Publications by authors named "Luiz Augusto Basso"

84 Publications

Application of cellulosic materials as supports for single-step purification and immobilization of a recombinant β-galactosidase via cellulose-binding domain.

Int J Biol Macromol 2022 Jan 7;199:307-317. Epub 2022 Jan 7.

Laboratório de Biotecnologia de Alimentos, Brazil; Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil. Electronic address:

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of β-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/g, expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na, K, Mg, and Ca), magnesium provided the highest increase in the enzymatic activity of β-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a β-galactosidase on cellulose via CBD.
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http://dx.doi.org/10.1016/j.ijbiomac.2022.01.006DOI Listing
January 2022

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

Microbiol Spectr 2021 12 22;9(3):e0000921. Epub 2021 Dec 22.

Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Partenon, Porto Alegre, Brazil.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The -encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate gene essentiality and vulnerability of its protein product, EPSPS, from () (EPSPS). We demonstrate that -deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for -knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected EPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (/) of recombinant wild-type (WT) and mutated versions of EPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, from M. smegmatis is essential, its essentiality is dependent on EPSPS activity, and EPSPS is vulnerable. We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.
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http://dx.doi.org/10.1128/Spectrum.00009-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8694188PMC
December 2021

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability.

Bioresour Technol 2022 Feb 6;345:126497. Epub 2021 Dec 6.

Laboratório de Biotecnologia de Alimentos, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil; Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil. Electronic address:

For the first time, this work reported the one-step purification and targeted immobilization process of a β-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after β-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between β-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. β-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of β-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.
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http://dx.doi.org/10.1016/j.biortech.2021.126497DOI Listing
February 2022

Synthesis and Antimycobacterial Activity of 3-Phenyl-1-indoles.

Molecules 2021 Aug 25;26(17). Epub 2021 Aug 25.

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681-Prédio 92A, Porto Alegre 90616-900, RS, Brazil.

Tuberculosis has been described as a global health crisis since the 1990s, with an estimated 1.4 million deaths in the last year. Herein, a series of 20 1-indoles were synthesized and evaluated as in vitro inhibitors of (Mtb) growth. Furthermore, the top hit compounds were active against multidrug-resistant strains, without cross-resistance with first-line drugs. Exposing HepG2 and Vero cells to the molecules for 72 h showed that one of the evaluated structures was devoid of apparent toxicity. In addition, this 3-phenyl-1-indole showed no genotoxicity signals. Finally, time-kill and pharmacodynamic model analyses demonstrated that this compound has bactericidal activity at concentrations close to the Minimum Inhibitory Concentration, coupled with a strong time-dependent behavior. To the best of our knowledge, this study describes the activity of 3-phenyl-1-indole against Mtb for the first time.
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http://dx.doi.org/10.3390/molecules26175148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433792PMC
August 2021

Effects of tafenoquine against active, dormant and resistant Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2021 05 13;128:102089. Epub 2021 May 13.

Programa de Pós-Graduação em Biotecnologia, Centro de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, Brazil; Laboratório de Biotecnologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, Brazil; Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Brazil. Electronic address:

Antimalarial drugs have been suggested as promising scaffolds with anti-tubercular activities. In this work, we demonstrated, for the first time, the effectiveness of tafenoquine against mycobacteria. Firstly, tafenoquine inhibited the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis with lower MICs values as compared to other antimalarial drugs, such as mefloquine, chloroquine, and primaquine. Importantly, tafenoquine was active against three multi-drug resistant strains of M. tuberculosis with MIC values similar to pan-sensitive strains, suggesting that tafenoquine is capable of evading the major mechanisms of resistance found in drug-resistant clinical isolates of M. tuberculosis. Importantly, tafenoquine displayed a synergistic effect when combined with mefloquine. In addition, tafenoquine displayed an improved activity compared to the groups treated with both isoniazid and rifampicin in the six-week nutrient starved M. tuberculosis cultures. This finding suggests that further investigations of tafenoquine against dormant mycobacteria are worth pursuing. Moreover, different concentrations of tafenoquine ranging from 1.25 to 80 μM displayed different effects against M. tuberculosis, from moderate (reduction of a 1.8 log CFU/mL) to potent bactericidal (reduction of a 4.2 log CFU/mL) activities. Tafenoquine may represent a hit for further drug optimization and for future clinical development as a new anti-mycobacterial agent, especially in cases of resistant and/or dormant forms of tuberculosis.
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http://dx.doi.org/10.1016/j.tube.2021.102089DOI Listing
May 2021

Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Biomed Pharmacother 2021 Jul 6;139:111672. Epub 2021 May 6.

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900 Porto Alegre, RS, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biopha.2021.111672DOI Listing
July 2021

8-Mercaptoguanine-based inhibitors of dihydroneopterin aldolase: synthesis, inhibition and docking studies.

J Enzyme Inhib Med Chem 2021 Dec;36(1):847-855

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from (FolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against FolB. The compounds showed IC values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of FolB inhibitors.
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http://dx.doi.org/10.1080/14756366.2021.1900157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993393PMC
December 2021

Ultrasound-Assisted Synthesis of 4-Alkoxy-2-methylquinolines: An Efficient Method toward Antitubercular Drug Candidates.

Molecules 2021 Feb 24;26(5). Epub 2021 Feb 24.

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900 Porto Alegre, Rio Grande do Sul, Brazil.

Tuberculosis (TB) has been described as a global health crisis since the second half of the 1990s. (Mtb), the etiologic agent of TB in humans, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2019, it was estimated that around 10 million individuals were contaminated by this bacillus and about 1.2 million succumbed to the disease. In recent years, our research group has reported the design and synthesis of quinoline derivatives as drug candidates for the treatment of TB. These compounds have demonstrated potent and selective growth inhibition of drug-susceptible and drug-resistant Mtb strains. Herein, a new synthetic approach was established providing efficient and rapid access (15 min) to a series of 4-alkoxy-6-methoxy-2-methylquinolines using ultrasound energy. The new synthetic protocol provides a simple procedure utilizing an open vessel system that affords the target products at satisfactory yields (45-84%) and elevated purities (≥95%). The methodology allows the evaluation of a larger number of molecules in assays against the bacillus, facilitating the determination of the structure-activity relationship with a reduced environmental cost.
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http://dx.doi.org/10.3390/molecules26051215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956363PMC
February 2021

Therapeutic effect of uridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo.

Eur J Pharmacol 2021 Jan 22;890:173670. Epub 2020 Oct 22.

Laboratório de Pesquisa Em Biofísica Celular e Inflamação, Pontifícia Universidade Católica Do Rio Grande Do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, 90619-900, Brazil.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis.
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http://dx.doi.org/10.1016/j.ejphar.2020.173670DOI Listing
January 2021

Anti-tubercular profile of new selenium-menadione conjugates against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain and multidrug-resistant clinical isolates.

Eur J Med Chem 2021 Jan 23;209:112859. Epub 2020 Sep 23.

Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de Química, Campus Do Valonguinho, CEP 24020-150, Niterói, RJ, Brazil. Electronic address:

Tuberculosis (TB) is one of the most fatal diseases and is responsible for the infection of millions of people around the world. Most recently, scientific frontiers have been engaged to develop new drugs that can overcome drug-resistant TB. Following this direction, using a designed scaffold based on the combination of two separate pharmacophoric groups, a series of menadione-derived selenoesters was developed with good yields. All products were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv and attractive results were observed, especially for the compounds 8a, 8c and 8f (MICs 2.1, 8.0 and 8.1 μM, respectively). In addition, 8a, 8c and 8f demonstrated potent in vitro activity against multidrug-resistant clinical isolates (CDCT-16 and CDCT-27) with promising MIC values ranging from 0.8 to 3.1 μM. Importantly, compounds 8a and 8c were found to be non-toxic against the Vero cell line. The SI value of 8a (>23.8) was found to be comparable to that of isoniazid (>22.7), which suggests the possibility of carrying out advanced studies on this derivative. Therefore, these menadione-derived selenoesters obtained as hybrid compounds represent promising new anti-tubercular agents to overcome TB multidrug resistance.
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http://dx.doi.org/10.1016/j.ejmech.2020.112859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510590PMC
January 2021

CPBMF65, a synthetic human uridine phosphorylase-1 inhibitor, reduces HepG2 cell proliferation through cell cycle arrest and senescence.

Invest New Drugs 2020 12 4;38(6):1653-1663. Epub 2020 May 4.

Laboratório de Pesquisa em Biofísica Celular e Inflamação, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.
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http://dx.doi.org/10.1007/s10637-020-00941-2DOI Listing
December 2020

Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents.

Eur J Med Chem 2020 Apr 21;192:112179. Epub 2020 Feb 21.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address:

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure-activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 μM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.
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http://dx.doi.org/10.1016/j.ejmech.2020.112179DOI Listing
April 2020

Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction.

Enzyme Microb Technol 2020 Mar 12;134:109468. Epub 2019 Nov 12.

Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, PO Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil. Electronic address:

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109468DOI Listing
March 2020

Resistance Reversed in KatG Mutants of Mycobacterium tuberculosis.

Trends Microbiol 2019 08 6;27(8):655-656. Epub 2019 Jun 6.

Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil; Escola de Ciências, PUCRS, Rio Grande do Sul, Brazil. Electronic address:

A peptidomimetic containing a thiazolo ring-fused 2-pyridone (C10) has now been reported to inhibit hypoxia-induced tolerance to isoniazid (INH) in Mycobacterium tuberculosis (Flentie et al., Proc. Natl. Acad. Sci. U. S. A., 2019). The C10 compound could also potentiate the bactericidal activity in aerobically grown bacilli, prevented selection of drug-resistant strains, and reversed INH resistance in katG (catalase-peroxidase) mutants.
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http://dx.doi.org/10.1016/j.tim.2019.05.008DOI Listing
August 2019

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

J Med Chem 2019 02 17;62(3):1231-1245. Epub 2019 Jan 17.

Laboratório de Produtos Naturais e Sintéticos, Instituto de Biotecnologia , Universidade de Caxias do Sul , 95070-560 Caxias do Sul , RS , Brazil.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01305DOI Listing
February 2019

1H-Benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones: Design, synthesis and antitubercular activity.

Eur J Med Chem 2018 Jul 2;155:153-164. Epub 2018 Jun 2.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address:

Using a classical hybridization approach, a series of 1H-benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones were synthesized (39 examples) and evaluated as inhibitors of Mycobacterium tuberculosis growth. Chemical modification studies yielded potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.24 μM against M. tuberculosis H37Rv strain. Further, the synthesized compounds were active against four drug-resistant strains containing different levels of resistance for the first line drugs. These molecules were devoid of apparent toxicity to HepG2, HaCat, and Vero cells with IC > 30 μM. Viability in mammalian cell cultures was evaluated using MTT and neutral red assays. In addition, some 3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or morphological alteration in zebrafish (Danio rerio) toxicity models. 3,4-Dihydroquinazolin-4-ones 9q and 9w were considered the lead compounds of these series of molecules with MIC values of 0.24 μM and 0.94 μM against M. tuberculosis H37Rv, respectively. Taken together, these data indicate that this class of compounds may furnish candidates for future development of novel anti-TB drugs.
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http://dx.doi.org/10.1016/j.ejmech.2018.06.005DOI Listing
July 2018

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

Microb Pathog 2018 Jun 30;119:60-64. Epub 2018 Mar 30.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto de Pesquisas Biomédicas, Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, Brazil.

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.
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http://dx.doi.org/10.1016/j.micpath.2018.03.056DOI Listing
June 2018

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

J Inorg Biochem 2018 02 21;179:71-81. Epub 2017 Nov 21.

CNRS, LCC (Laboratoire de Chimie de Coordination), 205, route de Narbonne, BP 44099, F-31077 Toulouse, Cedex 4, France; Université de Toulouse, UPS, INPT, F-31077 Toulouse, Cedex 4, France. Electronic address:

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by HO and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using HO with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.
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http://dx.doi.org/10.1016/j.jinorgbio.2017.11.013DOI Listing
February 2018

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

Mem Inst Oswaldo Cruz 2017 Nov;112(11):785-789

Pontifícia Universidade Católica do Rio Grande do Sul, Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Porto Alegre, RS, Brasil.

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.
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http://dx.doi.org/10.1590/0074-02760170105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661903PMC
November 2017

Activity of 2-(quinolin-4-yloxy)acetamides in Mycobacterium tuberculosis clinical isolates and identification of their molecular target by whole-genome sequencing.

Int J Antimicrob Agents 2018 Mar 23;51(3):378-384. Epub 2017 Aug 23.

Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Av. Ipiranga 6681 - Prédio 92A Tecnopuc, Porto Alegre, RS 90619-900, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, Brazil.

The 2-(quinolin-4-yloxy)acetamides (QOAs) have been reported to be promising molecules for tuberculosis treatment. Recent studies demonstrated their potent antimycobacterial activity, biological stability and synergism with rifampicin. The identification of the molecular target is an essential step towards the development of a novel drug candidate. Here, we report the target identification of the QOAs. We found that these compounds are active against Mycobacterium tuberculosis clinical isolates resistant to isoniazid, rifampicin, ethambutol, streptomycin and ethionamide. The initial evidence that DNA gyrase might be the target of QOAs, based on high minimum inhibitory concentration (MIC) values against ofloxacin-resistant clinical isolates and structural similarities with fluoroquinolones, was discarded by experiments performed with M. tuberculosis GyrA point mutant, DNA gyrase supercoiling inhibition assay and overexpression of DNA gyrase. We selected spontaneous mutants for our lead compound 21 and observed that these strains were also resistant to all QOA derivatives. The genomes of the spontaneous mutants were sequenced, and the results revealed a single mutation in qcrB gene (T313A), which indicates that the QOAs target the cytochrome bc complex. The protein-compound interaction was further investigated by molecular docking. These findings reinforce the relevance of these compounds as promising candidates for the treatment of multidrug-resistant tuberculosis.
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http://dx.doi.org/10.1016/j.ijantimicag.2017.08.023DOI Listing
March 2018

Toxicological profile of IQG-607 after single and repeated oral administration in minipigs: An essential step towards phase I clinical trial.

Regul Toxicol Pharmacol 2017 Nov 23;90:78-86. Epub 2017 Aug 23.

Centro de Pesquisas Em Biologia Molecular e Funcional (CPBMF) and Instituto Nacional de Ciência e Tecnologia Em Tuberculose (INCT-TB), Pontifícia Universidade Católica Do Rio Grande Do Sul (PUCRS), Av. Ipiranga 6681 - Prédio 92A Tecnopuc, 90619-900 Porto Alegre, Brazil; Programa de Pós-Graduação Em Biologia Celular e Molecular, PUCRS, Porto Alegre, Brazil.

IQG-607 is an anti-tuberculosis drug candidate, with a promising safety and efficacy profile in models of tuberculosis infection both in vitro and in vivo. Here, we evaluated the safety and the possible toxic effects of IQG-607 after acute and 90-day repeated administrations in minipigs. Single oral administration of IQG-607 (220 mg/kg) to female and male minipigs did not result in any morbidity or mortality. No gross lesions were observed in the minipigs at necropsy. Repeated administration of IQG 607 (65, 30, or 15 mg/kg), given orally, for 90 days, in both male and female animals did not cause any mortality and no significant body mass alteration. Diarrhea and alopecia were the clinical signs observed in animals dosed with IQG-607 for 90 days. Long-term treatment with IQG-607 did not induce evident alterations of blood cell counts or any hematological parameters. Importantly, the repeated schedule of administration of IQG-607 resulted in increased cholesterol levels, increased glucose levels, decrease in the globulin levels, and increased creatinine levels over the time. Most necropsy and histopathological alterations of the organs from IQG-607-treated groups were also observed for the untreated group. In addition, pharmacokinetic parameters were evaluated. IQG-607 represents a potential candidate molecule for anti-tuberculosis drug development programs. Its promising in vivo activity and mild to moderate toxic events detected in this study suggest that IQG-607 represents a candidate for clinical development.
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http://dx.doi.org/10.1016/j.yrtph.2017.08.015DOI Listing
November 2017

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis.

Mem Inst Oswaldo Cruz 2017 Mar;112(3):203-208

Pontifícia Universidade Católica do Rio Grande do Sul, Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Porto Alegre, RS, Brasil.

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.
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http://dx.doi.org/10.1590/0074-02760160462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319374PMC
March 2017

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

FEMS Microbiol Lett 2017 02;364(4)

National Institute of Science and Technology in Tuberculosis, Research Center in Molecular and Functional Biology, Pontifical Catholic University of Rio Grande do Sul-PUCRS, CEP 90619-900, Porto Alegre, RS, Brazil.

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.
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http://dx.doi.org/10.1093/femsle/fnx023DOI Listing
February 2017

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

Eur J Med Chem 2017 Jan 23;126:491-501. Epub 2016 Nov 23.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil; Programa de Pós-graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejmech.2016.11.048DOI Listing
January 2017

Mefloquine and its oxazolidine derivative compound are active against drug-resistant Mycobacterium tuberculosis strains and in a murine model of tuberculosis infection.

Int J Antimicrob Agents 2016 Aug 7;48(2):203-7. Epub 2016 Jun 7.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF) and Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681 - Prédio 92A Tecnopuc, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, Brazil. Electronic address:

Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5-8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.
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http://dx.doi.org/10.1016/j.ijantimicag.2016.04.029DOI Listing
August 2016

2-(Quinolin-4-yloxy)acetamides Are Active against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains.

ACS Med Chem Lett 2016 Mar 11;7(3):235-9. Epub 2016 Jan 11.

Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil.

2-(Quinolin-4-yloxy)acetamides have been described as potent in vitro inhibitors of Mycobacterium tuberculosis growth. Herein, additional chemical modifications of lead compounds were carried out, yielding highly potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.05 μM. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to Vero and HaCat cells (IC50s ≥ 20 μM). In addition, the 2-(quinolin-4-yloxy)acetamides showed intracellular activity against the bacilli in infected macrophages with action similar to rifampin, low risk of drug-drug interactions, and no sign of cardiac toxicity in zebrafish (Danio rerio) at 1 and 5 μM. Therefore, these data indicate that this class of compounds may furnish candidates for future development to, hopefully, provide drug alternatives for tuberculosis treatment.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789679PMC
March 2016

Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for growth.

Biochem Biophys Rep 2015 Dec 8;4:277-282. Epub 2015 Oct 8.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil.

Guanosine monophosphate synthetase (GMPS), encoded by gene, is a key enzyme for guanine nucleotide biosynthesis in . The gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct deletion in has been described so far. Here, we demonstrated that the gene is essential for H37Rv growth. The lethal phenotype of gene disruption was avoided by insertion of a copy of the ortholog gene from indicating that this GMPS protein is functional in . Protein validation of the essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from and GMPS proteins. These results validate GMPS as a molecular target for drug design against , and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.
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http://dx.doi.org/10.1016/j.bbrep.2015.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669397PMC
December 2015

Piperazine derivatives: synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies.

Eur J Med Chem 2015 Jan 18;90:436-47. Epub 2014 Nov 18.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil; Programa de Pós-graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address:

The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates. MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis system, and is the bona fide target for isoniazid, the most prescribed drug for tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and screened as MtInhA inhibitors, which resulted in the identification of compounds with IC50 values in the submicromolar range. A structure-activity relationship (SAR) evaluation indicated the importance of the chemical environment surrounding the carbonyl group for inhibition. In addition, the structure of one selected compound was supported by crystallographic studies, and experimental geometrical values were compared with semi-empirical quantum chemical calculations. Furthermore, the mode of inhibition and inhibitory dissociation constants were determined for the nine most active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-CoA) substrate binding site.
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http://dx.doi.org/10.1016/j.ejmech.2014.11.034DOI Listing
January 2015

[Fe(CN)5(isoniazid)](3-): an iron isoniazid complex with redox behavior implicated in tuberculosis therapy.

J Inorg Biochem 2014 Nov 12;140:236-44. Epub 2014 Aug 12.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom. Electronic address:

Tuberculosis has re-emerged as a worldwide threat, which has motivated the development of new drugs. The antituberculosis complex Na3[Fe(CN)5(isoniazid)] (IQG607) in particular is of interest on account of its ability to overcome resistance. IQG607 has the potential for redox-mediated-activation, in which an acylpyridine (isonicotinoyl) radical could be generated without assistance from the mycobacterial KatG enzyme. Here, we have investigated the reactivity of IQG607 toward hydrogen peroxide and superoxide, well-known intracellular oxidizing agents that could play a key role in the redox-mediated-activation of this compound. HPLC, NMR and electronic spectroscopy studies showed a very fast oxidation rate for bound isoniazid, over 460-fold faster than free isoniazid oxidation. A series of EPR spin traps were used for detection of isonicotinoyl and derived radicals bound to iron. This is the first report for an isonicotinoyl radical bound to a metal complex, supported by (14)N and (1)H hyperfine splittings for the POBN and PBN trapped radicals. POBN and PBN exhibited average hyperfine coupling constants of aN=15.6, aH=2.8 and aN=15.4, aH=4.7, respectively, which are in close agreement to the isonicotinoyl radical. Radical generation is thought to play a major role in the mechanism of action of isoniazid and this work provides strong evidence for its production within IQG607, which, along with biological and chemical oxidation data, support a redox-mediated activation mechanism. More generally the concept of redox activation of metallo prodrugs could be applied more widely for the design of therapeutic agents with novel mechanisms of action.
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http://dx.doi.org/10.1016/j.jinorgbio.2014.08.002DOI Listing
November 2014

Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide.

Tuberculosis (Edinb) 2014 Jul 9;94(4):421-7. Epub 2014 May 9.

Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul - PUCRS, Avenida Ipiranga, 6681, TecnoPUC 92A, 90619-900 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre, RS, Brazil. Electronic address:

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.
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http://dx.doi.org/10.1016/j.tube.2014.04.008DOI Listing
July 2014
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