Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate.

Design of Novel Inhibitors of Human Thymidine Phosphorylase: Synthesis, Enzyme Inhibition, in Vitro Toxicity, and Impact on Human Glioblastoma Cancer.

Overexpressed human thymidine phosphorylase (hTP) has been associated with cancer aggressiveness and poor prognosis by triggering proangiogenic and antiapoptotic signaling. Designed as transition-state analogues by mimicking the oxacarbenium ion, novel pyrimidine-2,4-diones were synthesized and evaluated as inhibitors of hTP activity. The most potent compound (8g) inhibited hTP in the submicromolar range with a noncompetitive inhibition mode with both thymidine and inorganic phosphate substrates. Furthermore, compound 8g was devoid of apparent toxicity to a panel of mammalian cells, showed no genotoxicity signals, and had low probability of drug-drug interactions and moderate in vitro metabolic rates. Finally, treatment with 8g (50 mg/(kg day)) for 2 weeks (5 days/week) significantly reduced tumor growth using an in vivo glioblastoma model. To the best of our knowledge, this active compound is the most potent in vitro hTP inhibitor with a kinetic profile that cannot be reversed by the accumulation of any enzyme substrates.

Assessing the role of deoD gene in Mycobacterium tuberculosis in vitro growth and macrophage infection.

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP), encoded by deoD gene (Rv3307), is an enzyme from the purine salvage pathway, which has been widely studied as a molecular target for the development of inhibitors with potential antimycobacterial activity. However, the role of MtPNP in tuberculosis pathogenesis and dormancy is still unknown. The present work aims to construct a deoD knockout strain from M. tuberculosis, to evaluate the role of MtPNP in the growth of M. tuberculosis under oxygenated condition and in a dormancy model, and to assess whether deoD gene is important for M. tuberculosis invasion and growth in macrophages. The construction of a knockout strain for deoD gene was confirmed at DNA level by PCR and protein level by Western blot and LC-MS/MS. The deoD gene is not required for M. tuberculosis growth and survival under oxygenated and hypoxic conditions. The disruption of deoD gene did not affect mycobacterial ability to invade and grow in RAW 264.7 cells under the experimental conditions employed here.

Effect of the bradykinin 1 receptor antagonist SSR240612 after oral administration in Mycobacterium tuberculosis-infected mice.

The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B1R-/-, B2R-/- and double B1R/B2R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B1R and B2R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B2R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B1R/B2R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B1R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg9-BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B1R (des-Arg9-BK and SSR240612) and B2R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

New insights into the SAR and drug combination synergy of 2-(quinolin-4-yloxy)acetamides against Mycobacterium tuberculosis.

2-(Quinolin-4-yloxy)acetamides have been described as potent and selective in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Herein, a new series of optimized compounds were found to demonstrate highly potent antitubercular activity, with minimum inhibitory concentration (MIC) values against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains in the submicromolar range. Furthermore, the most active compounds had no apparent toxicity to mammalian cells, and they showed intracellular activities similar to those of isoniazid and rifampin in a macrophage model of Mtb infection. Use of the checkerboard method to investigate the association profiles of lead compounds with first- and second-line antituberculosis drugs showed that 2-(quinolin-4-yloxy)acetamides have a synergistic effect with rifampin. Ultimately, the good permeability, moderate rates of metabolism and low risk of drug-drug interactions displayed by some of the synthesized compounds indicate that 2-(quinolin-4-yloxy)acetamides may yield candidates to use in the development of novel alternative therapeutics for tuberculosis treatment.

Protective effects of resveratrol on hepatotoxicity induced by isoniazid and rifampicin via SIRT1 modulation.

Acute liver injury was induced in male BALB/c mice by coadministering isoniazid and rifampicin. In this work, the effects of resveratrol (1) were investigated in the hepatotoxicity caused by isoniazid-rifampicin in mice. Compound 1 was administered 30 min prior to isoniazid-rifampicin. Serum biochemical tests, liver histopathological examination, oxidative stress, myeloperoxidase activity, cytokine production (TNF-α, IL-12p70, and IL-10), and mRNA expression of SIRT1-7 and PPAR-γ/PGC1-α were evaluated. The administration of 1 significantly decreased aspartate transaminase and alanine aminotransferase levels, myeloperoxidase activity, and cytokine levels. Furthermore, 1 reverted the decrease of catalase and glutathione activities and ameliorated the histopathological alterations associated with antituberculosis drugs. Modulation of SIRT1 and PPAR-γ/PGC1-α expression is likely involved in the protective effects of 1. The results presented herein show that 1 was able to largely prevent the hepatotoxicity induced by isoniazid and rifampicin in mice, mainly by modulating SIRT1 mRNA expression.

IQG-607 abrogates the synthesis of mycolic acids and displays intracellular activity against Mycobacterium tuberculosis in infected macrophages.

In this work, the antitubercular activity of a pentacyano(isoniazid)ferrate(II) compound (IQG-607) was investigated using a macrophage model of Mycobacterium tuberculosis infection. Importantly, treatment of M.-tuberculosis-infected macrophages with IQG-607 significantly diminished the number of CFU compared with the untreated control group. The antitubercular activity of IQG-607 was similar to that observed for the positive control drugs isoniazid and rifampicin. Nevertheless, higher concentrations of IQG-607 produced a significantly greater reduction in bacterial load compared with the same concentrations of isoniazid. Analysis of the mechanism of action of IQG-607 revealed that the biosynthesis of mycolic acids was blocked. The promising activity of IQG-607 in infected macrophages and the experimental determination of its mechanism of action may help in further studies aimed at the development of a new antimycobacterial agent.

Implication of purinergic P2X7 receptor in M. tuberculosis infection and host interaction mechanisms: a mouse model study.

In the present study, we analyzed the role of purinergic P2X7 receptor in Mycobacterium tuberculosis infection and host interaction mechanisms in vitro and in vivo. For experimental procedures, a macrophage murine cell line RAW 264.7, and male Swiss, wild-type C57BL/6 and P2X7 receptor knockout (P2X7R−/−) mice were used throughout this study. We have demonstrated that treatment of RAW 264.7 cells with ATP (3 and 5 mM) resulted in a statistically significant reduction of M. tuberculosis-colony-forming units. The purinergic P2X7 receptor expression was found significantly augmented in the lungs of mice infected with M. tuberculosis H37Rv. Infected wild-type mice showed a marked increase in the spleen weight, in comparison to non-infected animals. Furthermore, M. tuberculosis-infected P2X7R−/− mice showed an increase of M. tuberculosis burden in lung tissue, when compared to infected wild-type mice. In P2X7R−/− spleens, we observed a significant decrease in the populations of Treg (CD4+Foxp3+), T cells (CD4+, CD8+CD25+ and CD4+CD25+), dendritic cells (CD11c+) and B220+ cells. However, a significant increase in CD11b+ cells was observed in P2X7R−/− mice, when compared to wild-type animals. In the lungs, P2X7R−/− M. tuberculosisinfected mice exhibited pulmonary infiltrates containing an increase of Treg cells (CD4+Foxp3+), T cells (CD4+ and CD8+) and a decrease in the B220+ cells, when compared with wild-type M. tuberculosis-infected mice. The findings observed in the present study provide novel evidence on the role of P2X7 receptors in the pathogenesis of tuberculosis.

Cysteamine prevents inhibition of thiol-containing enzymes caused by cystine or cystine dimethylester loading in rat brain cortex.

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Considering that thiol-containing enzymes are critical for several metabolic pathways, our main objective was to investigate the effects of cystine or cystine dimethylester load on the thiol-containing enzymes creatine kinase and pyruvate kinase, in the brain cortex of young Wistar rats. The animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester or 1 micromol/g body weight of cystine and/or 0.46 micromol/g body weight of cysteamine from the 16th to the 20th postpartum day and sacrificed after 12 h. Cystine or cystine dimethylester administration inhibited the two enzyme activities. Co-administration of cysteamine, the drug used to treat cystinotic patients, normalized the two enzyme activities. Lactate dehydrogenase activity, a nonthiol-containing enzyme was not affected by cystine dimethylester administration. Cystine inhibits creatine kinase and pyruvate activities possibly by oxidation of the sulfhydryl groups of the enzymes. Considering that creatine kinase and pyruvate kinase, like other thiol-containing enzymes, are crucial for energy homeostasis and antioxidant defenses, the enzymes inhibition caused by cystine released from lysosomes could be one of the mechanisms of tissue damage in patients with cystinosis.

Cystine inhibits creatine kinase activity in pig retina.

BACKGROUND: Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. METHODS: Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. RESULTS: Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. CONCLUSIONS: Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Inhibition of creatine kinase activity by cystine in the kidney of young rats.

Nephropathic cystinosis is a lethal genetic disease caused by a lysosomal transport disorder leading to intralysosomal cystine accumulation in all tissues. Cystinosis is the most common inherited cause of Fanconi syndrome, but the mechanisms by which cystine causes tissue damage are not fully understood. Thiol-containing enzymes are critical for renal energy metabolism and may be altered by disulfides like cystine. Therefore, in the present study our main objective was to investigate the in vivo and in vitro effects of cystine on creatine kinase, which contains critical thiol groups in its structure, in the kidney of young Wistar rats. We observed that cystine inhibited in vivo and in vitro the enzyme activity and that this inhibition was prevented by cysteamine and glutathione. The results suggest oxidation of essential sulfhydryl groups necessary for creatine kinase function by cystine. Considering that creatine kinase and other thiol-containing enzymes are crucial for renal energy metabolism, and programmed cell death occurs in situations of energy deficiency, the enzyme inhibition caused by cystine released from lysosomes might be a mechanism of tissue damage in patients with cystinosis.

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex.

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.

Frequência de ovos de Toxocara spp. em três parques públicos da cidade de Porto Alegre, Rio Grande do Sul, Brasil / Frequency of Toxocara spp in three public parks of Porto Alegre city-Brazil

Com o objetivo de realizar um monitoramento mensal para verificar a presença e a frequência de ovos de Toxacara spp, foram coletadas, entre fevereiro de 2002 e janeiro de 2003, 216 amostras de areia em 18 caixas, de praças de recreação localizadas em três parques públicos da cidade de Porto Alegre, Rio Grande do Sul, Brasil. As amostras foram processadas em laboratório pela técnica de Faust modificada, sendo que os resultados mostraram ema frequência de ovos de Toxocara spp em 77,7 por cento das caixas examinadas. Esses dados indicam a necessidade da implantação de medidas sanitárias e educativas visando à prevenção do risco de contaminação da população, especialmente a infantil, por parasitos causadores da larva migrans visceral.