title: High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors.
abstract: The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.
authors.affiliation: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, United States of America.
authors.forename: Umesh K
authors.initials: UK
authors.lastname: Bageshwar
authors.affiliation: Broad Institute, Cambridge, MA, United States of America.
authors.forename: Lynn
authors.initials: L
authors.lastname: VerPlank
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, United States of America.
authors.forename: Dwight
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, United States of America.
authors.forename: Wen
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, United States of America.
authors.forename: Shruthi
authors.initials: S
authors.lastname: Hamsanathan
authors.affiliation: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, United States of America.
authors.forename: Neal
authors.initials: N
authors.lastname: Whitaker
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, United States of America.
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.affiliation: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, United States of America.
authors.forename: Siegfried M
authors.initials: SM
authors.lastname: Musser
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli