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

Abstract

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 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 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 α or β 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.

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