New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

The extravasation of leukocytes from the peripheral blood stream to inflammatory sites is a key feature in the innate immune response to infection (1). Different chemoattractants (e.g. N-formylated peptides, C5a, interleukin-8, leukotriene B 4 , and platelet-activating factor) and chemokines induce leukocyte infiltration and activation through binding to G proteincoupled seven-transmembrane cell-surface receptors (2, 3). The chemoattractant-mediated dissociation of G␣ i2 from the G␤␥ subunit complex results in the activation of several downstream signaling effector enzymes that promote intracellular calcium mobilization, modifications in the metabolism of phosphoinositides, and activation of mitogen-activated protein kinases (4). The integration by the cell of the different chemoattractant-activated signaling pathways results in directed cell migration, recruitment of new receptors from the granules to the cell surface, release of proteolytic enzymes, production of large amounts of superoxide by the neutrophil NADPH oxidase, and increased gene transcription (5-8). The extent of the cellular response is dependent on the identity of the agonist and on the level of expression and desensitization of the receptors involved in the activation process (9).Two synthetic hexapeptides, Trp-Lys-Tyr-Met-Val-Met-NH 2 (WKYMVM)andTrp-Lys-Tyr-Met-Val-D-Met-NH 2 (WKYMVm), that stimulate phosphoinositide hydrolysis in myeloid cells were identified by screening a peptide library (10, 11). The D-methionine-containing hexapeptide (WKYMVm) was found to be a very potent activator of several leukocyte effector functions such as chemotaxis, mobilization of complement receptor-3, and activation of the NADPH oxidase (11). The peptide WKYMVm activates neutrophils through both the N-formyl peptide receptor (FPR) 1 and FPRL1 (N-formyl peptide receptor-like-1) (12, 13). The latter was originally cloned from human phagocytes by low-stringency hybridization of a cDNA library with the FPR cDNA sequence, and it was initially defined as an orphan receptor (14 -16). FPRL1 was later referred to as the LXA 4 receptor since it was shown to bind lipoxin A 4 with high affinity (17). In addition, several different peptides/proteins have been reported to stimulate this receptor. These include a leucine zipper-like domain of the HIV-1

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.