Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors |
| |
Authors: | Segers Kenneth Klaassen Hugo Economou Anastasios Chaltin Patrick Anné Jozef |
| |
Affiliation: | aLaboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroederstraat 10, 3000 Leuven, Belgium;bCenter for Innovation and Stimulation of Drug Discovery, Minderbroedersstraat 12, 3000 Leuven, Belgium;cInstitute of Molecular Biology and Biotechnology, Foundation of Research and Technology-Hellas, and Department of Biology, University of Crete, Vassilika Vouton, 71110 Iraklio, Crete, Greece;dCentre for Drug Design and Development, KULeuven R&D, Katholieke Universiteit Leuven, Minderbroedersstraat 8A, 3000 Leuven, Belgium |
| |
Abstract: | A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors. |
| |
Keywords: | Bacterial protein secretion SecA High-throughput screening Inhibitor ATPase Antibiotics |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|