title: Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors.
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.
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroederstraat 10, 3000 Leuven, Belgium. Kenneth.Segers@rega.kuleuven.be
authors.forename: Kenneth
authors.initials: K
authors.lastname: Segers
authors.forename: Hugo
authors.initials: H
authors.lastname: Klaassen
authors.forename: Anastasios
authors.initials: A
authors.lastname: Economou
authors.forename: Patrick
authors.initials: P
authors.lastname: Chaltin
authors.forename: Jozef
authors.initials: J
authors.lastname: Anné
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; D054852:Small Molecule Libraries; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins