Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D. F. 04510, México.
Biochimie. 2011 Feb;93(2):286-95. doi: 10.1016/j.biochi.2010.09.022. Epub 2010 Oct 7.
Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)(+)-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites-diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO(2)), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO(2))-as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO(2)>DSF>MeDTC-SO(2)>MeDDTC-SO>MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)(+), (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO(2) and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO(2) and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.
来自机会性病原体铜绿假单胞菌(PaBADH)的甜菜碱醛脱氢酶催化甜菜碱醛的不可逆、NAD(P)(+)依赖氧化,产生甘氨酸甜菜碱,一种渗透保护剂。PaBADH 参与胆碱的分解代谢,可能参与细菌感染人体组织时所面临的渗透和氧化应激的防御。鉴于感染组织中存在丰富的胆碱或胆碱前体,PaBADH 是一个潜在的药物靶点,因为其抑制作用将导致细胞内有毒的甜菜碱醛积累。我们测试了硫醇试剂双硫仑(DSF)和五种 DSF 代谢物-二乙基二硫代氨基甲酸盐(DDC)、S-甲基-N,N-二乙基二硫代氨基甲酰磺酸盐(MeDDTC-SO)和磺酰胺(MeDDTC-SO(2))以及 S-甲基-N,N-二乙基硫代氨基甲酰磺酸盐(MeDTC-SO)和磺酰胺(MeDTC-SO(2))作为 PaBADH 和铜绿假单胞菌生长的抑制剂。作为体外 PaBADH 抑制剂,它们的效力顺序为:MeDDTC-SO(2)>DSF>MeDTC-SO(2)>MeDDTC-SO>MeDTC-SO。DDC 不能使酶失活。DSF 代谢物对 PaBADH 的失活作用:(i)不受 NAD(P)(+)影响,(ii)不能被二硫苏糖醇逆转,(iii)不影响酶的四级结构。在所测试的 DSF 代谢物中,MeDTC-SO(2)和 MeDDTC-SO 在含有胆碱的培养基中产生显著的原位 PaBADH 失活和铜绿假单胞菌生长抑制作用,在该培养基中 PaBADH 的表达被诱导。它们在缺乏胆碱的培养基中没有效果,表明 PaBADH 是它们的主要细胞内靶标,并且生长抑制是由于甜菜碱醛的积累所致。这两种化合物在体外和原位对酶失活的动力学非常相似,表明它们不受细胞摄取的限制。MeDDTC-SO(2)和 DSF 在原位没有抑制作用,可能是因为它们对细胞内非必需硫醇的高反应性导致其耗尽。我们的结果支持 PaBADH 是治疗铜绿假单胞菌感染的有前途的靶标,并且一些 DSF 代谢物可能对此有所帮助。