A target-specific cellular assay for screening of topoisomerase I inhibitors

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 (Delta topI) were labeled with S65 T and wild-type green fluorescent protein (GFP), respectively, We showed th at the growth of such a pair of S., cerevisiae strains labeled with this GF P combination can be independently quantified after both strains were mixed , When growth of the mixture of wild-type and Delta topI strain was monitor ed in the presence of compounds, only growth of the wild-type strain was in hibited by the topoisomerase I-specific drug camptothecin, In contrast, amp hotericin B, a broad-spectrum antifungal drug, inhibited growth of both str ains. 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 w ild-type but not the Delta topI strain. Thus, by using a Delta topI strain as internal control in the same assay mixture, the number of candidate topo isomerase I inhibitors to be retested could be reduced by more than 90%, Fu rther applications of this type of S, cerevisiae-based cellular high-throug hput assays will be discussed.