TRICSS - a multi-user high-throughput platform to quantify biological interactions in solution

Communication between and within cells occurs by the production of ligands (proteins, peptides, fatty acids, steroids and other low-molecular-weight compounds) that binds in a specific and selective way to a receiving molecule, otherwise known as a receptor. When a ligand binds to its respective receptor, it initiates several different types of responses which are vital for cellular proliferation, migration, survival, and differentiation in every living organism. This principle has been exploited in the medical and industrial sectors, where a drug or a chemical substance of interest is designed to bind a target, causing a desired biological response (e.g., antiandrogens are a class of drugs used in the treatment of prostate cancer that act by targeting and blocking the androgen receptor and/or suppressing hormone production). The interaction of ligands with their receptors can be characterized in terms of a binding affinity, which is the measure of the strength of the interaction. Understanding binding affinity is key to define, model, and ultimately precisely manipulate biological processes. However, detecting binding events and precisely measuring strength of interactions can be technically challenging and time consuming. Together with our academic and industrial collaborators, we want to purchase and use a novel plate reader, currently not available in the UK, called the Dianthus. The Dianthus is the first ever multi-user, high-throughput screening platform to use two biophysical modalities to detect and quantify with high sensitivity true binding events among a diverse range of ligands and receptors. The reliability and reproducibility of data generated by the Dianthus makes this technology highly attractive to users from academia and industry alike. In fact, knowledge of binding affinities is central to every drug discovery process to help design drugs that bind their targets selectively and specifically. Thus, the Dianthus will be vital in supporting and pushing the boundaries of many UK bioscience programmes, ultimately giving us new insights into biological systems in ways we may not have thought possible.

The Dianthus is the first ever plate-based and microfluidics-free screening platform to use two biophysical modalities to measure the strength of molecular interactions: (1) Temperature Related Intensity Change (TRIC) of fluorescence, which is a measure for the decrease or increase of the fluorescence of a molecule in solution upon heating as a function of time, allows the identification and fine-mapping of low- and high-affinity interactions; (2) Spectral Shift (SS), which is the measure of the change in fluorescence emission at 650 and 670 nm as a consequence of the change in chemical environment of the fluorophore caused by ligand binding, enables affinity measurements and thermodynamic characterisation without the explicit need for site-specific labeling or ligand-induced conformational change. The nature of the methods requires the target molecules to be chemically modified with red fluorophores, while ligands need to be non-fluorescent in the investigated spectral range. The inherent flexibility and high sensitivity derived from using these two biophysical modalities will allow us to study ligand binding in a wide range of systems, fostering multidisciplinary national and industrial collaborations. We have the expertise (technical and facility management) and research environment (i) to conduct the original research projects detailed in this application, (ii) to address all technological limitations/bottlenecks of other biophysical measurement techniques (ITC, BLI, SPR), and (iii) to revolutionise our approach to ligand screening and drug discovery.