Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of M. abscessus infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of M. abscessus in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat M. abscessus skin infections requires further evaluations.IMPORTANCEMycobacterium abscessus causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against M. abscessus in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against M. abscessus skin infections.

High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

Tailoring selective triclosan azo-adducts: Design, synthesis, and anti-mycobacterial evaluation.

A series of triclosan azo-adducts were synthesized to investigate their structure-activity relationship against Mycobacterium tuberculosis and non-tuberculous mycobacteria. The series' most potent compound was four and sixteen times more active than triclosan and rifabutin against drug-resistant Mycobacterium abscessus, respectively, while being less cytotoxic to human macrophages than triclosan on day one. Additionally, one of the azo-adducts was twice as efficient against M. tuberculosis as triclosan and twice as effective against Mycobacterium marinum as isoniazid. Furthermore, the synthesized azo-adducts were equally effective against M. abscessus strains overexpressing InhA, suggesting that these compounds work through a distinct mechanism.

Silencing essential gene expression in Mycobacterium abscessus during infection.

IMPORTANCE: Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3, encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.

New therapeutic strategies for Mycobacterium abscessus pulmonary diseases - untapping the mycolic acid pathway.

INTRODUCTION: Treatment options against Mycobacterium abscessus infections are very limited. New compounds are needed to cure M. abscessus pulmonary diseases. While the mycolic acid biosynthetic pathway has been largely exploited for the treatment of tuberculosis, this metabolic process has been overlooked in M. abscessus, although it offers many potential drug targets for the treatment of this opportunistic pathogen. AREAS COVERED: Herein, the authors review the role of the MmpL3 membrane protein and the enoyl-ACP reductase InhA involved in the transport and synthesis of mycolic acids, respectively. They discuss their importance as two major vulnerable drug targets in M. abscessus and report the activity of MmpL3 and InhA inhibitors. In particular, they focus on NITD-916, a direct InhA inhibitor against M. abscessus, particularly warranted in the context of multidrug resistance. EXPERT OPINION: There is an increasing body of evidence validating the mycolic acid pathway as an attractive drug target to be further exploited for M. abscessus lung disease treatments. The NITD-916 studies provide a proof-of-concept that direct inhibitors of InhA are efficient in vitro, in macrophages and in zebrafish. Future work is now required to improve the activity and pharmacological properties of these inhibitors and their evaluation in pre-clinical models.

In Vitro and In Vivo Efficacy of NITD-916 against Mycobacterium fortuitum.

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

Efficacy and Mode of Action of a Direct Inhibitor of Mycobacterium abscessus InhA.

There is an unmet medical need for effective treatments against Mycobacterium abscessus pulmonary infections, to which cystic fibrosis (CF) patients are particularly vulnerable. Recent studies showed that the antitubercular drug isoniazid is inactive against M. abscessus due to the incapacity of the catalase-peroxidase to convert the pro-drug into a reactive metabolite that inhibits the enoyl-ACP reductase InhA. To validate InhAMAB as a druggable target in M. abscessus, we assayed the activity of NITD-916, a 4-hydroxy-2-pyridone lead candidate initially described as a direct inhibitor of InhA that bypasses KatG bioactivation in Mycobacterium tuberculosis. The compound displayed low MIC values against rough and smooth clinical isolates in vitro and significantly reduced the bacterial burden inside human macrophages. Moreover, treatment with NITD-916 reduced the number and size of intracellular mycobacterial cords, regarded as markers of the severity of the infection. Importantly, NITD-916 significantly lowered the M. abscessus burden in CF-derived lung airway organoids. From a mechanistic perspective, NITD-916 abrogated de novo synthesis of mycolic acids and NITD-916-resistant spontaneous mutants harbored point mutations in InhAMAB at residue 96. That NITD-916 targets InhAMAB directly without activation requirements was confirmed genetically and by resolving the crystal structure of the protein in complex with NADH and NITD-916. These findings collectively indicate that InhAMAB is an attractive target to be exploited for future chemotherapeutic developments against this difficult-to-treat mycobacterium and highlight the potential of NITD-916 derivatives for further evaluation in preclinical settings.

Designing quinoline-isoniazid hybrids as potent anti-tubercular agents inhibiting mycolic acid biosynthesis.

Isoniazid is a cornerstone of modern tuberculosis (TB) therapy and targets the enoyl ACP reductase InhA, a key enzyme in mycolic acid biosynthesis. InhA is still a promising target for the development of new anti-TB drugs. Herein, we report the design, synthesis, and anti-tubercular activity of new isoniazid hybrids. Among these, 1H-1,2,3 triazole-tethered quinoline-isoniazid conjugates 16a to 16g exhibited high activity against Mycobacterium tuberculosis with minimal inhibitory concentrations in the 0.25-0.50 µg/mL range and were bactericidal in vitro. Importantly, these compounds were well tolerated at high doses on mammalian cells, leading to high selectivity indices. The hybrids were dependent on functional KatG production to inhibit mycolic acid biosynthesis. Moreover, overexpression of InhA in M. tuberculosis resulted in high resistance levels to 16a-16g and reduced mycolic acid biosynthesis inhibition, similar to isoniazid. Overall, these findings suggest that the synthesized quinoline-isoniazid hybrids are promising anti-tubercular molecules, which require further pre-clinical evaluation.

[Mycobacterium abscessus, a model of resistance to multiple antibiotic classes]. / Mycobacterium abscessus, un modèle de résistance aux différentes classes d'antibiotiques.

Mycobacterium abscessus is an environmental fast-growing, non-tuberculous mycobacterium responsible for severe lung infections, especially in patients with underlying lung disorders such as cystic fibrosis. The standard chemotherapy combines a b-lactam (imipenem or cefoxitin), an aminoglycoside (amikacin) and a macrolide (clarithromycin or azithromycin). However, resistance of this bacterium to most antibiotic classes, including nearly all anti-tubercular drugs, leads frequently to treatment failure and considerably reduces the therapeutic arsenal available to the clinician. A comprehensive understanding of the innate and acquired resistance mechanisms is thus necessary to counteract M. abscessus lung infections.

TITLE: Mycobacterium abscessus, un modèle de résistance aux différentes classes d'antibiotiques. ABSTRACT: Mycobacterium abscessus est une bactérie non tuberculeuse, environnementale, à croissance rapide, qui est responsable d'infections pulmonaires sévères, notamment chez les patients atteints de mucoviscidose. Le traitement actuel combine l'utilisation d'une b-lactamine et d'un aminoglycoside associés à un macrolide. Cette bactérie est polyrésistante à la plupart des antibiotiques utilisés en clinique. Les mécanismes de résistance, innés ou acquis, qu'elle a développés, conduisent fréquemment à des échecs thérapeutiques, ce qui limite considérablement les moyens de lutte disponibles pour le clinicien. Une compréhension globale des mécanismes de résistance de cette bactérie s'avère ainsi nécessaire pour contrer les infections pulmonaires qu'elle provoque.

Mycobacteriophage-antibiotic therapy promotes enhanced clearance of drug-resistant Mycobacterium abscessus.

Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper.

Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.