Biophysical Assays
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Biophysical Assays

Biophysical assays enable an understanding of drug-target interactions at the molecular level. Primary and secondary biophysical screening selection is performed to determine the residence time, mechanism of action, and kinetics of the drug, all of which are critical for the therapeutic effect of the drug. Therefore, biophysical screening is essential for drug discovery.

Introduction of Biophysical Assays

Biophysical assays are an integral part of any protein-centered drug discovery project, and it is also a key aspect to improve the success rate of drug discovery. Biophysical assays have been successfully used for assay development, primary screening, hit confirmation, and detailed mechanistic characterization of compound binding. The valuable combination of biophysics and HTS facilitates the establishment of high-quality, high-throughput assays through the characterization of proteins and tool ligands, as well as the evaluation of HTS output through orthogonal applications of biophysical methods to screen for target involvement.

There are some biophysical methods to determine binding kinetics, affinity and specificity, secondary structure and thermodynamic profiles, or structural insights into the molecular interaction of a small molecule or peptide with its target.

Thermal Shift Assay

Thermal shift assay (TSA), including Thermofluor and differential scanning fluorimetry, has been used to study protein stability, such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutations. It has been widely used to optimize buffer conditions for soluble proteins in X-ray crystallography.

Microscale Thermophoresis

Microscale thermophoresis (MST) is a technique for the biophysical analysis of biomolecular interactions. The MST detects the movement of fluorescent molecules under microscopic temperature gradients, which is generated by focusing an infrared laser beam on a segment of a microliter volume capillary. MST allows direct measurement of interactions in solution without the immobilization on the surface (immobilization-free technology).

Mass Spectrometry

There are two ionization modes in mass spectrometry (MS), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). MS analysis can provide information on all stages of biological therapy development, from the earliest stages of drug discovery to the latest stages of formulation development. There are three wide applications in the development of biological therapies:

  1. Identification
  2. Monitoring of molecular mass changes caused by chemical modification or degradation of molecules
  3. Monitoring of molecular structure changes that can be revealed by techniques such as H/D exchange or rapid photochemical oxidation of proteins (FPOP)

Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a quantitative biophysical technique, which describes the binding interactions between two or more molecules in solution. It is most commonly used to study the binding of small molecules (such as drug compounds) to larger macromolecules (proteins, DNA, etc.). It fully characterizes the thermodynamics of ligand-target binding, enabling the determination of Kd, reaction stoichiometry, enthalpy (∆H) and entropy (∆S).

Nuclear Magnetic Resonance (NMR)

Nuclear magnetic resonance (NMR) is one of the most versatile biophysical methods, which can be characterized by ligand-protein interactions through ligand observation or protein observation.

X-Ray Crystallography

X-Ray crystallography (XRC) is the experimental science of determining the atomic and molecular structure of crystals. X-ray crystallography can rapidly test compounds and produce crystal structures.

Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a highly sensitive technique for accurately measuring the interaction of two biomolecules. The method requires immobilization of the biomolecule to the surface. SPR can evaluate the affinity, kinetics and thermodynamics of ligand-target interactions with high sensitivity and label-free.

Dynamic Light Scattering

Dynamic light scattering (DLS) is a physical technique used to determine the particle size distribution of small particles in suspension or polymers in solution. DLS is based on the fact that aggregates have low diffusion coefficient and fluctuations of scattered light have different properties. The DLS instruments are commercially available as plate readers for 96-, 384- and even 1024-well plates.

Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) is a thermoanalytical technique, which is the major measurement method of thermal stability. In this technique, the heat difference required to increase the temperature of a sample and reference is measured as a function of temperature.


Biophysical assays have developed from being used to solve isolated problems with tool compounds or target proteins (experienced in some earlier projects) to becoming an essential and integral component in the workflow of establishing and conducting hit identification and characterization throughout the whole project.

Biophysics has been completely integrated into the discovery of small molecule drugs. The improvement of sensitivity and capabilities of existing technologie makes it possible to use biophysical assays at all stages of the drug discovery process, from target protein QC, to stress test pilot screening, to hit finding, to hit list validation, and even to the characterization of compound MoAs.


  1. Genick CC; et al. Biophysics: for HTS hit validation, chemical lead optimization, and beyond. Expert Opin Drug Discov. 2017 Sep; 12(9): 897-907.
  2. Razinkov VI; et al. Methods of high throughput biophysical characterization in biopharmaceutical development. Curr Drug Discov Technol. 2013 Mar; 10(1): 59-70.
  3. Holdgate G; et al. Biophysical methods in early drug discovery. ADMET DMPK. 2019 Dec 11; 7(4): 222-241.
  4. Genick CC; et al. Applications of Biophysics in High-Throughput Screening Hit Validation. J Biomol Screen. 2014 Jun; 19(5): 707-14.