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Development and Implementation of a Highly Miniaturized Confocal 2D-FIDA–Based High-Throughput Screening Assay to Search for Active Site Modulators of the Human Heat Shock Protein 90ß

Virginie Riou

Novartis Institute for Biomolecular Research, Discovery Technologies, Basel, Switzerland

Joseph Schoepfer

Novartis Institute for Biomolecular Research, Oncology Research, Basel, Switzerland

Johannes Ottl

Novartis Institute for Biomolecular Research, Discovery Technologies, Basel, Switzerland

Kurt Müller

Novartis Institute for Biomolecular Research, Discovery Technologies, Vienna, Austria

Patrick Chene

Novartis Institute for Biomolecular Research, Oncology Research, Basel, Switzerland

Lorenz M. Mayr

Ireos Filipuzzi

Novartis Institute for Biomolecular Research, Discovery Technologies, Basel, Switzerland

The beta isoform of the heat shock protein 90 (Hsp90ß) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90ß are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90ß. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90ß inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90ß. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II^TM from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Key Words: Hsp90ß • radicicol • 17-AAG • 2D-FIDA • confocal nanoscreening • miniaturized uHTS

Journal of Biomolecular Screening, Vol. 9, No. 7, 569-577 (2004)
DOI: 10.1177/1087057104265538


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