Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.
The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.
Lo
Mei-Chu
MC
Biophysics/Enzymology-Chemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA. lom@wyeth.com
Aulabaugh
Ann
A
Jin
Guixian
G
Cowling
Rebecca
R
Bard
Jonathan
J
Malamas
Michael
M
Ellestad
George
G
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D010450:Endopeptidases; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D011489:Protein Denaturation; D011506:Proteins / Q000037:antagonists & inhibitors*; D013696:Temperature; D013997:Time Factors
D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases