Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries

Abstract

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αIIbβ3 as Novel Antiplatelet Agents

“…Determining a direct measurement of a molecule’s binding interaction with its target is a vital parameter for characterizing a chemical probe. We employed microscale thermophoresis (MST) for this purpose, leveraging its sensitivity and capacity to deliver quantitative data on the binding interaction . For the MST experiments, we chose to pursue NCGC00351170 and an inactive analog NCGC00351154.…”

“…We employed microscale thermophoresis (MST) for this purpose, leveraging its sensitivity and capacity to deliver quantitative data on the binding interaction. 35 For the MST experiments, we chose to pursue NCGC00351170 and an inactive analog NCGC00351154. We began by testing increasing amounts of the compound(s) and incubating them with the fluorescently labeled CIB1-(His) 6 , followed by the detection of the MST traces on the NT automated instrument (for more details see Materials and Methods Section).…”

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions

“…These factors need to be considered to obtain reliable data using DSF 13,14 . To overcome these limitations, labelling of target proteins with fluorophore tags was recently introduced to DSF for better understanding of proteins unfolding in complex physiological systems [15][16][17] . While being a valuable technique, it cannot be universally applied since it needs to be customized for each specific protein being studied.…”