title: Fluorescein analogues inhibit SecA ATPase: the first sub-micromolar inhibitor of bacterial protein translocation.
abstract: SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for antimicrobial agents. A series of fluorescein analogues were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose bengal (RB) and erythrosin B (EB) were found to be potent inhibitors SecA with IC(50) values of 0.5 μM and 2 μM, respectively. RB and EB inhibit the catalytic SecA ATPase more effectively than the F(1) F(0) -proton ATPase. We used three assays to test the effect of these compounds on full-length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC(50) values: translocation ATPase<membrane ATPase<intrinsic ATPase. Very importantly, the potency of these fluorescein analogues in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC(50) values of approximately 0.25 μM, making RB the most potent inhibitor of SecA ATPase and SecA-dependent protein translocation reported thus far. The ability of these compounds to inhibit SecA also directly translates into antibacterial effects. Our findings show the value of fluorescein analogues as probes for mechanistic studies of SecA functions and for the potential development of new antimicrobial agents with SecA as the target.
authors.affiliation: Department of Biology, Georgia State University, 490 Petit Science Center, 161 Jesse Hill Jr. Drive, Atlanta, GA 30303, USA.
authors.forename: Ying-Ju
authors.initials: YJ
authors.lastname: Huang
authors.forename: Hongyun
authors.initials: H
authors.lastname: Wang
authors.forename: Fen-Biao
authors.initials: FB
authors.lastname: Gao
authors.forename: Minyong
authors.initials: M
authors.lastname: Li
authors.forename: Hsiuchin
authors.initials: H
authors.lastname: Yang
authors.forename: Binghe
authors.initials: B
authors.lastname: Wang
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D004923:Erythrosine / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D019793:Fluorescein / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D021381:Protein Transport / Q000187:drug effects; D012395:Rose Bengal / Q000737:chemistry / Q000494:pharmacology*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C078167:OMPA outer membrane proteins; D012395:Rose Bengal; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D004923:Erythrosine; D019793:Fluorescein