Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

Supramolecular assemblies of rifampicin and cationic bilayers: preparation, characterization and micobactericidal activity

Background: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. Methods: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (zeta) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations. Results: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 mu g/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species. Conclusions: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Sulfonyl-hydrazones of cyclic imides derivatives as potent inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase B (PtpB)

Searching lead compounds for new antituberculosis drugs, the activity of synthetic sulfonamides and sulfonyl-hydrazones were assayed for their potential inhibitory activity towards a protein tyrosine phosphatase from Mycobacterium tuberculosis - PtpB. Four sulfonyl-hydrazones N-phenylmaleimide derivatives were active (compounds 14, 15, 19 and 21), and the inhibition of PtpB was found to be competitive with respect to the substrate p-nitrophenyl phosphate. Structure-based molecular docking simulations were performed and indicated that the new inhibitor candidates showed similar binding modes, filling the hydrophobic pocket of the protein by the establishment of van der Waals contacts, thereby contributing significantly to the complex stability.

Decrease in Mycobacterium tuberculosis specific immune responses in patients with untreated psoriasis living in a tuberculosis endemic area

Tuberculosis has emerged as a major concern in patients with immuno-mediated diseases, including psoriasis, undergoing treatment with biologicals. However, it is not known whether the chronically activated immune system of psoriasis patients interferes with their Mycobacterium tuberculosis (Mtb)-specific immunity, especially in tuberculosis-endemic areas like Brazil. We evaluated T-cell responses to a Mtb lysate and to the recombinant Mtb proteins ESAT-6 and Ag85B of tuberculin skin test (TST) positive and TST negative patients with severe or mild/moderate, untreated psoriasis in three different assays: lymphocyte proliferation, enzyme immunoassay for interferon (IFN)-gamma and interleukin (IL)-10 production by peripheral blood mononuclear cells and overnight enzyme immunospot (ELISpot) for enumerating IFN-gamma-secreting cells. In our cohort, a low proportion (29%) of the severe psoriasis patients tested were TST-positive. IFN-gamma and IL-10 secretion and T-cell proliferation to Mtb antigens were reduced in TST-negative but not in TST-positive patients with severe psoriasis when compared to healthy controls with the same TST status. Similarly, severe psoriasis patients had decreased cytokine secretion and proliferative response to phytohemagglutinin. However, most psoriasis patients and healthy controls showed detectable numbers of IFN-gamma-secreting effector-memory T-cells in response to Mtb antigens by ELISpot. TST-negative, mild/moderate psoriasis patients had responses that were mostly intermediary between TST-negative controls and severe psoriasis patients. Thus, patients with severe psoriasis possess decreased anti-Mtb central memory T-cell responses, which may lead to false-negative results in the diagnosis of TB infection, but retain T-cell memory-effector activity against Mtb antigens. We hypothesize that the latter may confer some protection against tuberculosis reactivation.

In vitro uptake and antimycobacterial activity of liposomal usnic acid formulation

The cellular uptake and antimycobacterial activity of usnic acid (UA) and usnic acid-loaded liposomes (UA-LIPOs) were assessed on J774 macrophages. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of UA and UA-LIPO against Mycobacterium tuberculosis were determined. Concentrations required to inhibit 50% of cell proliferation (IC(50)) were 22.5 (+/- 0.60) and 12.5 (+/- 0.26) mu g/ml, for UA and UA-LIPO, respectively. The MICs of UA and UA-LIPO were 6.5 and 5.8 mu g/mL, respectively. The MBC of UA-LIPO was twice as low (16 mu g/mL) as that of UA (32 mu g/mL). An improvement in the intracellular uptake of UA-LIPO was found (21.6 x 10(4) +/- 28.3 x 10(2) c.p.s), in comparison with UA (9.5 x 10(4) +/- 11.4 x 10(2) c.p.s). In addition, UA-LIPO remains much longer inside macrophages (30 hours). All data obtained from the encapsulation of usnic acid into liposomes as a drug delivery system (DDS) indicate a strong interaction between UA-liposomes and J774 macrophages, thereby facilitating UA penetration into cells. Considering such a process as ruling the Mycobacterium-transfection by magrophages, we could state that associating UA with this DDS leads to an improvement in its antimycobacterial activity.

Structure activity relationship, acute toxicity and cytotoxicity of antimycobacterial neolignan analogues

Objectives The study`s aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. Methods The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). Key findings Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). Conclusions LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.

Participação da apolipoproteína-E na atividade microbicida “in vitro” contra o Mycobacterium tuberculosis

A parede celular de Mycobacterium tuberculosis (Mtb) é constituída por 60% de lipídios, impedindo a passagem de uma grande quantidade de substâncias, além de desempenhar um importante papel na imunopatogênese. A apresentação desses antígenos aos linfócitos se dá por meio de moléculas do tipo CD1.Por sua vez a Apolipoproteína-E (ApoE), glicoproteína amplamente distribuída nos tecidos, pode facilitar a apresentação de lipídios pelo CD1. A ApoE possui três principais alelos ApoE- 2, 3 e 4, que codificam três isoformas de proteínas, tipos 2, 3 e 4, que possuem diferentes estruturas e funções. A presença de determinadas isoformas da ApoE está associada a doenças infecciosas, como herpes labial, dano hepático severo causado pelo vírus da hepatite C, diarréia infantil e tuberculose pulmonar. Neste contexto, avaliamos a participação da ApoE na atividade microbicida in vitro frente ao Mtb. Para tanto, foram arrolados 13 indivíduos PPD-, 17 indivíduos PPD+ e 4 indivíduos com tuberculose pulmonar ativa. O uso de plasma humano depletado de ApoE nos experimentos de atividade microbicida in vitro mostraram um aumento significante (p=0,02) no número de micobactérias (431.5 ± 81.92 UFC) quando comparado ao grupo controle (313.0 ± 74.61 UFC). Esses resultados foram confirmados por um modelo experimental utilizando esplenócitos de camundongos de camundongos C57BL/6 (815.9 ± 76.32 UFC) e animais APOE nocaute (1133 ± 86.85 UFC) (p = 0.021). Quanto à produção de IL-10, no grupo PPD+, observamos que o grupo com depleção de ApoE (866.7 ± 447.8) apresentou uma produção menor desta citocina com relação ao controle infectado (1089 ± 481.3) (p=0,023). Já em relação ao IFN-, em ambos os grupos observou-se, após 72 horas, uma tendência à diminuição da produção dessa citocina no grupo com depleção, com relação ao grupo controle. Esses dados sugerem que a ApoE tem papel distinto na ativação da resposta imune e sua ausência pode prejudicar a resposta imune frente à tuberculose

QSAR modeling of antitubercular activity of diverse organic compounds

Tuberculosis (TB) is a worldwide infectious disease that has shown over time extremely high mortality levels. The urgent need to develop new antitubercular drugs is due to the increasing rate of appearance of multi-drug resistant strains to the commonly used drugs, and the longer durations of therapy and recovery, particularly in immuno-compromised patients. The major goal of the present study is the exploration of data from different families of compounds through the use of a variety of machine learning techniques so that robust QSAR-based models can be developed to further guide in the quest for new potent anti-TB compounds. Eight QSAR models were built using various types of descriptors (from ADRIANA.Code and Dragon software) with two publicly available structurally diverse data sets, including recent data deposited in PubChem. QSAR methodologies used Random Forests and Associative Neural Networks. Predictions for the external evaluation sets obtained accuracies in the range of 0.76-0.88 (for active/inactive classifications) and Q(2)=0.66-0.89 for regressions. Models developed in this study can be used to estimate the anti-TB activity of drug candidates at early stages of drug development (C) 2011 Elsevier B.V. All rights reserved.

Design, synthesis and biologival evaluation of novel isoniazid derivatives with potente antitubercular activity

The disturbing emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) has been driving the scientific community to urgently search for new and efficient antitubercular drugs. Despite the various drugs currently under evaluation, isoniazid is still the key and most effective component in all multi-therapeutic regimens recommended by the WHO. This paper describes the QSAR-oriented design, synthesis and in vitro antitubercular activity of several potent isoniazid derivatives (isonicotinoyl hydrazones and isonicotinoyl hydrazides) against H37Rv and two resistant Mtb strains. QSAR studies entailed RFs and ASNNs classification models, as well as MLR models. Strict validation procedures were used to guarantee the models' robustness and predictive ability. Lipophilicity was shown not to be relevant to explain the activity of these derivatives, whereas shorter N-N distances and lengthy substituents lead to more active compounds. Compounds I, 2, 4, 5 and 6, showed measured activities against H37Rv higher than INH (i.e., MIC <= 0.28 mu M), while compound 9 exhibited a six fold decrease in MIC against the katG (S315T) mutated strain, by comparison with INH (Le., 6.9 vs. 43.8 mu M). All compounds were ineffective against H37Rv(INH) (Delta katG), a strain with a full deletion of the katG gene, thus corroborating the importance of KatG in the activation of INH-based compounds. The most potent compounds were also shown not to be cytotoxic up to a concentration 500 times higher than MIC. (C) 2014 Elsevier Masson SAS. All rights reserved.

Radiometric studies of mycobacterium tuberculosis

An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C) acetate, (U-14C) glycerol, (1-14C) palmitic acid, 1-14C) lauric acid, (U-14C) L-malic acid, (U-14C) D-glucose, and (U-14C) D-glucose, but not (1-14C) L-glucose, (U-14C) glycine, or (U-14C) pyruvate to 14CO2. By using either 14C-for-mate, (1-14C) palmitic acid, or (1-14C) lauric acid, 10(7) organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Modulatory activity of antioxidants against the toxicity of Rifampicin in vivo

The World Health Organization (WHO) has shown concern about the burden of tuberculosis in the developing countries. Even though rifampicin is an effective drug in the management of tuberculosis, it has been documented to have some toxic effects in humans. Therefore, this study intends to investigate the modulatory effect of vitamins C and E on the hepatotoxicity, sperm quality and brain toxicity of Rifampicin. Forty Wistar albino rats were used, 10 animals per group. Group 1 animals received 0.3 mL of distilled water, the Group 2 animals received the therapeutic dose of rifampicin, Group 3 animals received therapeutic doses of rifampicin plus vitamin E, while Group 4 received therapeutic doses of rifampicin and vitamin C. The administration was performed orally during three months; the animals were sacrificed by cervical dislocation at the end of that period. Blood samples were collected and liver function and lipid profile was analyzed using fully automated clinical chemistry device. The liver, brain and reproductive organs underwent histopathological examination. Sperm samples were collected from the epididymis to achieve count and motility and morphological analysis. Results showed rifampicin alone to raise (p < 0.05) liver function enzymes (Aspartate amino transferase [AST], Serum alanine amino transferase [ALT] and Total Bilirubin) when compared with controls. While the vitamin E treated group showed remarkable protection, the vitamin C treated group showed questionable protection against the rifampicin induced liver damage. Sperm count results showed an important (p < 0.05) increase in the sperm quality in vitamin E and C treated groups. However, the vitamin E plus Rifampicin treated group showed increased lipid peroxidation. The histopathological findings revealed structural damages by rifampicin in liver, brain and epididymis while some remarkable architectural integrity was observed in the antioxidant-treated groups. It can be concluded that vitamin E or C improved sperm quality and protected against the brain damage caused by rifampicin. Moreover, vitamin E demonstrated remarkable hepatoprotection against rifampicin induced damage while vitamin C shows a questionable hepatoprotection.

Myeloid Sirtuin 2 expression does not impact long-term Mycobacterium tuberculosis control

Sirtuins (Sirts) regulate several cellular mechanisms through deacetylation of several transcription factors and enzymes. Recently, Sirt2 was shown to prevent the development of inflammatory processes and its expression favors acute Listeria monocytogenes infection. The impact of this molecule in the context of chronic infections remains unknown. We found that specific Sirt2 deletion in the myeloid lineage transiently increased Mycobacterium tuberculosis load in the lungs and liver of conditional mice. Sirt2 did not affect long-term infection since no significant differences were observed in the bacterial burden at days 60 and 120 post-infection. The initial increase in M. tuberculosis growth was not due to differences in inflammatory cell infiltrates in the lung, myeloid or CD4+ T cells. The transcription levels of IFN-?, IL-17, TNF, IL-6 and NOS2 were also not affected in the lungs by Sirt2-myeloid specific deletion. Overall, our results demonstrate that Sirt2 expression has a transitory effect in M. tuberculosis infection. Thus, modulation of Sirt2 activity in vivo is not expected to affect chronic infection with M. tuberculosis.

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.