title: Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University , 445 E 69th Street, New York, New York 10021, United States.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhao
authors.forename: Crystal M
authors.initials: CM
authors.lastname: Darby
authors.forename: Jennifer
authors.initials: J
authors.lastname: Small
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Bachovchin
authors.forename: Xiuju
authors.initials: X
authors.lastname: Jiang
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Burns-Huang
authors.forename: Helene
authors.initials: H
authors.lastname: Botella
authors.forename: Sabine
authors.initials: S
authors.lastname: Ehrt
authors.forename: Dale L
authors.initials: DL
authors.lastname: Boger
authors.forename: Erin D
authors.initials: ED
authors.lastname: Anderson
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Anna E
authors.initials: AE
authors.lastname: Speers
authors.forename: Virneliz
authors.initials: V
authors.lastname: Fernandez-Vega
authors.forename: Peter S
authors.initials: PS
authors.lastname: Hodder
authors.forename: Christina
authors.initials: C
authors.lastname: Eberhart
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Spicer
authors.forename: Carl F
authors.initials: CF
authors.lastname: Nathan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D048588:Benzoxazines / Q000737:chemistry / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology*; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D048588:Benzoxazines; D015335:Molecular Probes; D015842:Serine Proteinase Inhibitors; D057057:Serine Proteases