title: An easy and fast method for the evaluation of Staphylococcus epidermidis type I signal peptidase inhibitors.
abstract: In the framework of the search for new antimicrobial therapies to combat resistant bacteria, the type I signal peptidase (SPase I) serves as a potentially interesting target for the development of antibacterials with a new mode of action. Bacterial SPases I play a key role in protein secretion as they are responsible for the cleavage of signal peptides from secreted proteins. For the Gram-positive Staphylococcus epidermidis, an important source of biofilm-associated infections, three putative SPases I (denoted Sip1, Sip2, Sip3) have been described, of which Sip1 lacks the catalytic lysine. Here, we report the in vitro activity of purified Sip2 and Sip3 using pre-SceD as a native preprotein substrate of S. epidermidis and in a FRET-based assay. For the latter, a novel internally quenched fluorescent peptide substrate based on the signal peptide sequence of this native preprotein was developed and specific cleavage of this synthetic fluorogenic peptide substrate was demonstrated. The latter in vitro assay represents a rapid and reliable tool in future research for the identification and validation of potential SPase I inhibitors.
authors.affiliation: Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Katrijn
authors.initials: K
authors.lastname: Bockstael
authors.forename: Nick
authors.initials: N
authors.lastname: Geukens
authors.forename: C V Smitha
authors.initials: CV
authors.lastname: Rao
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.forename: Jozef
authors.initials: J
authors.lastname: Anné
authors.forename: Arthur
authors.initials: A
authors.lastname: Van Aerschot
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000302:isolation & purification; D013212:Staphylococcus epidermidis / Q000187:drug effects* / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase