Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global burden, responsible for over 1 million deaths annually. The emergence and spread of drug-resistant M. tuberculosis strains (MDR-, XDR- and TDR-TB) is the main challenge in global TB-control, requiring the development of novel drugs acting on new biotargets, thus able to overcome the drug-resistance. Tryptanthrin is a natural alkaloid, with great therapeutic potential due to its simple way of synthesis and wide spectrum of biological activities including high bactericidal activity on both drug-susceptible and MDR M. tuberculosis strains. InhA was suggested as the target of tryptanthrins by in silico modeling, making it a promising alternative to isoniazid, able to overcome drug resistance provided by katG mutations. However, neither the mechanism of action of tryptanthrin nor the mechanism of resistance to tryptanthrins was ever confirmed in vitro. We show that the MmpS5-MmpL5 efflux system is able to provide resistance to tryptanthrins using an in-house test-system. Comparative genomic analysis of spontaneous tryptanthrin-resistant M. smegmatis mutants showed that mutations in MSMEG_1963 (EmbR transcriptional regulator) lead to a high-level resistance, while those in MSMEG_5597 (TetR transcriptional regulator) to a low-level one. Mutations in an MFS transporter gene (MSMEG_4427) were also observed, which might be involved in providing a basal level of tryptanthrins-resistance.

The World Health Organization (WHO) reports that tuberculosis (TB) is one of the top 10 leading causes of global mortality. The increasing incidence of multidrug-resistant TB highlights the urgent need for an intensified quest to discover innovative anti-TB medications In this study, we investigated four new derivatives from the quinoxaline-2-carboxylic acid 1,4-dioxide class. New 3-methylquinoxaline 1,4-dioxides with a variation in substituents at positions 2 and 6(7) were synthesized via nucleophilic aromatic substitution with amines and assessed against a Mycobacteria spp. Compound 4 showed high antimycobacterial activity (1.25 μg/mL against M. tuberculosis) and low toxicity in vivo in mice. Selection and whole-genomic sequencing of spontaneous drug-resistant M. smegmatis mutants revealed a high number of single-nucleotide polymorphisms, confirming the predicted mode of action of the quinoxaline-2-carboxylic acid 1,4-dioxide 4 as a DNA-damaging agent. Subsequent reverse genetics methods confirmed that mutations in the genes MSMEG_4646, MSMEG_5122, and MSMEG_1380 mediate resistance to these compounds. Overall, the derivatives of quinoxaline-2-carboxylic acid 1,4-dioxide present a promising scaffold for the development of innovative antimycobacterial drugs.

The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.

According to the World Health Organization (WHO), antibiotic resistance is currently one of the most serious threats to human health, food security, and development. Tuberculosis (TB) remains one of the deadliest bacterial diseases. The primary challenge in treating tuberculosis infection is the emergence of strains with multidrug resistance (MDR) to 4-9 drugs. The emergence of bacterial strains with MDR is a consequence of patients’ insufficient adherence to treatment, interrupted therapy, improperly prescribed courses of chemotherapy, and, according to recent data, the accumulation of antibiotics in the environment, which can activate the natural drug resistance system in bacteria. The consequences of MDR to antibiotics include prolonged hospitalizations, increased medical expenses, and mortality. Therefore, the task is to develop new effective antibacterial agents with novel mechanisms to reduce the emergence of bacterial resistance. In this study, we investigated the mechanisms of action of new promising antimycobacterial derivatives of quinoxalin-1,4-dioxide on the model organism Mycobacterium smegmatis .

In 2022, the World Health Organization reported that tuberculosis (TB) was the second leading cause of death globally from a single infectious agent following COVID-19. The development of new antitubercular agents with novel mechanisms of action for use in complex TB therapy is considered a key approach to combating TB. In this study, we examined the gene expression profile of M. smegmatis when exposed to a promising antituberculosis agent, quinoxaline 1,4-dioxide (QdNO) 7-chloro-2-(ethoxycarbonyl)-3-methyl-6-(piperazin-1-yl)quinoxaline-1,4-dioxide-1 (LCTA-3368). We investigated how the bacterial response changed with different minimum inhibitory concentrations (MIC) (1/4 × MIC, 1/2 × MIC, and 1 × MIC) and durations (30 min and 90 min) of treatment with the drug. Our analysis revealed significant upregulation in genes involved in DNA repair and replication processes, as well as changes in the expression of 95 genes encoding proteins with oxidoreductase activity. We additionally showed that the concentration of reactive oxygen species increases in a dose-dependent manner upon exposure of M. smegmatis to LCTA-3368. These findings support the proposed mechanism of antibacterial action of QdNOs, which is associated with the formation of free radicals leading to DNA damage.

In 2022, the World Health Organization reported that tuberculosis (TB) was the second highest cause of death globally from a single infectious agent, following COVID-19. Development of new antitubercular agents with a novel mechanism of action for use in the complex TB therapy is considered one of the key approaches for fighting TB. In this study we examined the gene expression profile of M. smegmatis when exposed to a promising antituberculosis agent quinoxaline 1,4-dioxide (QdNO) 7-chloro-2-(ethoxycarbonyl)-3-methyl-6-(piperazin-1-yl)quinoxaline-1,4-dioxide-1 (LCTA-3368). We looked at how the bacterial response changed with different doses (1/4 × MIC, 1/2 × MIC, and 1 x MIC) and durations (30 min and 90 min) of treatment with the drug. Our analysis revealed significant upregulation of the expression of genes involved in DNA repair and replication processes, and changes in the expression of 95 genes encoding proteins with oxidoreductase activity. This confirmed the assumption about the mechanism of antibacterial action of QdNOs associated with the formation of free radicals leading to DNA damage.