Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Abstract Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid I...

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Autores principales:	Adrien Boes, Samir Olatunji, Tamimount Mohammadi, Eefjan Breukink, Mohammed Terrak
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/01e5e5b8ff56424899107e86065805e5
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spelling	oai:doaj.org-article:01e5e5b8ff56424899107e86065805e52021-12-02T18:03:47ZFluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ10.1038/s41598-020-63380-22045-2322https://doaj.org/article/01e5e5b8ff56424899107e86065805e52020-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-63380-2https://doaj.org/toc/2045-2322Abstract Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.Adrien BoesSamir OlatunjiTamimount MohammadiEefjan BreukinkMohammed TerrakNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-8 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Adrien Boes Samir Olatunji Tamimount Mohammadi Eefjan Breukink Mohammed Terrak Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
description	Abstract Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.
format	article
author	Adrien Boes Samir Olatunji Tamimount Mohammadi Eefjan Breukink Mohammed Terrak
author_facet	Adrien Boes Samir Olatunji Tamimount Mohammadi Eefjan Breukink Mohammed Terrak
author_sort	Adrien Boes
title	Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
title_short	Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
title_full	Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
title_fullStr	Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
title_full_unstemmed	Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ
title_sort	fluorescence anisotropy assays for high throughput screening of compounds binding to lipid ii, pbp1b, ftsw and murj
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/01e5e5b8ff56424899107e86065805e5
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_version_	1718378669494239232

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

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