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Influence of 6-aminonicotinamide (6AN) on Leishmania promastigotes evaluated by metabolomics: Beyond the pentose phosphate pathway
Chemico-Biological Interactions ( IF 4.7 ) Pub Date : 2018-08-29 , DOI: 10.1016/j.cbi.2018.08.014
Shawgi Hago Almugadam , Alessandro Trentini , Martina Maritati , Carlo Contini , Gianluca Rugna , Tiziana Bellini , Maria Cristina Manfrinato , Franco Dallocchio , Stefania Hanau

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (μM), although pre-treatment with 100 μM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.



中文翻译:

通过代谢组学评估6-氨基烟酰胺(6AN)对利什曼原虫前鞭毛体的影响:超越戊糖磷酸途径

6-氨基烟酰胺(6AN)是一种抗代谢物,用于抑制许多细胞系统中产生NADPH的戊糖磷酸途径(PPP),使其对氧化应激更加敏感。它通过NAD(P)+糖水解酶转化为6-氨基NAD和6-氨基NADP,由于无法参与氧化还原反应而导致PPP中间体的积累。一些寄生虫(例如恶性疟原虫球菌)高度敏感,但并非所有类型的细胞都对6AN表现出强响应性,这可能是由于靶向途径不同所致。例如,在细菌中,主要目标是用于NAD +生物合成的Preiss-Handler打捞途径。我们对测试动素体原生动物的6AN感兴趣利什曼原虫症是另一种通过代谢组学阐明6AN作用机理的模型。利什曼原虫前鞭毛体是生活在沙the中的生命周期阶段,与恶性疟原虫和哺乳动物细胞(μM)相比,其EC50(mM)高出三个数量级,尽管在亚致死氧化之前用100μM6AN进行了预处理挑战诱导了婴儿乳杆菌中的超加性细胞杀伤。通过代谢组学,我们未检测到6ANAD / P提示利什曼原虫中的NAD +糖水解酶可能无法像其他微生物一样有效地催化转糖苷作用。与报道的对6AN处理的癌细胞的作用相反,我们没有检测到6-磷酸葡萄糖酸盐(6 PG)的积累,表明6ANADP无法与PPP酶6 PG脱氢酶高亲和力结合。相比之下,6AN导致磷酸核糖焦磷酸(PRPP)的大量减少和核碱基的积累,这证明PPP在某种程度上受到了影响。更重要的是,我们发现烟酸盐的产生有所减少,这证明了该酶干扰了NAD +生物合成的Preiss-Handler打捞途径,很可能是抑制了烟碱酰胺酶催化的反应。因此,我们来自利什曼原虫的综合数据 该菌株虽然证实了对PPP的干扰,但也表明6AN损害了Preiss-Handler途径,强调了开发针对该最后一条途径的化合物的重要性。

更新日期:2018-08-29
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