Phosphoramidate Pronucleotides: A Comparison of the Phosphoramidase Substrate Specificity of Human and Escherichia coli Histidine Triad Nucleotide Binding Proteins

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• 作者：Tsui-Fen Chou ; Janina Baraniak ; Renata Kaczmarek ; Xin Zhou ; Jilin Cheng ; Brahma Ghosh ; Carston R. Wagner
• 刊名：Molecular Pharmaceutics
• 出版年：2007
• 出版时间：April 2007
• 年：2007
• 卷：4
• 期：2
• 页码：208 - 217
• 全文大小：259K
• 年卷期：v.4,no.2(April 2007)
• ISSN：1543-8392

文摘

To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety ofpronucleotide approaches have been developed. Our laboratory and others have demonstrated thatnucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphatenucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for theirbioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bondsbetween nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability ofnucleosides 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 fluorogenicindole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kineticparameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize theelemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosinephosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificityconstant (k_cat/K_m) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preferencewas 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 lesssterically hindering amine leaving group, tryptamine, with k_cat and K_m values comparable to those foundfor adenosine kinase. The apparent second-order rate constants (k_cat/K_m) for adenosine tryptaminephosphoramidate monoester were found to be 10⁷ M^-1 s^-1 for hHint1 and 10⁶ M^-1 s^-1 for E. coli hinT.Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observedhydrogen 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 tobe 3-4 orders of magnitude smaller than that for uridine tryptamine phosphoramidate for hHint1 and 2orders 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 foradenosine tryptamine phosphoramidate. Consequently, nucleoside phosphoramidates containing unhindered primary amines and either an or 2'-OH group should be easily bioactivated by Hints withefficiencies rivaling those for the 5'-monophosphorylation of nucleosides by nucleoside kinases. Thedifferential substrate specificity observed for human and E. coli enzymes represents a potential therapeuticrationale for the development of selective antibiotic phosphoramidate pronucleotides.