A high-throughput fluorescence chemical denaturation assay as a general screen for protein-ligand binding

被引：25

作者：

Mahendrarajah, Kumaran ^{[4
]}

Dalby, Paul A. ^{[3
]}

Wilkinson, Barrie ^{[2
]}

Jackson, Sophie E. ^{[4
]}

Main, Ewan R. G. ^{[1
]}

机构：

[1] Univ London, Sch Biol & Chem Sci, London E1 4NS, England

[2] Biotica, Cambridge CB10 1XL, England

[3] UCL, Dept Biochem Engn, London WC1E 7JE, England

[4] Univ Cambridge, Dept Chem, Cambridge CB2 1EW, England

来源：

ANALYTICAL BIOCHEMISTRY | 2011年 / 411卷 / 01期

基金：

英国生物技术与生命科学研究理事会;

关键词：

DRUG DISCOVERY; STABILITY; FKBP12; RAPAMYCIN; FK506;

D O I：

10.1016/j.ab.2010.12.001

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.

引用

页码：155 / 157

页数：3

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