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

• Published in: ACS chemical biology Vol. 10; no. 2; pp. 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: Journal Article
• Language: English
• Published: United States American Chemical Society 20-02-2015
• 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
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb500746z

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.