Details
| Original language | English |
|---|---|
| Pages (from-to) | 6345-6352 |
| Number of pages | 8 |
| Journal | Bioorganic and Medicinal Chemistry |
| Volume | 25 |
| Issue number | 24 |
| Publication status | Published - 7 Oct 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.
Keywords
- Fluorescence labelled ATP, Functional assay, HSP70/DnaK, Inhibitor screening, Natural products, Protein microarray
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Medicine
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Pharmacology, Toxicology and Pharmaceutics(all)
- Pharmaceutical Science
- Pharmacology, Toxicology and Pharmaceutics(all)
- Drug Discovery
- Biochemistry, Genetics and Molecular Biology(all)
- Clinical Biochemistry
- Chemistry(all)
- Organic Chemistry
Sustainable Development Goals
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In: Bioorganic and Medicinal Chemistry, Vol. 25, No. 24, 07.10.2017, p. 6345-6352.
Research output: Contribution to journal › Article › Research › peer review
}
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 -