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Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis

Academic Article
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Overview

related to degree

  • Anderson, Erin, Ph.D. in Organic Chemistry, Scripps Research 2007 - 2013
  • Bachovchin, Daniel A., Ph.D. in Chemistry, Scripps Research 2006 - 2011

authors

  • Zhao, N.
  • Darby, C. M.
  • Small, J.
  • Bachovchin, Daniel A.
  • Jiang, X.
  • Burns-Huang, K. E.
  • Botella, H.
  • Ehrt, S.
  • Boger, Dale
  • Anderson, Erin
  • Cravatt, Benjamin
  • Speers, Anna
  • Fernandez-Vega, V.
  • Hodder, Peter
  • Eberhart, C.
  • Rosen, Hugh
  • Spicer, Timothy
  • Nathan, C. F.

publication date

  • February 2015

journal

  • ACS Chemical Biology  Journal

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.
  • 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-Gu�rin (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.

subject areas

  • Bacterial Proteins
  • Benzoxazines
  • Gene Expression Regulation, Bacterial
  • Gene Expression Regulation, Enzymologic
  • Homeostasis
  • Hydrogen-Ion Concentration
  • Molecular Probes
  • Molecular Structure
  • Mycobacterium tuberculosis
  • Periplasm
  • Serine Proteases
  • Serine Proteinase Inhibitors
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Identity

PubMed Central ID

  • PMC4340348

International Standard Serial Number (ISSN)

  • 1554-8929

Digital Object Identifier (DOI)

  • 10.1021/cb500746z

PubMed ID

  • 25457457
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Additional Document Info

start page

  • 364

end page

  • 371

volume

  • 10

issue

  • 2

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