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Label Free Drug Screening

The basic premise for using thermofluor for drug screening assumes that high energy binding sites on proteins for small molecules (e.g. drugs) are functionally significant.
These include:

-Enzyme active sites

-Cofactor binding sites

-Allosteric effector binding sites

-Protein:Protein interaction sites that bind adaptor peptide domains (e.g. SH2, SH3, etc.)

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Thermoflour detection of specific ligand binding

The free energy of ligand binding to the native state of a protein (assuming a substantial enthalpy contribution) produces an increase (positive Delta Tm) in the protein's thermal stability that can be measured using thermofluor. Thermoflour technology has consequently been used in numerous drug-screening campaigns to find compounds that range from enzyme inhibitors to agents that interfere with protein-protein interactions.

For ligand binding experiments, it is necessary to saturate the target protein binding site, so that solution ligand concentrations typically have to be 50-100um to find binding “hits” in the 5-10uM range. As a result of this solubility requirement, thermofluor HTS campaigns of “drug-like” screening libraries are typically carried out with relatively pure single compounds to insure adequate solubility under most solution assay conditions.

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HT Screening Data

Most thermofluor screening campaigns produce “destabilizing” hits (e.g Delta Tm is negative) in addition to stabilizing hits (Delta Tm positive), presumably reflecting preferential ligand binding to partially unfolded protein states. Many such compounds may appear as active hits in corresponding enzymatic assays of target activity, but rarely develop extensive SAR relationships, and are often shown to act promiscuously against multiple protein targets.

High-density miniaturized thermal shift assays as a general strategy for drug discovery. Pantoliano MW, et.al. J Biomol Screen. 2001 Dec;6(6):429-40. [Original thermofluor reference] PMID: 11788061
Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Lo MC, et.al. Anal Biochem (2004) 332:153-159 [Binding affinities obtained from thermal shift assays are compared to isothermal titration calorimetry and IC(50) values from enzymatic assays.] PMID: 15301960
Universal screening methods and applications of ThermoFluor. Cummings MD, et.al. J Biomol Screen. 2006 Oct;11(7):854-63. Epub 2006 Aug 30. [Review] PMID: 16943390
Application of high-throughput isothermal denaturation to assess protein stability and screen for ligands. Senisterra GA, et.al. J Biomol Screen (2008) 13:337-342 [High-throughput isothermal denaturation (ITD) analysis of citrate synthase and malate dehydrogenase stability.] PMID: 18448703
ThermoFAD, a Thermofluor-adapted flavin ad hoc detection system for protein folding and ligand binding. Forneris F, et.al. FEBS J. 2009 May;276(10):2833-40. PMID: 19459938
A High-Throughput Screening Assay for Simultaneous Selection of Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose-5-Phosphate Synthase (Dxs) or 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr). Humnabadkar V, et.al. J Biomol Screen. 2011 Mar;16(3):303-12 [Drug Screening] PMID: 21335601
Protein thermal shifts to identify low molecular weight fragments. Kranz JK, Schalk-Hihi C. Methods Enzymol. 2011;493:277-98. [Thermofluor screening overview] PMID: 21371595
Identification of Inhibitors that Block Vaccinia Virus Infection by Targeting the DNA Synthesis Processivity Factor D4. Nuth M, et.al. J Med Chem. 2011 Mar 25. [Drug Screening] PMID: 21438571
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