title: Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors.
abstract: The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.
authors.affiliation: Department of Biological Reagents, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Totoritis
authors.forename: Chaya
authors.initials: C
authors.lastname: Duraiswami
authors.forename: Amy N
authors.initials: AN
authors.lastname: Taylor
authors.forename: John J
authors.initials: JJ
authors.lastname: Kerrigan
authors.forename: Nino
authors.initials: N
authors.lastname: Campobasso
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Smith
authors.forename: Paris
authors.initials: P
authors.lastname: Ward
authors.forename: Bryan W
authors.initials: BW
authors.lastname: King
authors.forename: Monique
authors.initials: M
authors.lastname: Murrayz-Thompson
authors.forename: Amber D
authors.initials: AD
authors.lastname: Jones
authors.forename: Glenn S
authors.initials: GS
authors.lastname: Van Aller
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Aubart
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zalacain
authors.forename: Sara H
authors.initials: SH
authors.lastname: Thrall
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Meek
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000493:pharmacokinetics; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D002384:Catalysis; D020134:Catalytic Domain / Q000187:drug effects; D002712:Chlorides / Q000737:chemistry / Q000494:pharmacology; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement / Q000379:methods; D006877:Hydroxamic Acids / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D007553:Isotope Labeling; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D012997:Solvents; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D002712:Chlorides; D006877:Hydroxamic Acids; D012997:Solvents; D000581:Amidohydrolases; C084507:peptide deformylase; D015032:Zinc; C010531:actinonin