Microarray-based screening of heat shock protein inhibitors

•A protein microarray for screening of heat shock protein inhibitors was developed utilizing human HSP90 as a model protein.•Utilizing this microarray, novel geldanamycin/reblastatin derivatives were successfully screened against HSP90.•Thereby, novel inhibitors were identified with IC 50 values as low as 0.5 nM.•The capacity for multiplexing was demonstrated by simultaneous screening against human HSP90 alpha and HtpG of Helicobacter pylori.•The microarray is highly miniaturized and allows for cost-effective screening with low amounts of inhibitors and protein.

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z*-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family.