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 (kcat/Km) 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 kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) 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 (kcat/Km) 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 (kcat/Km) 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.

Histidine Triad Nucleotide-binding Protein 1 (HINT-1) Phosphoramidase Transforms Nucleoside 5′-O-Phosphorothioates to Nucleoside 5′-O-Phosphates

“…It has been demonstrated for the phosphoramidase activity of Hint-1 that the enzyme prefers purine-based substrates rather than pyrimidines and that a hydroxyl group at the ribose 2Ј position of the substrate is essential for interactions with the ␥-oxygen of Asp-43 (rHint-1) of the enzyme in the binding pocket (45). We observed a similar tendency for rHint-1 desulfurating activity.…”

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

“…Because binding of PSI-352707 to Hint1 was very weak, we were not able to determine the kinetic parameters for PSI-352707 with this enzyme (K m Ͼ1 mM). Chou et al (21) have studied Hint1 substrate specificity using different phosphoramidates and phosphoramidothiolates, and the reported K m values ranged from 0.13 to 215 M. The weak binding affinity for PSI-352707 may be due to the 2Ј-fluoro-2Ј-methyl disubstitution on the sugar moiety.…”

“…Because Hint proteins have been shown to possess phosphoramidase activity (15,21,22), recombinant human Hint1 and Hint3 were cloned, purified, and tested for the ability to convert PSI-352707 to the corresponding monophosphate, PSI-7411. The activity of purified Hint1 and Hint3 was first tested with the control compound AMP-NH 2 , and both enzymes were able to deaminate AMP-NH 2 to AMP (data not shown).…”

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

“…It was then subjected to chromatography on a SiO 2 gel column; the product was eluted with CHCl 3 /MeOH/ H 2 O (5:3:0.5). The relevant fractions were concentrated in vacuo, the residue was dissolved in water (1 ml) and lyophilized to afford the title compound as a white solid (14 mg, yield 14%); 1 kinetic studies were carried out with tryptamine 5Ј-adenosine phosphoramidate (TpAd) or AIPA as substrate and with the fluorescent activity assay as described (5). Excitation wavelength was set at 280 nm, fluorescence emission was measured at 360 nm, and excitation and emission slits were set at 10 nm for concentrations of substrates ranging from 0.05 to 1 M for wild-type and 5 nm for concentrations ranging from 10 to 50 M for V97D, V97E, and V97R mutants.…”

“…Hints are homodimeric proteins that have been found to be purine phosphoramidases (2)(3)(4)(5). Recently, we have demonstrated that lysyl-adenylate (lysyl-AMP) generated by bacterial and human lysyl-tRNA synthetases (LysRS) are also substrates for the respective bacterial and human Hints (6).…”