Cloning, expression, and characterization of<i>Mycobacterium tuberculosis</i>dihydrofolate reductase

White, E. Lucile; Ross, L. J. N.; Cunningham, Amanda; Escuyer, Vincent

doi:10.1016/s0378-1097(04)00038-2

Cited by 44 publications

(37 citation statements)

References 12 publications

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“…5b), which is similar to the optimum pH for recombinant human DHFR (White et al, 2004). DHFR was most active at the higher pH values, and the activity at pH 5 was 40 % of that at pH 10.…”

Section: Effects Of Temperature Ph and Metal Ions On The Activity Ofsupporting

confidence: 59%

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Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

et al. 2016

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Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH 4 ), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH 4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH 4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH 4 salvage pathway in a fungus.

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“…5b), which is similar to the optimum pH for recombinant human DHFR (White et al, 2004). DHFR was most active at the higher pH values, and the activity at pH 5 was 40 % of that at pH 10.…”

Section: Effects Of Temperature Ph and Metal Ions On The Activity Ofsupporting

confidence: 59%

“…alpina DHFR, with 50 % and 100 % inhibition at 10 mM, respectively. This is in contrast to the DHFRs from Mycobacterium tuberculosis, Haloferax volcanii and Plasmodium vivax (Leartsakulpanich et al, 2002;White et al, 2004;Wright et al, 2002).…”

Section: Effects Of Temperature Ph and Metal Ions On The Activity Ofmentioning

confidence: 68%

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

et al. 2016

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“…While TMP is a specific inhibitor of bacterial Dhfr, MTX inhibits the DHFR enzymes of Plasmodium berghei, Escherichia coli, and humans (17). MTX is actually a better inhibitor of the M. tuberculosis Dhfr enzyme (IC 50 ϭ 8.3 nM) than TMP is (24). MTX has a chemical structure very similar to that of folic acid and binds to Dhfr 1,000 times more tightly than folate does.…”

Section: Tuberculosis Is Resistant To Tmp and Susceptible To Smx Thementioning

confidence: 99%

The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

Vilchèze

Jacobs

2012

Antimicrob Agents Chemother

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ABSTRACTThe challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistantMycobacterium tuberculosisstrains. While TMP alone was not effective againstM. tuberculosis, the combination of TMP and SMX was bacteriostatic againstM. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDRM. tuberculosisstrains. Treatment ofM. tuberculosiswith TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistancein vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMXin vivoto prevent the emergence of drug-resistantM. tuberculosis.

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“…No entanto, a maioria desses compostos não apresentam ação seletiva na enzima DHFR do Mycobacterium, exercendo também atividade inibitória na enzima humana. 132,133 Nesse contexto, foi descrito através do screening de uma biblioteca com mais 265 mil compostos do Instituto Nacional do Câncer (NCI-USA) potenciais inibidores seletivos da enzima DHFR micobacteriana. O composto 52 (Figura 20) …”

Section: Dhfrunclassified

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

2017

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POTENTIAL MOLECULAR TARGETS FOR ANTITUBERCULOSIS DRUG DISCOVERY. Tuberculosis (TB) is an infectious disease caused by mycobacteria from the Mycobacterium genus, mainly by Mycobacterium tuberculosis (MTB). The World HealthOrganization (WHO) aims to reduce the number of TB cases worldwide in the coming years. Nevertheless, the increasing number of multidrug-resistant (MDR-TB) and extensive-drug resistance (XDR-TB) strains, and the ineffectiveness of the current treatment in latent tuberculosis are challenges to be overcome. In this review, we will demonstrate the recent advances in the tuberculosis drug discovery, focusing the research of new molecular targets in the Mycobacterium tuberculosis. Among the promising targets described herein, we highlight those, which act in different pathways in the mycobacteria, such as energy metabolism, cell wall biosynthesis, DNA synthesis, iron metabolism and transport through membranes. Furthermore, bioactive compounds discovered using phenotypic assays screening and validated through genetic approaches are also presented.Keywords: medicinal chemistry; tuberculosis; Mycobacterium tuberculosis; molecular targets; new drugs. INTRODUÇÃOA tuberculose (TB) é uma doença infecciosa causada por micobactérias do complexo Mycobacterium, no entanto, a espécie responsável pelo maior número de casos de morbidade e mortalidade é o Mycobacterium tuberculosis (MTB). 1 Ocupando o ranking mundial como a principal causa de mortes causadas por doenças infecciosas, nos últimos anos a tuberculose ultrapassou, em número de casos, a infecção causada pelo vírus da imunodeficiência humana (HIV). 2,3 O último levantamento realizado em 2015 pela Organização Mundial da Saúde (OMS) apontou 9.6 milhões de novos casos no mundo e 1.5 milhões de mortes causadas pela doença. 3 Somado a esses dados, o surgimento e aumento de cepas de M. tuberculosis multirresistente aos fármacos (MDR-TB) e extensivamente resistente aos fármacos (XDR-TB) vêm alarmando as autoridades de todo o mundo. Essas formas de tuberculose apresentam baixas taxas de cura e maiores taxas de mortalidade devido às dificuldades de tratamento. 4 Além disso, já foram relatados na clínica casos de tuberculose totalmente resistente aos fármacos (TDR-TB). 5,6 Ao longo dos últimos anos, é possível constatar algumas evoluções no desenvolvimento de compostos candidatos a fármacos que possam atuar contra a TB. 7 Após um intervalo de mais de 50 anos sem novos medicamentos para o tratamento da TB, a bedaquilina foi aprovada em 2012 pela agência norte-americana U.S. Food and Drug Administration (FDA) para o tratamento de MDR-TB. Atualmente, diversos candidatos a fármacos estão em estudos clínicos, [8][9][10][11][12] 39 Atualmente, diversos métodos e estratégias são utilizados para o desenvolvimento de novos fármacos contra a TB, como por exemplo: 1) abordagens genéticas para identificação de novos alvos; 2) ensaios de triagem em larga escala utilizando a micobactéria como plataforma; 3) ferramentas de biologia estrutural e triagem virtual e; 4) modificações ...

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Cloning, expression, and characterization ofMycobacterium tuberculosisdihydrofolate reductase

Cited by 44 publications

References 12 publications

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

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