L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

Antimicrobial combinations against Helicobacter pylori including benzoxadiazol-based flavodoxin inhibitors: in vitro characterization.

IMPORTANCE: The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

In vitro synergy screens of FDA-approved drugs reveal novel zidovudine- and azithromycin-based combinations with last-line antibiotics against Klebsiella pneumoniae.

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options available. Drug repurposing could accelerate the development of new urgently needed successful interventions. This work aimed to identify and characterise novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We first performed a semi-qualitative high-throughput synergy screen (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) against a FDA-library containing 1430 clinically approved drugs; a total of 109 compounds potentiated any of the last-line antibiotics. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays, obtaining 15.09% and 65.85% confirmation rates, respectively. Accordingly, TKA were used for synergy classification based on determination of bactericidal activities at 8, 24 and 48 h, selecting 27 combinations against K. pneumoniae. Among them, zidovudine or azithromycin combinations with last-line antibiotics were further evaluated by TKA against a panel of 12 MDR/XDR K. pneumoniae strains, and their activities confronted with those clinical combinations currently used for MDR enterobacteria treatment; these combinations showed better bactericidal activities than usual treatments without added cytotoxicity. Our studies show that sHTSS paired to TKA are powerful tools for the identification and characterisation of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of zidovudine- and azithromycin-based combinations for the treatment of these infections.

Repurposing ß-Lactams for the Treatment of Mycobacterium kansasii Infections: An In Vitro Study.

Mycobacterium kansasii (Mkn) causes tuberculosis-like lung infection in both immunocompetent and immunocompromised patients. Current standard therapy against Mkn infection is lengthy and difficult to adhere to. Although ß-lactams are the most important class of antibiotics, representing 65% of the global antibiotic market, they have been traditionally dismissed for the treatment of mycobacterial infections, as they were considered inactive against mycobacteria. A renewed interest in ß-lactams as antimycobacterial agents has shown their activity against several mycobacterial species, including M. tuberculosis, M. ulcerans or M. abscessus; however, information against Mkn is lacking. In this study, we determined the in vitro activity of several ß-lactams against Mkn. A selection of 32 agents including all ß-lactam chemical classes (penicillins, cephalosporins, carbapenems and monobactams) with three ß-lactamase inhibitors (clavulanate, tazobactam and avibactam) were evaluated against 22 Mkn strains by MIC assays. Penicillins plus clavulanate and first- and third-generation cephalosporins were the most active ß-lactams against Mkn. Combinatorial time-kill assays revealed favorable interactions of amoxicillin-clavulanate and cefadroxil with first-line Mkn treatment. Amoxicillin-clavulanate and cefadroxil are oral medications that are readily available, and well tolerated with an excellent safety and pharmacokinetic profile that could constitute a promising alternative option for Mkn therapy.

Embedding Biomimetic Magnetic Nanoparticles Coupled with Peptide AS-48 into PLGA to Treat Intracellular Pathogens.

Among the strategies employed to overcome the development of multidrug-resistant bacteria, directed chemotherapy combined with local therapies (e.g., magnetic hyperthermia) has gained great interest. A nano-assembly coupling the antimicrobial peptide AS-48 to biomimetic magnetic nanoparticles (AS-48-BMNPs) was demonstrated to have potent bactericidal effects on both Gram-positive and Gram-negative bacteria when the antimicrobial activity of the peptide was combined with magnetic hyperthermia. Nevertheless, intracellular pathogens remain challenging due to the difficulty of the drug reaching the bacterium. Thus, improving the cellular uptake of the nanocarrier is crucial for the success of the treatment. In the present study, we demonstrate the embedding cellular uptake of the original nano-assembly into THP-1, reducing the toxicity of AS-48 toward healthy THP-1 cells. We optimized the design of PLGA[AS-48-BMNPs] in terms of size, colloidal stability, and hyperthermia activity (either magnetic or photothermal). The stability of the nano-formulation at physiological pH values was evaluated by studying the AS-48 release at this pH value. The influence of pH and hyperthermia on the AS-48 release from the nano-formulation was also studied. These results show a slower AS-48 release from PLGA[AS-48-BMNPs] compared to previous nano-formulations, which could make this new nano-formulation suitable for longer extended treatments of intracellular pathogens. PLGA[AS-48-BMNPs] are internalized in THP-1 cells where AS-48 is liberated slowly, which may be useful to treat diseases and prevent infection caused by intracellular pathogens. The treatment will be more efficient combined with hyperthermia or photothermia.

Discovery of 3H-pyrrolo[2,3-c]quinolines with activity against Mycobacterium tuberculosis by allosteric inhibition of the glutamate-5-kinase enzyme.

The therapeutic potential of 3H-pyrrolo[2,3-c]quinolines-the main core of Marinoquinoline natural products-has been explored for the development of new anti-TB agents. The chemical modification of various positions in this scaffold has led to the discovery of two pyrroloquinolines (compounds 50 and 54) with good in vitro activity against virulent strains of Mycobacterium tuberculosis (H37Rv, MIC = 4.1 µM and 4.2 µM, respectively). Enzymatic assays showed that both derivatives are inhibitors of glutamate-5-kinase (G5K, encoded by proB gene), an essential enzyme for this pathogen involved in the first step of the proline biosynthesis pathway. G5K catalyzes the phosphoryl-transference of the γ-phosphate group of ATP to L-glutamate to provide L-glutamyl-5-phosphate and ADP, and also regulates the synthesis of L-proline. The results of various molecular dynamics simulation studies revealed that the inhibition of G5K would be caused by allosteric interaction of these compounds with the interface between enzyme domains, against different pockets and with distinct recognition patterns. The binding of compound 54 promotes long-distance conformational changes at the L-glutamate binding site that would prevent it from anchoring for catalysis, while compound 50 alters the ATP binding site architecture for recognition. Enzyme assays revealed that compound 50 caused a substancial increase in the Kmapp for ATP, while no significant effect was observed for derivative 54. This work also demonstrates the potential of the G5K enzyme as a biological target for the development of new anti-TB drugs.

Overcoming the Prokaryote/Eukaryote Barrier in Tuberculosis Treatment: A Prospect for the Repurposing and Use of Antiparasitic Drugs.

Antimicrobial resistance, the so-called silent pandemic, is pushing industry and academia to find novel antimicrobial agents with new mechanisms of action in order to be active against susceptible and drug-resistant microorganisms. In the case of tuberculosis, the need of novel anti-tuberculosis drugs is specially challenging because of the intricate biology of its causative agent, Mycobacterium tuberculosis. The repurposing of medicines has arisen in recent years as a fast, low-cost, and efficient strategy to identify novel biomedical applications for already approved drugs. This review is focused on anti-parasitic drugs that have additionally demonstrated certain levels of anti-tuberculosis activity; along with this, natural products with a dual activity against parasites and against M. tuberculosis are discussed. A few clinical trials have tested antiparasitic drugs in tuberculosis patients, and have revealed effective dose and toxicity issues, which is consistent with the natural differences between tuberculosis and parasitic infections. However, through medicinal chemistry approaches, derivatives of drugs with anti-parasitic activity have become successful drugs for use in tuberculosis therapy. In summary, even when the repurposing of anti-parasitic drugs for tuberculosis treatment does not seem to be an easy job, it deserves attention as a potential contributor to fuel the anti-tuberculosis drug pipeline.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.

Measuring Efflux and Permeability in Mycobacteria.

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.

In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target.

New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for the in silico search of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase module of the FADS, and some of them were able to inhibit growth of three bacterial species: C. ammoniagenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae, where the last two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets.

Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA).

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition.

Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles.

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2'-N-acetyltransferase [AAC(2')], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2') enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2') and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2') and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets.

Matryoshka-type gastro-resistant microparticles for the oral treatment of Mycobacterium tuberculosis.

AIM: Production of Matryoshka-type gastroresistant microparticles containing antibiotic-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (NP) against Mycobacterium tuberculosis. MATERIALS & METHODS: The emulsification and evaporation methods were followed for the synthesis of PLGA-NPs and methacrylic acid-ethyl acrylate-based coatings to protect rifampicin from degradation under simulated gastric conditions. RESULTS & CONCLUSION: The inner antibiotic-loaded NPs here reported can be released under simulated intestinal conditions whereas their coating protects them from degradation under simulated gastric conditions. The encapsulation does not hinder the antituberculosis action of the encapsulated antibiotic rifampicin. A sustained antibiotic release could be obtained when using the drug-loaded encapsulated NPs. Compared with the administration of the free drug, a more effective elimination of M. tuberculosis was observed when applying the NPs against infected macrophages. The antibiotic-loaded PLGA-NPs were also able to cross an in vitro model of intestinal barrier.

Polypeptidic Micelles Stabilized with Sodium Alginate Enhance the Activity of Encapsulated Bedaquiline.

The coating of polypeptidic micelles with sodium alginate is described as a strategy to improve the stability of micelles for drug delivery. Bedaquiline, approved in 2012 for the treatment of multidrug resistant tuberculosis, has been used as an example of hydrophobic drug to study the loading efficiency, the release of the encapsulated drug in different media, and the in vitro antimicrobial activity of the system. Alginate coating prevents the burst release of the drug from micelles upon dilution and leads to a sustained release in all tested media. In view of possible oral administration, the alginate coated micelles show better stability in gastric and intestinal simulated media. Notably, the encapsulated bedaquiline shows increased in vitro activity against Mycobacterium tuberculosis compared to free bedaquiline.

Synthesis and biological activity of dehydrophos derivatives.

The synthesis of dehydrophos derivatives featuring modified peptide chains, characterized by the presence of substituents in the vinyl moiety, or possessing a phosphonic acid monoalkyl ester other than the monomethyl ester one, has been accomplished by a versatile procedure based on Horner-Wadsworth-Emmons olefination with suitable aldehydes and on the selective hydrolysis of the dialkyl phosphonate group. Such derivatives have been tested against a series of bacterial strains, using the naturally occurring peptide, dehydrophos, for comparison. Thus, the effects of the aforementioned structural variations on antimicrobial activity have been studied.

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase.

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 µg/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 µg/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 µg/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis.

The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95-15.6 µM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 µM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development.

Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis.

The increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains and the very few drugs available for treatment are promoting the discovery and development of new molecules that could help in the control of this disease. Bacteriocin AS-48 is an antibacterial peptide produced by Enterococcus faecalis and is active against several Gram-positive bacteria. We have found that AS-48 was active against Mycobacterium tuberculosis, including H37Rv and other reference and clinical strains, and also against some nontuberculous clinical mycobacterial species. The combination of AS-48 with either lysozyme or ethambutol (commonly used in the treatment of drug-susceptible tuberculosis) increased the antituberculosis action of AS-48, showing a synergic interaction. Under these conditions, AS-48 exhibits a MIC close to some MICs of the first-line antituberculosis agents. The inhibitory activity of AS-48 and its synergistic combination with ethambutol were also observed on M. tuberculosis-infected macrophages. Finally, AS-48 did not show any cytotoxicity against THP-1, MHS, and J774.2 macrophage cell lines at concentrations close to its MIC. In summary, bacteriocin AS-48 has interesting antimycobacterial activity in vitro and low cytotoxicity, so further studies in vivo will contribute to its development as a potential additional drug for antituberculosis therapy.