Biophysical Assay Techniques in Drug Discovery

Biophysical assays can be tailored to integrated drug discovery projects. Biophysical techniques can be used for standalone projects to definitively measure and characterize the binding of one molecule to another.

The biophysical methods available can be applied to your projects with a wide variety of molecular characteristics – from protein-protein interactions to peptides and large lead-like or natural product compounds, all the way to fragment screening.

The flexibility of biophysical screening techniques means they can be applied to projects of any nature to answer key questions such as:

  • Do your molecules bind to the target?
  • Where do they bind?
  • How do they bind?

Biophysical assays can drive the start of a drug discovery project: initial hit finding, fragment-based drug discovery, and high-throughput screening. They may also be involved at later stages of a project: lead optimization, affinity determination, kinetic analysis, and thermodynamic analysis.

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Biophysical Assays Available at Charles River

Surface Plasmon Resonance (SPR) Biophysical Assay

Surface Plasmon Resonance (SPR) is a powerful biophysical technique used to study the binding behavior of immobilized macromolecules and analytes in solution. The technique makes it possible to measure interactions in real time with high sensitivity. SPR has become the gold standard for studying the kinetics of biomolecular interactions in biomedical research and drug discovery. The interactions that can be studied are diverse and include protein–protein, protein–small molecule, protein–nucleic acid, protein–carbohydrate as well as nucleic acid-small molecule.

Nuclear Magnetic Resonance (NMR)

Nuclear Magnetic Resonance (NMR) spectroscopy is a biophysical technique used to study the properties, structure, and dynamics of molecules at the atomic level. NMR is commonly used to investigate interactions of biological molecules, especially proteins, nucleic acids (DNA and RNA), and carbohydrates in solution and solid states. Unlike other structural biology techniques, such as X-ray crystallography, NMR biophysical screening does not require the sample to be crystallized, allowing the study of molecules in environments that closely mimic their natural, physiological conditions.

Thermal Shift Assay (TSA)

Thermal shift assay (TSA) is well-regarded to quickly identify hits against a target or protein buffer composition optimization. A typical application is to measure the ability of a compound to stabilize a protein – the assumption that compound binding increases the melting temperature of the protein. The same concept can be applied to protein-protein interactions. TSA is often used to validate orthogonal biophysical techniques, such as SPR biophysical assays. Using TSA can benefit your project as it is inexpensive to run, extremely high throughput, and has potentially low protein consumption.

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Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC), a highly sensitive solution-based method, is often described as the "gold standard" for measuring the interaction between two molecules. As well as measuring affinity and stoichiometry, ITC is unique amongst other common biophysical techniques for also measuring the thermodynamics of an interaction – the enthalpy (ΔH), entropy (ΔS), and Gibbs free energy change (ΔG). ITC is best used to complement and validate orthogonal biophysical techniques, such as SPR.

Charles River has an experienced Biophysical Assays team who will support you in your decision of which biophysical techniques is best placed to progress your project.

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Frequently Asked Questions (FAQs) About Biophysical Assays

  • Do I need to provide protein for biophysical screening studies?

    No. While we are happy to work with reagents provided by clients, there is the option to buy commercially available protein, or we can offer a protein production service.

  • What ITC instrument is available at Charles River?

    We have a VP-ITC machine (Malvern Instruments). VP-ITC offers the ability to investigate any biomolecular interaction. Its fast, convenient label-free in-solution analysis delivers the information needed to characterize the molecular interactions of proteins, antibodies, nucleic acids, and other biomolecules. The system is widely used in research environments for lead optimization, hit validation, assessing the driving mechanisms of binding, and studying enzyme kinetics and inhibitor design.

  • What TSA instrument is available at Charles River?

    We use the Bio-Rad CFX Opus 386 real time PCR machine, employing SYPRO orange dye to bind to hydrophobic regions of denatured proteins following temperature ramping.

  • How much protein is required for biophysical assays?

    For TSA studies very little protein is required, typically only 1-2 μg per sample within the 384-well plate. For ITC the VP-ITC equipment requires ~2 mls of a 10-30 μM protein solution per run, dependent on the expected affinity of the protein-ligand interaction. 

    For SPR, as little as 100 μg can be sufficient, but this is dependent on the method of immobilization and the tags present. More protein is usually required if it is to be used as analyte (e.g., for protein-protein interactions).