Oxathiazolones Selectively Inhibit the Human Immunoproteasome over the Constitutive Proteasome

Selective inhibitors for the human immunoproteasome LMP7 (β5i) subunit over the constitutive proteasome hold promise for the treatment of autoimmune and inflammatory diseases and hematologic malignancies. Here we report that oxathiazolones inhibit the immunoproteasome β5i with up to 4700-fold selectivity over the constitutive proteasome, are cell permeable, and inhibit proteasomes inside cells

Synthetic Calanolides with Bactericidal Activity against Replicating and Nonreplicating Mycobacterium tuberculosis

It is urgent to introduce new drugs for tuberculosis to shorten the prolonged course of treatment and control drug-resistant Mycobacterium tuberculosis (Mtb). One strategy toward this goal is to develop antibiotics that eradicate both replicating (R) and nonreplicating (NR) Mtb. Naturally occurring (+)-calanolide A was active against R-Mtb. The present report details the design, synthesis, antimycobacterial activities, and structure–activity relationships of synthetic calanolides. We identified potent dual-active nitro-containing calanolides with minimal in vitro toxicity that were cidal to axenic Mtb and Mtb in human macrophages, while sparing Gram-positive and -negative bacteria and yeast. Two of the nitrobenzofuran-containing lead compounds were found to be genotoxic to mammalian cells. Although genotoxicity precluded clinical progression, the profound, selective mycobactericidal activity of these calanolides will be useful in identifying pathways for killing both R- and NR-Mtb, as well as in further structure-based design of more effective and drug-like antimycobacterial agents

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

Identification of a Mycothiol-Dependent Nitroreductase from <i>Mycobacterium tuberculosis</i>

The success of <i>Mycobacterium tuberculosis</i> (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c. We show that Rv2466c is a mycothiol-dependent nitroreductase of Mtb and can reduce the nitro group of a novel mycobactericidal compound using mycothiol as a cofactor. In addition to its function as a nitroreductase, Rv2466c confers partial protection to menadione stress