Mycobacterium tuberculosis is the cause of one of the most deadly diseases of mankind, and despite the availability of effective treatments, tuberculosis (TB) remains a major public health threat. The difficult challenges in treating multiple-drugresistant (MDR) and extensively drug-resistant (XDR) TB and the importance of shortening the duration of treatment to improve patients' compliance make the discovery of new anti-TB drugs imperative (1-5). Attempts to discover new TB drugs and targets via large-scale screening against intact mycobacteria have largely been confined to synthetic compound libraries and to date have yielded only one new clinical TB drug, the diarylquinoline bedaquiline (6, 7). Although very potent, to be of maximum benefit, bedaquiline, a diarylquinoline, and nitroimidazoles (8) require new companion drugs to be used in a multidrug regimen.While the intensive search for antibiotics from soil microorganisms in the mid-20th century yielded several clinically useful TB drugs, the pathogenic nature of M. tuberculosis and its extremely slow growth rate did not allow classical agar diffusion tests and excluded M. tuberculosis from the initial target panel. The discovery of TB drugs of natural origin at that time therefore relied upon the detection of activity against nonmycobacteria in agar diffusion assays followed by bioassay-guided isolation of the active principle, again using nonmycobacteria. Activity against M. tuberculosis was only assessed once the active principle was purified.Because M. tuberculosis is uniquely susceptible to a number of antimicrobial agents, a high-throughput screening (HTS) of actinomycete extracts directly against the virulent H37Rv strain was conducted, and this campaign revealed selective anti-TB peptides produced by a genetically distinct Nonomuraea species, strain MJM5123. Here, we describe the activity profile of ecumicin, its efficacy in infected mice, the identification of its molecular target, and the elucidation of its unusual mechanism of action. MATERIALS AND METHODSHigh-throughput screening. Approximately 7,000 actinomycete cultures isolated from Korea, China, Nepal, the Philippines, Vietnam, Antarctica, and the Arctic Circle and maintained at Myongji University, South Korea, were fermented in 20-ml cultures in glucose-soybean starch (GSS) medium (rich medium), Bennett's medium (normal medium), and dextrinyeast-corn steep liquor (DYC) medium (minimal medium) (see Table S1 in the supplemental material). The mycelia and culture medium supernatants were separated and extracted with methanol and ethyl acetate, respectively. Nine extracts were thus generated from each microbial isolate.

Protein tyrosine phosphatases are often exploited and subverted by pathogenic bacteria to cause human diseases. The tyrosine phosphatase mPTPB from Mycobacterium tuberculosis is an essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host. Consequently, there is considerable interest in understanding the mechanism by which mPTPB evades the host immune responses, and in developing potent and selective mPTPB inhibitors as unique antituberculosis (antiTB) agents. We uncovered that mPTPB subverts the innate immune responses by blocking the ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by activating the Akt pathway. We identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. We demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics.combinatorial chemistry | pathogen-host interaction | phosphatase inhibitor | signaling mechanism

dMmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.

A series of biphenyl analogues of the new tuberculosis drug PA-824 was prepared, primarily by coupling the known (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol with iodobenzyl halides, followed by Suzuki coupling of these iodides with appropriate arylboronic acids or by assembly of the complete biaryl side chain prior to coupling with the above alcohol. Antitubercular activity was determined under both replicating (MABA) and nonreplicating (LORA) conditions. para-Linked biaryls were the most active, followed by meta-linked and then ortho-linked analogues. A more detailed study of a larger group of para-linked analogues showed a significant correlation between potency (MABA) and both lipophilicity (CLOGP) and the electron-withdrawing properties of terminal ring substituents ( summation operatorsigma). Selected compounds were evaluated for their efficacy in a mouse model of acute Mycobacterium tuberculosis infection. In vivo activity correlated well with the stability of compounds to microsomal metabolism. Three compounds bearing combinations of lipophilic, electron-withdrawing groups achieved >200-fold higher efficacies than the parent drug.

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