title: Isozyme-specific ligands for O-acetylserine sulfhydrylase, a novel antibiotic target.
abstract: The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.
authors.affiliation: Department of Food Sciences, University of Parma, Parma, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spyrakis
authors.forename: Ratna
authors.initials: R
authors.lastname: Singh
authors.forename: Pietro
authors.initials: P
authors.lastname: Cozzini
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Enea
authors.initials: E
authors.lastname: Salsi
authors.forename: Paolo
authors.initials: P
authors.lastname: Felici
authors.forename: Samanta
authors.initials: S
authors.lastname: Raboni
authors.forename: Paolo
authors.initials: P
authors.lastname: Benedetti
authors.forename: Gabriele
authors.initials: G
authors.lastname: Cruciani
authors.forename: Glen E
authors.initials: GE
authors.lastname: Kellogg
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001419:Bacteria / Q000201:enzymology / Q000254:growth & development; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D003547:Cysteine Synthase; D003545:Cysteine