Abstract
Differential scanning fluorimetry (DSF) is an accessible, rapid, and economical biophysical technique that has seen many applications over the years, ranging from protein folding state detection to the identification of ligands that bind to the target protein. In this review, we discuss the theory, applications, and limitations of DSF, including the latest applications of DSF by ourselves and other researchers. We show that DSF is a powerful high-throughput tool in early drug discovery efforts. We place DSF in the context of other biophysical methods frequently used in drug discovery and highlight their benefits and downsides. We illustrate the uses of DSF in protein buffer optimization for stability, refolding, and crystallization purposes and provide several examples of each. We also show the use of DSF in a more downstream application, where it is used as an in vivo validation tool of ligand-target interaction in cell assays. Although DSF is a potent tool in buffer optimization and large chemical library screens when it comes to ligand-binding validation and optimization, orthogonal techniques are recommended as DSF is prone to false positives and negatives.
Highlights
Biophysics drives modern drug discovery efforts, allowing rapid and high-throughput data acquisition to screen through large compound libraries in an effort to identify new bioactive molecules
The protein unfolding thermodynamic parameter ΔTm is monitored as the primary indicator to justify stability changes of the target protein, no matter whether targets were in a purified form, in lysate, cells, or even tissues
Emerged label-free nanoDSF approaches especially obviate the need for dyes, allowing the same approach to be applied to membrane protein research, simultaneously addressing problems caused by the interaction between dye and the hydrophobic surface of proteins, or the detergent additives applied and interactions between the dye and other molecules in a screen
Summary
Biophysics drives modern drug discovery efforts, allowing rapid and high-throughput data acquisition to screen through large compound libraries in an effort to identify new bioactive molecules. Measurements of the Tm of a protein in the presence and absence of environment changes or ligands result in an estimate of the thermal shift (ΔTm) deriving from these differences (Scott et al 2016) (Fig. 1b) This shift is typically an indicator of complex formation and/or thermal stabilization. Aggregation and/or sample instability may lead to the presence of multiple species of target protein within the experiment, both leading to increased fluorescence background from any conformational variability and resulting in variable thermal stability profiles of the different order oligomers (Fig. 1c). In 2014, a label-free DSF technique marketed as nanoDSF was developed (Alexander et al 2014) This approach removes the requirement for an extrinsic dye or fusion tag, instead of relying on the change of intrinsic tryptophan fluorescence at 330 nm and 350 nm (Fig. 1e). Biolayer interferometry (Wartchow et al 2011) provides similar binding information to that obtained by SPR, with advantages in signal stability arising from the use of interferometry patterns
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