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A Target-Specific Cellular Assay for Screening of Topoisomerase I Inhibitors
Spaltmann1, Lohrmann, Ziegelbauer
1Bayer AG, Institut für Antiinfektiva Forschung, Wuppertal, Germany.
Abstract:
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 (DeltatopI) 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 DeltatopI 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 DeltatopI strain. Thus, by using a DeltatopI 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.
Insights
A new high-throughput assay using Saccharomyces cerevisiae detects topoisomerase I inhibitors. This method significantly reduces the number of compounds needing retesting by over 90%.
Area of Science:
- Biochemistry and Molecular Biology
- Drug Discovery and Development
- Yeast Genetics and Cell Biology
Background:
- Topoisomerase I is a key enzyme in DNA topology regulation, targeted by anticancer drugs and a potential target for antifungal agents.
- Existing methods for identifying topoisomerase I inhibitors can be labor-intensive and may yield a high number of false positives.
Purpose of the Study:
- To develop a cellular, target-specific high-throughput assay for identifying topoisomerase I inhibitors.
- To utilize a Saccharomyces cerevisiae (yeast) system with differentially labeled wild-type and topoisomerase I deletion mutant strains for efficient screening.
Main Methods:
- Development of a dual-strain yeast assay using wild-type and DeltatopI (topoisomerase I deletion mutant) Saccharomyces cerevisiae strains, each labeled with a distinct green fluorescent protein (GFP) variant.
- Independent quantification of the growth of both yeast strains when mixed and exposed to various compounds.
- Screening of compound libraries to identify inhibitors specifically targeting topoisomerase I.
Main Results:
- The assay successfully differentiated between topoisomerase I-specific inhibitors (e.g., camptothecin, inhibiting only the wild-type strain) and broad-spectrum agents (e.g., amphotericin B, inhibiting both strains).
- Only 0.06% of screened compounds inhibited the wild-type strain but not the DeltatopI strain, compared to 0.9% inhibiting both.
- The DeltatopI strain as an internal control reduced the number of candidate topoisomerase I inhibitors requiring retesting by over 90%.
Conclusions:
- The developed Saccharomyces cerevisiae-based cellular high-throughput assay is highly effective for specific detection of topoisomerase I inhibitors.
- This assay significantly enhances screening efficiency by minimizing false positives and reducing the workload for subsequent validation steps.
- The methodology offers a robust platform for discovering novel anticancer and antifungal agents targeting topoisomerase I.