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.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence

“…ETZ does not alter M. tuberculosis cytoplasmic pH homeostasis (Fig. 1D) and is thus acting by a mechanism that is distinct from compounds found to modulate cytoplasmic pH homeostasis (40)(41)(42). ETZ therefore represents a first-in-class agent and strategy to inhibit M. tuberculosis pH-dependent adaptation and virulence.…”

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

“…For example, our data show that activity of MarP (Rv3671c) remains largely unchanged throughout replication and non-replication, suggesting that MarP has a role in maintaining persistence. Indeed, MarP is required for replication in mice specifically during persistence (Vandal et al, 2008) and is now under active development as a drug target (Zhao et al, 2015). Given that we accurately predict persistence targets, these SHs should be further tested by genetic methods for essentiality during non-replication.…”

Targeting Phenotypically TolerantMycobacterium tuberculosis

“…The most commonly used models for nonreplicating mycobacteria are: hypoxia (the “Wayne model”) and the “low oxygen recovery assay” (“LORA”) (29, 95, 96); carbon starvation (54, 122); nutrient starvation (12, 52); stationary phase (105); maintenance of intrabacterial pH under acidic culture conditions (120, 123–127); biofilms (102, 128–131); depleting strain SS18b of streptomycin (103, 104, 132, 133); and a multi-stress model that combines acidic pH (pH 5.0), mild hypoxia (1% O 2 ), nitric oxide and other reactive nitrogen intermediates (0.5 mM NaNO 2 ), and a fatty acid carbon source (0.05% butyrate) (53, 94, 100, 134, 135). There are variations of these models, including an “acidic Wayne model” that combines hypoxia with mild acidity (131), and a nutrient-poor, multi-stress model in which cells are cultured at low pH (pH 5.0) under mild hypoxia or tissue-level normoxia (5% O 2 ) and supra-physiologic levels of CO 2 (10% CO 2 ) (136).…”