Table 2.

Recent applications of DSF.

Test system	Study design	Study purpose, outcome	References
Protein Therapeutics	Tm determination for sets of protein variants or same protein across different buffer or formulation conditions	Selection of best variant for further development, selection of storage conditions, improvement of formulations	[19,30,43–46]
HTS	DSF-driven screens of small molecule libraries followed by orthogonal validation assays, e.g. DoT1L, monoglyceride lipase, HIV-1 NC, MLL/WDR5, FGF22	Discovery of novel ligands for select protein targets, accompanied recently by public deposition of large datasets	[9,17,48,51,53]
Profiling	A set of small molecule ligands tested against a series of typically related proteins, e.g. kinases, PARPs	Provides selectivity/promiscuity profiles of bioactive small molecules to understand off-target effects and improve development	[8,52]
Discovery of Chemical Chaperones	Use of DSF to initially discover and/or to further characterize small molecule chaperones of mutant proteins in rare genetic diseases, e.g. correctors of ΔF508/CFTR, chaperones of glucocerebrosidase	Discovery of novel ligands for select protein targets, particularly relevant in cases where noninhibitory protein stabilizers are desired	[54,56,58]
Stabilization by Cofactors or Other Proteins	Profiling proteins for stabilization by protein binding partners and by combinations of cofactors and/or reaction substrates and products, e.g. PPI between E. coli MBP and Off730, Vibrio SDRvv cofactor, Ca2+ stabilization of vWF	Provides insights into reaction mechanism, mechanisms of folding and stabilization of multidomain proteins, aids in the discovery of native cofactors of new proteins, ascertains formation of protein- protein complexes	[36,60–62]
Mechanism of Action of Inhibitors	Cofactor and substrate co- dependency studies of protein stabilization to identify competitive- vs-noncompetitive modes, and to identify unusual modes of stabilization such as adduct formation, e.g. MOA of 15-PGDH inhibitors, PTC124-AMP adduct, GST MOA studies	Provides initial insights into MOA, useful when the assay for the underlying enzymatic reaction is difficult to scale up for full enzyme kinetics study	[63,65,68]