation is a multi-well (6 - 384) microplate reader that measures fluorescence across the entire spectral range; it is capable of monitoring absorbance, fluorescent intensity, luminescence, time-resolved fluorescence and fluorescence polarisation. It allows the simultaneous transfer of liquids via a 8- or 16-channel pipettor whilst monitoring the accompanying fast kinetic response associated with the drug application. The ability to synchronise fluid applications and simultaneously monitor the response under different conditions makes the FlexStation the only apparatus that can make the meaningful concentration- response measurements that are needed in FBDD. The use of the FlexStation dramatically accelerates my ability to screen fragment libraries for drug candidates and assess the newly synthesised drugs that are developed from them.

For my work the FlexStation provides a simple, rapid, and cost-effective method for the early identification of novel drug leads and the elimination of poor candidates. It is robust, sensitive, flexible, scalable, and allows drugs to be tested on functional receptors in live cells, which makes the experiments physiologically relevant. I currently screen using ligand-gated ion channels, but voltage-gated channels and GPCRs can also be tested, making the FlexStation highly versatile and widely applicable.

P2X1.jpg

In silico Modelling and Docking: In silico methods can be used to predict the orientation of ligands in their binding sites. Using experimental methods such mutagenesis coupled with radioligand binding, flow cytometry or electrophysiology it is possible to test these predictions by determining which of several predicted binding site residues effect ligand- receptor interactions when they are substituted.

I use crystal structures to construct homology models into which I dock identified ligands. Typically, alignments of the receptor of interest and a template file are made using FUGUE, models constructed using Modeller and the stereo-chemical quality assessed using PROCHECK, RAMPAGE and ANOLEA. Into these models ligands are docked using GOLD suite. Potential ligand-receptor interactions are identified by creating a 5 sphere around docked ligands and the predictions evaluated by making point mutations to the receptor. Docking typically identifies 10-15 amino acids to be verified by experimentation, and alanine and amino acids with conserved chemical properties (e.g. Tyr -> Phe) are often used to subtly examine the predicted interactions. Coupled with SAR, docking also allows hypothesis testing of suitable locations upon the ligand to which fluorescent moieties can be attached without significantly altering ligand properties.

Electro.jpg

Electrophysiology: Electrophysiology yields high quality pharmacological data. I use electrophysiology to measure the function of voltage-gated and ligand-gated ion channels, and the properties of ligands. Importantly, electrophysiology also provides vital supporting evidence for the other experimental techniques I use.

Whole cell patch-clamp allows me to measure the function and pharamcology of single or multiple ion channels on the surface of stable or transiently transfected HEK 293 cells. As the same cells can also be used to probe binding with radioligand methods and flow cytometry, patch-clamp provides a valuable tool that ties together these binding experiments with the functional response of receptors in the same cells.

Two-electrode voltage-clamp is suitable for measuring ion currents passing through channels expressed on the surface of Xenopus oocytes following the injection of RNA. I use this system to express a wide diversity of receptors and have used the method extensively to characterise the function and pharamcology of ligand-gated ion channels. Because RNA translation is efficient in these cells, the method is particualrly well-suited to studies of poorly expressing receptors or mutagenesis studies.

Fluorescence.jpg Scopolamine.jpg

Flourescence: Fluorescence can be used to directly label receptors or the ligands that bind to them. Traditionally high-affinity, radiolabeled ligands were used to characterise ligand binding, but fluorescent ligands are providing new opportunities for quantifying binding interactions. These fluorescent approaches can be non-destructive and readily adapted to high-throughput methods that provide fast, economical and information rich outputs.

I use flow cytometry to provide a quantitative assessment of ligand binding. This provides both an alternative, and a complementary, approach to radioligand methods. It affords opportunities to monitor ligand actions at purified receptors, expressed receptors on live cells, or receptors in vivo. Typically I use novel fluorescent ligands (conjugated to fluorophores such as fluorescein, Alexa dyes, silica-rhodamine) that have been synthesiesd by my collaborator, and I assess their properties using flow cytometry and several of the other methods described on this page. By creating fluorescently-labelled ligands that have different emission spectra to fluorescently-labelled receptors I am able to simultaneous assess receptor expression and ligand binding using different laser-lines. For examining ligand receptor interactions, flow cytometry can be a truly high-throughput method that can be performed on live cells. Similar to methods such as radioligand binding, fluorescence intensity and fluorescence polarisation, it is possible to evaluate affinities of labeled and unlabeled compounds and their rate constants, as well as providing a practical solution for assessing ligand binding at mutant receptors.

Confocal microscopy enables ligand binding to be directly observed. In the most simple of experiments, fluorescent ligands provide a means of labelling receptors without the need for antibodies, and it is possible to probe competition binding between these and non-labelled ligands on live cells. In vivo, the use of high wavelength fluorophores (e.g. Alex-647) has several advantages, including deeper tissue penetration, improved signal-to-noise ratios and less cytotoxicity. With the use of time-lapse confocal microscopy, rates of association and dissociation can be measured.

Fluorescence intensity measures the light emitted from an excited fluorophore. Excitation and emission maxima are at different wavelengths (Stokes’ shift) and with appropriate filters excitation light can be eliminated to allow measurements of the emitted light alone. This can vary according to the physical surroundings of the fluorophore such as changes in pH, the polarity of the surrounding medium or environment the fluorophore is bound in, and intramolecular quenching mechanisms, such as photoinduced electron transfer (PET). In contrast to these simple intensity measurements, fluorescence polarsation measures the light emitted from a fluorescent ligand in horizontal and vertical planes after excitation with plane-polarized light in one of the planes. Because polarisation is a general property of fluorescent molecules and does not depend on the detected signal fluorescence emission intensity, polarisation-based readouts are less affected by fluorophore concentrations, the nature of the dye and environmental interferences, such as pH changes, than assays based on fluorescence intensity measurements. I use fluorescence polarisation to measure the binding of fluorescent ligands, inlcuding properties such as the affinity and kinetics of ligand-receptor interactions.



These experiments are undertaken with the kind support of my collaborators. Some of these can be found here:

Martin Lochner

James Brozik

Richard Farndale

Sussex Drug Discovery Centre

Gavin Jarvis

Tony Jackson

Marc-David Ruepp