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

• Main Authors: Michael H. Ronzetti (Author), Bolormaa Baljinnyam (Author), Zina Itkin (Author), Sankalp Jain (Author), Ganesha Rai (Author), Alexey V. Zakharov (Author), Utpal Pal (Author), Anton Simeonov (Author)
• Format: Book
• Published: Frontiers Media S.A., 2022-11-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 1663-9812; 10.3389/fphar.2022.1040039
• Keywords: high-throughput sceening; differential scanning fluorimetry; biophysical screening; small molecule screening; biochemistry; Therapeutics. Pharmacology; RM1-950
• Source: Frontiers in Pharmacology, Vol 13 (2022)

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.