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New oxazolidinones are in clinical development for the treatment of tuberculosis and nontuberculous mycobacterial (NTM) infections, as a replacement for linezolid and tedizolid, which cause mitochondrial toxicity after prolonged treatment. Here, we carried out side-by-side measurements of mitochondrial protein synthesis inhibition and activity against clinically relevant mycobacterial pathogens of approved and novel oxazolidinones.

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.

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 N-substituted 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.

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. Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus ex vivo and in vivo.

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. Systematic pairwise combination of the four β-lactams revealed strong bactericidal synergy for each of sulopenem, tebipenem and cefuroxime combined with amoxicillin in the presence of avibactam. These all-oral β-lactam combinations warrant clinical evaluation.

[Image: see text] Mycobacterium abscessus causes difficult-to-cure pulmonary infections. The bacterium is resistant to most anti-infective agents, including first line antituberculosis (anti-TB) drugs. MMV688844 (844) is a piperidine-4-carboxamide (P4C) with bactericidal properties against M. abscessus. We recently identified DNA gyrase as the molecular target of 844. Here, we present in silico docking and genetic evidence suggesting that P4Cs display a similar binding mode to DNA gyrase as gepotidacin. Gepotidacin is a member of the Novel Bacterial Topoisomerase Inhibitors (NBTIs), a new class of nonfluoroquinolone DNA gyrase poisons. Thus, our work suggests that P4Cs present a novel structural subclass of NBTI. We describe structure–activity relationship studies of 844 leading to analogues showing increased antibacterial activity. Selected derivatives were tested for their inhibitory activity against recombinant M. abscessus DNA gyrase. Further optimization of the lead structures led to improved stability in mouse plasma and increased oral bioavailability.

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 N-substituted 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.

New, more-effective drugs for the treatment of lung disease caused by nontuberculous 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 Mycobacterium 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 antibiofilm 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 promoter-based DNA damage reporter strains showed induction of recA promoter activity in the wild type but not in the 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.

Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosisin 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 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., 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.