Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

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 disru...

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Bibliographic Details
Published in:	ACS chemical biology 2015-02, Vol.10 (2), p.364-371
Main Authors:	Zhao, Nan, Darby, Crystal M, Small, Jennifer, Bachovchin, Daniel A, Jiang, Xiuju, Burns-Huang, Kristin E, Botella, Helene, Ehrt, Sabine, Boger, Dale L, Anderson, Erin D, Cravatt, Benjamin F, Speers, Anna E, Fernandez-Vega, Virneliz, Hodder, Peter S, Eberhart, Christina, Rosen, Hugh, Spicer, Timothy P, Nathan, Carl F
Format:	Article
Language:	eng
Subjects:	Bacterial Proteins - genetics Bacterial Proteins - metabolism Benzoxazines - chemistry Benzoxazines - pharmacology Gene Expression Regulation, Bacterial - physiology Gene Expression Regulation, Enzymologic - physiology Homeostasis Hydrogen-Ion Concentration Letters Molecular Probes - chemistry Molecular Probes - metabolism Molecular Structure Mycobacterium bovis Mycobacterium tuberculosis Mycobacterium tuberculosis - cytology Mycobacterium tuberculosis - enzymology Mycobacterium tuberculosis - genetics Mycobacterium tuberculosis - metabolism Periplasm - enzymology Serine Proteases - genetics Serine Proteases - metabolism Serine Proteinase Inhibitors - chemistry Serine Proteinase Inhibitors - pharmacology
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Summary:	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.
ISSN:	1554-8929 1554-8937