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.