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

authored by

Sona Mohammadi-Ostad-Kalayeh, Vjaceslavs Hrupins, Sabine Helmsen, Christin Ahlbrecht, Frank Stahl, Thomas Scheper, Matthias Preller, Frank Surup, Marc Stadler, Andreas Kirschning, Carsten Zeilinger

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.

Organisation(s)

Institute of Cell Biology and Biophysics
Centre of Biomolecular Drug Research (BMWZ)
Institute of Technical Chemistry
Institute of Organic Chemistry

External Organisation(s)

Hannover Medical School (MHH)
Helmholtz Centre for Infection Research (HZI)
Deutsches Elektronen-Synchrotron (DESY)

Type

Article

Journal

Bioorganic and Medicinal Chemistry

Volume

25

Pages

6345-6352

No. of pages

8

ISSN

0968-0896

Publication date

07.10.2017

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Biochemistry, Molecular Medicine, Molecular Biology, Pharmaceutical Science, Drug Discovery, Clinical Biochemistry, Organic Chemistry

Sustainable Development Goals

SDG 3 - Good Health and Well-being

Electronic version(s)

https://doi.org/10.1016/j.bmc.2017.10.003 (Access: Closed)