Target engagement and drug residence time can be observed in living cells with BRET

被引：188

作者：

Robers, Matthew B. ^{[1
]}

Dart, Melanie L. ^{[1
]}

Woodroofe, Carolyn C. ^{[2
]}

Zimprich, Chad A. ^{[1
]}

Kirkland, Thomas A. ^{[2
]}

Machleidt, Thomas ^{[1
]}

Kupcho, Kevin R. ^{[1
]}

Levin, Sergiy ^{[2
]}

Hartnett, James R. ^{[1
]}

Zimmerman, Kristopher ^{[1
]}

Niles, Andrew L. ^{[1
]}

Ohana, Rachel Friedman ^{[1
]}

Daniels, Danette L. ^{[1
]}

Slater, Michael ^{[1
]}

Wood, Monika G. ^{[1
]}

Cong, Mei ^{[1
]}

Cheng, Yi-Qiang ^{[3
]}

Wood, Keith V. ^{[1
]}

机构：

[1] Promega Corp, Fitchburg, WI 53711 USA

[2] Promega Biosci Inc, San Luis Obispo, CA 93401 USA

[3] Univ N Texas, Hlth Sci Ctr, Dept Pharmaceut Sci, UNT Syst Coll Pharm, Ft Worth, TX USA

来源：

NATURE COMMUNICATIONS | 2015年 / 6卷

关键词：

Chemical sciences; Medicinal chemistry; Chemical biology; HISTONE DEACETYLASE INHIBITOR; ENERGY-TRANSFER METHODS; 2 CATALYTIC DOMAINS; IN-VITRO; HDAC INHIBITORS; LIGAND-BINDING; ASSAY; THAILANDEPSINS; DISPLACEMENT; DEPSIPEPTIDE;

D O I：

10.1038/ncomms10091

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

引用

页数：24

共 55 条

[31] HIS REPORTER DISPLACEMENT ASSAY FOR FRAGMENT SCREENING AND FRAGMENT EVOLUTION TOWARD LEADS WITH OPTIMIZED BINDING KINETICS, BINDING SELECTIVITY, AND THERMODYNAMIC SIGNATURE [J].