Several key antituberculosis drugs, including pyrazinamide, with a molecular mass of 123.1 g/mol, are smaller than the usual drug-like molecules. Current drug discovery efforts focus on the screening of larger compounds with molecular masses centered around 400 to 500 g/mol. Fragment (molecular mass < 300 g/mol) libraries have not been systematically explored for antitubercular activity. Here we screened a collection of 1,000 fragments, present in the Maybridge Ro3 library, for whole-cell activity against Mycobacterium tuberculosis. Twenty-nine primary hits showed dose-dependent growth inhibition equal to or better than that of pyrazinamide. The most potent hit, indole propionic acid [IPA; 3-(1H-indol-3-yl)propanoic acid], a metabolite produced by the gut microbiota, was profiled in vivo. The molecule was well tolerated in mice and showed adequate pharmacokinetic properties. In a mouse model of acute M. tuberculosis infection, IPA reduced the bacterial load in the spleen 7-fold. Our results suggest that IPA should be evaluated as an add-on to current regimens and that fragment libraries should be further explored to identify antimycobacterial lead candidates.

Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosis in vitro and in vivo. However, its mechanism of action is unknown. IPA is the deamination product of tryptophan (Trp) and thus a close structural analog of this essential aromatic amino acid. De novo Trp biosynthesis in M. tuberculosis is regulated through feedback inhibition: Trp acts as an allosteric inhibitor of anthranilate synthase TrpE, which catalyzes the first committed step in the Trp biosynthesis pathway. Hence, we hypothesized that IPA may mimic Trp as an allosteric inhibitor of TrpE and exert its antimicrobial effect by blocking synthesis of Trp at the TrpE catalytic step. To test our hypothesis, we carried out metabolic, chemical rescue, genetic, and biochemical analyses. Treatment of mycobacteria with IPA inhibited growth and reduced the intracellular level of Trp, an effect abrogated upon supplementation of Trp in the medium. Missense mutations at the allosteric Trp binding site of TrpE eliminated Trp inhibition and caused IPA resistance. In conclusion, we have shown that IPA blocks Trp biosynthesis in M. tuberculosis via inhibition of TrpE by mimicking the physiological allosteric inhibitor of this enzyme. IMPORTANCE New drugs against tuberculosis are urgently needed. The tryptophan (Trp) analog indole propionic acid (IPA) is the first antitubercular metabolite produced by human gut bacteria. Here, we show that this antibiotic blocks Trp synthesis, an in vivo essential biosynthetic pathway in M. tuberculosis. Intriguingly, IPA acts by decoupling a bacterial feedback regulatory mechanism: it mimics Trp as allosteric inhibitor of anthranilate synthase, thereby switching off Trp synthesis regardless of intracellular Trp levels. The identification of IPA’s target paves the way for the discovery of more potent TrpE ligands employing rational, target-based lead optimization.

New, more effective drugs for the treatment of lung disease caused by non-tuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against M. tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and anti-biofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10−8/CFU and mapped to gyrA and gyrB encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild type enzyme but not the P4C resistant mutant. P4C resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in recA promotor-based DNA damage reporter strains showed induction of recA promoter activity in wild type but not in P4C resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.

Most antibiotics are produced by soil microbes and typically interfere with macromolecular synthesis processes as their antibacterial mechanism of action. These natural products are often large and suffer from poor chemical tractability. Here, we discuss discovery, mechanism of action, and the therapeutic potentials of an unusual antibiotic, indole propionic acid (IPA). IPA is produced by the human gut microbiota. The molecule is small, chemically tractable, and targets amino acid biosynthesis. IPA is active against a broad spectrum of mycobacteria, including drug resistant Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM). Interestingly, the microbiota-produced metabolite is detectable in the serum of healthy individuals, tuberculosis (TB) patients, and several animal models. Thus, the microbiota in our gut may influence susceptibility to mycobacterial diseases. If a gut-lung microbiome axis can be demonstrated, IPA may have potential as a biomarker of disease progression, and development of microbiota-based therapies could be explored. In addition to its antimycobacterial activity, the molecule displays antiinflammatory and antioxidant properties. This raises the possibility that IPA has therapeutic potential as both antibiotic and add-on host-directed drug for the treatment of TB in patient populations where disease morbidity and mortality is driven by excessive inflammation and tissue damage, such as TB-associated immune reconstitution inflammatory syndrome, TB-meningitis, and TB-diabetes.

Disruption of redox homeostasis in mycobacteria causes irreversible stress induction and cell death. Here, we report the dioxonaphthoimidazolium scaffold as a novel redox cycling antituberculosis chemotype with potent bactericidal activity against growing and nutrient-starved phenotypically drug-resistant nongrowing bacteria. Maximal potency was dependent on the activation of the redox cycling quinone by the positively charged scaffold and accessibility to the mycobacterial cell membrane as directed by the lipophilicity and conformational characteristics of the Nsubstituted side chains. Evidence from microbiological, biochemical, and genetic investigations implicates a redox-driven mode of action that is reliant on the reduction of the quinone by type II NADH dehydrogenase (NDH2) for the generation of bactericidal levels of the reactive oxygen species (ROS). The bactericidal profile of a potent water-soluble analogue 32 revealed good activity against nutrient-starved organisms in the Loebel model of dormancy, low spontaneous resistance mutation frequency, and synergy with isoniazid in the checkerboard assay.

Bioactive forms of oral β-lactams were screened in vitro against Mycobacterium abscessus with and without the bioactive form of the oral β-lactamase inhibitor avibactam ARX1796. Sulopenem was equally active without avibactam, while tebipenem, cefuroxime, and amoxicillin required avibactam for optimal activity.

Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus in vitro .

The combination of the β-lactam tebipenem and the β-lactamase inhibitor avibactam shows potent bactericidal activity against Mycobacterium abscessus in vitro . Here, we report that the combination of the respective oral prodrugs tebipenem-pivoxil and avibactam ARX-1796 showed efficacy in a mouse model of M. abscessus lung infection.