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Caracterização da NADH-menaquinona oxidorredutase do tipo II de M. tuberculosis: um novo alvo terapêutico no combate à tuberculose?
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A tuberculose pulmonar (TB) causa cerca de 2 milhões de mortes por ano, estimando-se ainda que um terço da população mundial esteja infetado por Mycobacterium tuberculosis. O tratamento da TB é moroso e exigente, levando muitas vezes ao seu abandono, resultando no aparecimento de estipes multirresistentes (MDR-TB) ou extensivamente resistentes (XDR-TB) aos fármacos utilizados. Estudos recentes identificaram a NADH-menaquinona oxidorredutase do tipo II (NDH-2) de M. tuberculosis como uma enzima-chave da cadeia respiratória deste organismo. A NDH-2 não tem homólogos na mitocôndria humana, o que a torna um excelente alvo para novos fármacos anti-TB.
O presente trabalho teve como objetivo a caracterização da NDH-2 como possível alvo terapêutico. Para tal, foram realizados ensaios de atividade enzimática da NDH-2, em ambiente membranar, na presença de compostos com atividade antimicobacteriana, nomeadamente, clofazimina (CFZ), flupentixol (FPX), tioridazina (TZ), verapamil (VP), PR e U4. Este estudo permitiu determinar os valores de concentração de composto que inibem 50% da atividade da enzima (IC50). Foi ainda realizado um estudo cinético mais detalhado para o PR com o objetivo de determinar os parâmetros cinéticos Vmax e KM.
Os resultados obtidos mostraram que todos os compostos utilizados inibem a atividade da NDH-2, apresentando os seguintes valores de IC50: PR (1,4 μM), CFZ (1,9 μM), FPX (2,8 μM), TZ (8,1 μM), U4 (46,5 μM) e VP (91,8 μM). Assim sendo, os três primeiros compostos parecem mais promissores na terapêutica antimicobacteriana.
Relativamente ao estudo cinético efetuado para o substrato NADH foi possível determinar o valor de Vmax (1,3 μmol min-1) e KM (61,9 μM) para a NDH-2 em ambiente membranar, na ausência de inibidor.
Pulmonary tuberculosis (TB) causes approximately 2 million deaths per year and it is estimated that a third of the world's population is infected by Mycobacterium tuberculosis. TB treatment is long and demanding which could lead to multidrug-resistant (MDR-TB) and extensively-drug resistant (XDR-TB) strains. Recent studies have identified the type II NADH:menaquinone oxidoreductase (NDH-2) of M. tuberculosis as a key enzyme of the respiratory chain of this organism. The NDH-2 does not have counterparts in human mitochondria, which makes it an excellent target for new anti-TB drugs. This study aimed to characterize NDH-2 as a possible therapeutic target. To do so, enzymatic activity assays of NDH-2 were performed, in membrane environment, in the presence of compounds with antimycobacterial activity, in particular, clofazimine (CFZ), flupentixol (FPX), thioridazine (TZ), verapamil (VP), PR and U4. This study allowed us to determine the concentration of compound which inhibits 50% of enzyme activity (IC50). It was also conducted a more detailed kinetic study for PR in order to determine the kinetic parameters Vmax and KM. The results showed that all compounds used inhibit the activity of NDH-2, with the following IC50 values: PR (1.4 μM), CFZ (1.9 μM), FPX (2.8 μM), TZ (8.1 μM), U4 (46.5 μM) e VP (91.8 μM). Hence, the first three compounds seem more promising in the treatment of TB. Regarding the kinetic study in presence of the PR, it was possible to determine the value of Vmax (1.3 μmol min-1) and KM (61.9 μM) for NDH-2 in the membrane environment and in the absence of inhibitor.
Pulmonary tuberculosis (TB) causes approximately 2 million deaths per year and it is estimated that a third of the world's population is infected by Mycobacterium tuberculosis. TB treatment is long and demanding which could lead to multidrug-resistant (MDR-TB) and extensively-drug resistant (XDR-TB) strains. Recent studies have identified the type II NADH:menaquinone oxidoreductase (NDH-2) of M. tuberculosis as a key enzyme of the respiratory chain of this organism. The NDH-2 does not have counterparts in human mitochondria, which makes it an excellent target for new anti-TB drugs. This study aimed to characterize NDH-2 as a possible therapeutic target. To do so, enzymatic activity assays of NDH-2 were performed, in membrane environment, in the presence of compounds with antimycobacterial activity, in particular, clofazimine (CFZ), flupentixol (FPX), thioridazine (TZ), verapamil (VP), PR and U4. This study allowed us to determine the concentration of compound which inhibits 50% of enzyme activity (IC50). It was also conducted a more detailed kinetic study for PR in order to determine the kinetic parameters Vmax and KM. The results showed that all compounds used inhibit the activity of NDH-2, with the following IC50 values: PR (1.4 μM), CFZ (1.9 μM), FPX (2.8 μM), TZ (8.1 μM), U4 (46.5 μM) e VP (91.8 μM). Hence, the first three compounds seem more promising in the treatment of TB. Regarding the kinetic study in presence of the PR, it was possible to determine the value of Vmax (1.3 μmol min-1) and KM (61.9 μM) for NDH-2 in the membrane environment and in the absence of inhibitor.
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Tuberculose Mycobacterium tuberculosis NADH-menaquinona oxidorredutase do tipo II alvo terapêutico atividade enzimática