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.

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.

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.

Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.

There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

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.

Total Synthesis of Ripostatin B and Structure-Activity Relationship Studies on Ripostatin Analogs.

Described is the total synthesis of the myxobacterial natural product ripostatin B and of a small number of analogs. Ripostatin B is a polyketide-derived 14-membered macrolide that acts as an inhibitor of bacterial RNA-polymerase, but is mechanistically distinct from rifamycin-derived RNA-polymerase inhibitors that are in use for tuberculosis treatment. The macrolactone ring of ripostatin B features two stereocenters and a synthetically challenging doubly skipped triene motif, with one of the double bonds being in conjugation with the ester carbonyl. Appended to the macrolactone core are an extended hydroxy-bearing phenylalkyl side chain at C13 and a carboxymethyl group at C3. The triene motif was established with high efficiency by ring-closing olefin metathesis, which proceeded in almost 80% yield. The side chain-bearing stereocenter α to the ester oxygen was formed in a Paterson aldol reaction between a methyl ketone and a ß-chiral ß-hydroxy aldehyde with excellent syn selectivity (dr >10:1). The total synthesis provided a blueprint for the synthesis of analogs with modifications in the C3 and C13 side chains. The C3-modified analogs showed good antibacterial activity against efflux-deficient Escherichia coli but, as ripostatin B, were inactive against Mycobacterium tuberculosis, in spite of significant in vitro inhibition of M. tuberculosis RNA-polymerase.

Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.

The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.

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.

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.

Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis.

Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.

Analysis of mutations in streptomycin-resistant strains reveals a simple and reliable genetic marker for identification of the Mycobacterium tuberculosis Beijing genotype.

The Mycobacterium tuberculosis pandemic is a major health problem, further complicated by an increasing incidence of drug-resistant isolates and the existence of highly transmissible strains, such as those in the Beijing family. Streptomycin (STR)-resistant M. tuberculosis clinical isolates have been analyzed to look for mutations in the rpsL, rrs, and gidB genes. In addition, the Rv1258c gene, which encodes Tap, an efflux pump that transports STR, has been sequenced. Mutations affecting codons 43 and 88 of the rpsL gene were found in 44.4% of the strains, and 16.7% of the strains carried mutations in the rrs gene, both of which probably contribute to STR resistance. Many strains presented with mutations in the gidB gene, but the implication of those mutations in STR resistance remains unclear. Interestingly, a cytosine nucleotide insertion between positions 580 and 581 (denominated Tap(580)) in the Rv1258c gene has been found in all Beijing isolates included in this study, suggesting that it might be a novel polymorphism specific to the Beijing family of M. tuberculosis. A simple and fast restriction fragment length polymorphism (RFLP)-PCR method for detecting the Tap(580) insertion has been developed and used to screen a collection of 220 DNA samples obtained from cultures of M. tuberculosis isolates and 30 respiratory specimens. In all cases, the Beijing and non-Beijing representative samples were identified correctly. Tap(580) is a novel polymorphism specific to the highly transmissible Beijing family, which allows for fast detection of these strains even at the very early stages of infection.

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.

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.

Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.

Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that disruption of the Rv1258c gene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression of tap altered susceptibility to many clinically approved antibiotics in M. bovis BCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanide m-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.

Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages.

Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.

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.

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.

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.

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.

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.