Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autoren

Externe Organisationen

  • Medizinische Hochschule Hannover (MHH)
  • Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
  • Deutsches Elektronen-Synchrotron (DESY)
Forschungs-netzwerk anzeigen

Details

OriginalspracheEnglisch
Seiten (von - bis)6345-6352
Seitenumfang8
FachzeitschriftBioorganic and Medicinal Chemistry
Jahrgang25
Ausgabenummer24
PublikationsstatusVeröffentlicht - 7 Okt. 2017

Abstract

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein–protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies.

ASJC Scopus Sachgebiete

Ziele für nachhaltige Entwicklung

Zitieren

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors. / Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine et al.
in: Bioorganic and Medicinal Chemistry, Jahrgang 25, Nr. 24, 07.10.2017, S. 6345-6352.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Mohammadi-Ostad-Kalayeh, S, Hrupins, V, Helmsen, S, Ahlbrecht, C, Stahl, F, Scheper, T, Preller, M, Surup, F, Stadler, M, Kirschning, A & Zeilinger, C 2017, 'Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors', Bioorganic and Medicinal Chemistry, Jg. 25, Nr. 24, S. 6345-6352. https://doi.org/10.1016/j.bmc.2017.10.003
Mohammadi-Ostad-Kalayeh, S., Hrupins, V., Helmsen, S., Ahlbrecht, C., Stahl, F., Scheper, T., Preller, M., Surup, F., Stadler, M., Kirschning, A., & Zeilinger, C. (2017). Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors. Bioorganic and Medicinal Chemistry, 25(24), 6345-6352. https://doi.org/10.1016/j.bmc.2017.10.003
Mohammadi-Ostad-Kalayeh S, Hrupins V, Helmsen S, Ahlbrecht C, Stahl F, Scheper T et al. Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors. Bioorganic and Medicinal Chemistry. 2017 Okt 7;25(24):6345-6352. doi: 10.1016/j.bmc.2017.10.003
Mohammadi-Ostad-Kalayeh, Sona ; Hrupins, Vjaceslavs ; Helmsen, Sabine et al. / Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors. in: Bioorganic and Medicinal Chemistry. 2017 ; Jahrgang 25, Nr. 24. S. 6345-6352.
Download
@article{fdc95f6db8a14484946965005f6a4fcf,
title = "Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors",
abstract = "A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein–protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies.",
keywords = "Fluorescence labelled ATP, Functional assay, HSP70/DnaK, Inhibitor screening, Natural products, Protein microarray",
author = "Sona Mohammadi-Ostad-Kalayeh and Vjaceslavs Hrupins and Sabine Helmsen and Christin Ahlbrecht and Frank Stahl and Thomas Scheper and Matthias Preller and Frank Surup and Marc Stadler and Andreas Kirschning and Carsten Zeilinger",
note = "Funding Information: This work was supported by the Nieders{\"a}chsische Krebsgesellschaft e.V. The authors would like to thank Liz Skellam for reading and comments on the manuscript. Publisher Copyright: {\textcopyright} 2017 Elsevier Ltd Copyright: Copyright 2020 Elsevier B.V., All rights reserved.",
year = "2017",
month = oct,
day = "7",
doi = "10.1016/j.bmc.2017.10.003",
language = "English",
volume = "25",
pages = "6345--6352",
journal = "Bioorganic and Medicinal Chemistry",
issn = "0968-0896",
publisher = "Elsevier Ltd.",
number = "24",

}

Download

TY - JOUR

T1 - Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors

AU - Mohammadi-Ostad-Kalayeh, Sona

AU - Hrupins, Vjaceslavs

AU - Helmsen, Sabine

AU - Ahlbrecht, Christin

AU - Stahl, Frank

AU - Scheper, Thomas

AU - Preller, Matthias

AU - Surup, Frank

AU - Stadler, Marc

AU - Kirschning, Andreas

AU - Zeilinger, Carsten

N1 - Funding Information: This work was supported by the Niedersächsische Krebsgesellschaft e.V. The authors would like to thank Liz Skellam for reading and comments on the manuscript. Publisher Copyright: © 2017 Elsevier Ltd Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2017/10/7

Y1 - 2017/10/7

N2 - A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein–protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies.

AB - A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein–protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies.

KW - Fluorescence labelled ATP

KW - Functional assay

KW - HSP70/DnaK

KW - Inhibitor screening

KW - Natural products

KW - Protein microarray

UR - http://www.scopus.com/inward/record.url?scp=85031322335&partnerID=8YFLogxK

U2 - 10.1016/j.bmc.2017.10.003

DO - 10.1016/j.bmc.2017.10.003

M3 - Article

C2 - 29042222

AN - SCOPUS:85031322335

VL - 25

SP - 6345

EP - 6352

JO - Bioorganic and Medicinal Chemistry

JF - Bioorganic and Medicinal Chemistry

SN - 0968-0896

IS - 24

ER -

Von denselben Autoren