Theory and applications of differential scanning fluorimetry in early-stage drug discovery

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.

Three Essential Resources to Improve Differential Scanning Fluorimetry (DSF) Experiments

“…Because DSF relies on the use of dye fluorescence as a proxy for protein unfolding, identifying and minimizing sources of protein-independent fluorescence is a critical step. Indeed, the fluorescence of SYPRO Orange is known to be sensitive to excipients that are common in biological buffers, such as glycerol, detergents, lipids and EDTA 34,43 . Here, we focus on two especially pernicious and common sources of protein-independent fluorescence: dye binding to colloidal aggregates and dye binding to the plastic used in manufacture of some microtiter plates.…”

“…The Tma is then calculated as the midpoint of the resulting fluorescence versus temperature curve, and higher Tmas suggest a more stable native protein. Because Tmas are broadly sensitive to changes in the biochemical state of the native protein, such as the binding of ligands [1][2][3][4][5][6][7] , mutations 8,9 , and proteinprotein interactions 10,11 , DSF is widely used to monitor diverse biochemical events via the associated change in Tma (∆Tma) 12,13 .…”

“…For example, DSF may rapidly identify stabilizing buffer condition,, enabling structural characterizations [17][18][19] or formulations of biotherapeutics 20 . High-throughput DSF screens are used to identify candidate binders to proteins of interest independent of enzymatic activity 7,12,21 . DSF has also been used to search for small-molecule correctors for genetically destabilized proteins [22][23][24][25] which underlie many misfolding diseases 13,26,27 such as cystic fibrosis 28,29 , Gaucher's disease 30 and Fabry's disease 31 .…”

“…For example, our lab conducted a primary chemical screen by DSF to identify ligands for cataract-associated forms of alpha B-crystallin 32 . These applications and others have been excellently reviewed 12,33 .…”

A new decade for Biophysical Reviews and a look into the future of biophysics

“…Jumping up one level in the scale of observation, the next review article by Gao et al also deals with the subject of protein unfolding, but in this case, their focus is on the technique of differential scanning fluorimetry (DSF), one of the most commonly used procedures for recording a temperature-induced unfolding transition (Gao et al 2020). By reviewing examples of DSF-based studies utilizing both the extrinsic and intrinsic fluorescence modes, the authors outline experimental considerations and contrast the advantages of DSF against other experimental procedures for monitoring protein stability.…”