title: Small-molecule inhibitors of the pseudaminic acid biosynthetic pathway: targeting motility as a key bacterial virulence factor.
abstract: Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Ménard
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Schoenhofen
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Limei
authors.initials: L
authors.lastname: Tao
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Annie
authors.initials: A
authors.lastname: Aubry
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Patrice
authors.initials: P
authors.lastname: Bouchard
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Reid
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Paule
authors.initials: P
authors.lastname: Lachance
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Twine
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Fulton
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Qizhi
authors.initials: Q
authors.lastname: Cui
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Hervé
authors.initials: H
authors.lastname: Hogues
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Enrico O
authors.initials: EO
authors.lastname: Purisima
authors.affiliation: Biologics Program, Human Health Therapeutics, National Research Council Canada, Montreal, Quebec, Canada traian.sulea@nrc-cnrc.gc.ca susan.logan@nrc-cnrc.gc.ca.
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada traian.sulea@nrc-cnrc.gc.ca susan.logan@nrc-cnrc.gc.ca.
authors.forename: Susan M
authors.identifier: http://orcid.org/0000-0001-6400-5829
authors.initials: SM
authors.lastname: Logan
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability; D055808:Drug Discovery; D005407:Flagella / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005408:Flagellin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation / Q000187:drug effects; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D057166:High-Throughput Screening Assays; D062105:Molecular Docking Simulation; D009068:Movement / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013400:Sugar Acids / Q000378:metabolism*; D014584:User-Computer Interface; D014774:Virulence
chemical_list: C513444:5,7-diacetamido-3,5,7,9-tetradeoxynonulosonic acid; D000900:Anti-Bacterial Agents; D054852:Small Molecule Libraries; D013400:Sugar Acids; D005408:Flagellin