title: A Target-Based Whole Cell Screen Approach To Identify Potential Inhibitors of Mycobacterium tuberculosis Signal Peptidase.
abstract: The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure-activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism.
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Shilah A
authors.initials: SA
authors.lastname: Bonnett
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Juliane
authors.initials: J
authors.lastname: Ollinger
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Susantha
authors.initials: S
authors.lastname: Chandrasekera
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Florio
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Theresa
authors.initials: T
authors.lastname: O'Malley
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Megan
authors.initials: M
authors.lastname: Files
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Jo-Ann
authors.initials: JA
authors.lastname: Jee
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: James
authors.initials: J
authors.lastname: Ahn
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Allen
authors.initials: A
authors.lastname: Casey
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Ovechkina
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: David
authors.initials: D
authors.lastname: Roberts
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Aaron
authors.initials: A
authors.lastname: Korkegian
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute , 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase
keywords: phenotypic screen; phenylhydrazones; signal peptidase; tuberculosis