Phosphoramidate pronucleotides:: A comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins
Phosphoramidate pronucleotides:: A comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins
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DOI：
10.1021/mp060070y
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发表时间：
2007-03-01
影响因子：
4.9
通讯作者：
Wagner, Carston R.
中科院分区：
文献类型：
--
作者：
Chou, Tsui-Fen;Baraniak, Janina;Wagner, Carston R.
为了促进以核苷酸为基础的治疗药物向细胞和组织的传递，各种各样的原核苷酸方法已经开发出来。我们的实验室和其他人已经证明，核苷磷酸酯可以在细胞内被激活为相应的5'-单磷酸核苷酸，组氨酸三联体核苷酸结合蛋白（提示）可能负责它们的生物激活。提示酶是一种保守而普遍存在的酶，它可以水解磷酸核苷5'-单磷酸和胺离去基之间的磷酸化键。基于核苷猝灭含有吲哚的共价连接胺的荧光的能力，开发了一种灵敏的、连续的荧光检测方法。合成了一系列天然荧光吲哚衍生物与核苷5′-单磷酸连接的底物，并对其在人类Hint1和大肠杆菌中水解的稳态动力学参数进行了评价。为了表征对反应的元素和立体化学影响，合成了两种磷酸氨基硫酸腺苷或鸟苷的p -非对映异构体，并研究了当磷酰氧被硫取代时，特异性常数（k(cat)/ k -m）降低了15-200倍。虽然没有观察到大肠杆菌对hinT的立体化学偏好，但hHint1对d-色氨酸磷酸酯的偏好是l-异构体的300倍。迄今为止评估的最有效的底物是那些含有较少的立体阻碍胺离开基团，色胺的底物，其k（cat）和k -m值与腺苷激酶的值相当。对hHint1和E. coli hinT的表观二级速率常数（k(cat)/ k -m）分别为10(7)M-1 s（-1）和10(6)M-1 s（-1）。人和大肠杆菌的酶都倾向于嘌呤而不是嘧啶类似物。与观察到的单磷酸腺苷2 '-OH基团与活性位点残基Asp43之间的氢键相一致，胸苷嘧啶色胺磷酸酯的二级速率常数（k(cat)/ k -m）比hHint1的尿嘧啶色胺磷酸酯小3-4个数量级，比大肠杆菌的hinT小2个数量级。然而，Ara-A -色胺磷酸酯被证明是一个很好的底物，其特异性常数（k(cat)/ k -m）仅比腺苷色胺磷酸酯低10倍。因此，含有不受阻碍的伯胺和α或β 2 '-OH基团的核苷磷酸酯应该很容易被提示生物激活，其效率与核苷激酶对核苷的5 '-单磷酸化相媲美。对人类和大肠杆菌酶所观察到的不同底物特异性代表了开发选择性抗生素磷酰胺原核苷酸的潜在治疗原理。
To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (k(cat)/K-m) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with k(cat) and K-m values comparable to those found for adenosine kinase. The apparent second-order rate constants (k(cat)/K-m) for adenosine tryptamine phosphoramidate monoester were found to be 10(7) M-1 s(-1) for hHint1 and 10(6) M-1 s(-1) for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2 '-OH group of adenosine monophosphate and the active site residue, Asp43, the second-order rate constant (k(cat)/K-m) for thymidine tryptamine phosphoramidate was found to be 3-4 orders of magnitude smaller than that for uridine tryptamine phosphoramidate for hHint1 and 2 orders of magnitude smaller than that for E. coli hinT. Ara-A tryptamine phosphoramidate was, however, shown to be a good substrate with a specificity constant (k(cat)/K-m) only 10-fold lower than the value for adenosine tryptamine phosphoramidate. Consequently, nucleoside phosphoramidates containing unhindered primary amines and either an alpha or beta 2 '-OH group should be easily bioactivated by Hints with efficiencies rivaling those for the 5 '-monophosphorylation of nucleosides by nucleoside kinases. The differential substrate specificity observed for human and E. coli enzymes represents a potential therapeutic rationale for the development of selective antibiotic phosphoramidate pronucleotides.