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

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) maintains its intrabacterial pH (pH_IB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that <i>Mtb</i> loses this ability when the mycobacterial acid resistance protease (<i>marP</i>) 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 <i>Mtb</i>’s pH_IB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient <i>Mtb</i>, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with <i>Mycobacterium bovis</i> variant <i>bacille Calmette-Guérin</i> (<i>BCG</i>) 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