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Journal of Biomolecular Screening
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Automated High Throughput Screening for Serine Kinase Inhibitors Using a LEADSeekerTM Scintillation Proximity Assay in the 1536-Well Format

Gabriele Sorg

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma KG, Biberach, Germany

Hans-Dieter Schubert

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma KG, Biberach, Germany

Frank H. Büttner

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma KG, Biberach, Germany

Ralf Heilker

Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma KG, Biberach, Germany

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [33P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [33P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z’ factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC50 values for both assay formats.

Journal of Biomolecular Screening, Vol. 7, No. 1, 11-19 (2002)
DOI: 10.1177/108705710200700103
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[Abstract] [PDF]