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A Target-Specific Cellular Assay for Screening of Topoisomerase I Inhibitors

Bayer AG, Institut fur Antiinfektiva Forschung, Wuppertal, Germany

Emanuel Lohrmann

Bayer AG, Molekulare Screening Technologie, Wuppertal, Germany

Karl Ziegelbauer

Bayer AG, Institut fur Antiinfektiva Forschung, Wuppertal, Germany, Bayer Yakuhin Ltd., Research Center Kyoto, Kizu-cho, Sorakugun, Kyoto, Japan

We have developed a cellular, target-specific high-throughput assay for the detection of topoisomerase I inhibitors. Topoisomerase I is a nonessential enzyme involved in controlling DNA topology. Topoisomerase I is the target of anticancer drugs such as camptothecin as well as a candidate target for new antifungal drugs. A wild-type Saccharomyces cerevisiae strain and its isogenic topoisomerase I deletion mutant (topI) were labeled with S65T and wild-type green fluorescent protein (GFP), respectively. We showed that the growth of such a pair of S. cerevisiae strains labeled with this GFP combination can be independently quantified after both strains were mixed. When growth of the mixture of wild-type and topI strain was monitored in the presence of compounds, only growth of the wild-type strain was inhibited by the topoisomerase I-specific drug camptothecin. In contrast, amphotericin B, a broad-spectrum antifungal drug, inhibited growth of both strains. The two strains were used to screen compound libraries. While 0.9% of all compounds inhibited growth of both strains, only 0.06% inhibited the wild-type but not the topI strain. Thus, by using a topI strain as internal control in the same assay mixture, the number of candidate topoisomerase I inhibitors to be retested could be reduced by more than 90%. Further applications of this type of S. cerevisiae-based cellular high-throughput assays will be discussed.

Journal of Biomolecular Screening, Vol. 4, No. 2, 93-100 (1999)
DOI: 10.1177/108705719900400209


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