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In drug discovery, biophysical approaches have become

attractive screening techniques. They are not only the

main primary hit finding methodologies, such as weakly

active fragments, but also as orthogonal approaches for

the validation of hit compounds found by conventional

biochemical or cellular assays. Among them, thermal shift

assay (TSA) is a reliable and simple technique to identify

active fragments and analyze the binding events of

ligands to the target by monitoring changes in protein

thermal stability.

Brief Introduction to Thermal Shift Assay (TSA)

The stability of a protein is related to its melting temperature, which is affected by

conditions such as buffer composition, pH values, amino acid mutations, or

ligand/fragment binding. Fluorescence-based thermal shift assay (usually referred

to as differential scanning fluorimetry or the thermofluor assay) is one of the

common TSA techniques and is a high-throughput and economical biophysical

approach for screening and validation of hits. In general, the fluorescence of the

protein solution is measured under a temperature gradient. As the temperature

rises, the hydrophobic core of the protein is exposed, and the added fluorescent

dye has affinity for the hydrophobic part of the protein. Therefore, this leads to an

increase in the fluorescence signal and can be used to determine the melting

temperature of the protein. The interaction between ligands and proteins can

significantly increase or decrease the melting temperature. Thus, a change of this

parameter offers crucial information about protein stabilization or destabilization.

In addition to TSA approaches, Creative Biostructure can also provide other

biophysical techniques for the identification, validation, affinity, selectivity, and

binding mechanisms of hits involved in early drug discovery. We support

combinatorial approaches, such as combining TSA assays with

surface plasmon resonance (SPR) for independent verification of biophysical

parameters. Please feel free to contact us if you would like to learn more.

References

1.Vollrath F.; et al. Differential scanning fluorimetry provides high throughput data on silk

protein transitions. Scientific Reports. 2014, 4: 5625.

2.Scott A D. Fluorescent thermal shift assays for identifying small molecule ligands. Biophysical

Techniques in Drug Discovery. 2017, 61: 208.

3.Kirsch P.; et al. Concepts and core principles of fragment-based drug design. Molecules. 2019,

4.Henderson M J.; et al. High-throughput cellular thermal shift assays in research and drug

discovery. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2020, 25(2): 137-147.

THANKS

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