HINT as putative phosphoramidase responsible of pro-tides activation: Molecular modelling studies [Abstract]

The ProTide approach was developed by McGuigan et al. as a method to improve the biological activity of poorly active nucleoside analogues by “kinase bypass” in which lipophilic nucleotide pro-drugs act as intracellular phosphate delivery motifs and achieve the generation of free nucleotides by nucleoside kinase-independent means. The activation of phosphoramidates is considered to be based upon two enzymatic cleavages: the hydrolysis of the amino acid ester moiety as the trigger of the process, and the Psingle bondN bond cleavage as the final step that would release the corresponding nucleoside analogue monophosphate. The latter step is carried out by an enzyme with phosphoramidase activity and it has been proposed that the protein responsible of the cleavage of the Psingle bondN bond belongs to the HINT family: adenosine monophosphoramidates AMP-NH2, AMP-N-ɛ-(N-α-acetyl lysine methyl ester) and AMP-para-nitroaniline were identified as rabbit Hint and yeast Hint1 substrates. Notably, AMP-N-alanine methyl ester was also reported as a substrate for the same enzymes, which might indicate their role in the activation of our phosphoramidates. We have recently reported some initial docking studies on a series BVDU phosphoramidate analogues in the human HINT-1 active site, which showed that indeed this enzyme could be able to bind and process the phosphoramidates. In the work presented here, we have extended the modelling simulations to different antiviral nucleoside analogues and examined their binding to HINT-1, as well as to the homology models of HINT-2 and HINT-3, with the aim of developing a predictive computational model which could be used in the design of more potent and selective phosphoramidate analogues (Fig. 1)

The application of phosphoramidate protide technology to acyclovir confers anti-HIV inhibition

Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the l-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl variation was tolerated. ACV ProTides with other amino acids, other than l-phenylalanine, showed no detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure−activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV agents and vice versa, so the assay became predictive

Sub micromolar inhibitors of HCV generated from inactive nucleosides by application of ProTide technology [Abstract]

We report the application of our phosphoramidate ProTide technology to various 4′-substituted ribonucleoside analogues, designed as potential inhibitors of hepatitis C virus (HCV) (Fig. 1). Thus, ProTides were prepared from 4′-azidouridine (AZU), -cytidine (AZC), -adenosine (AZA) and -5-methyluridine (AZMeU), besides other 4′-substituted uridines and cytidines. In each case, ProTide families included variations in the aryl, ester, and amino acid regions. A number of compounds showed potent inhibitory properties in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. Of particular note was the sub-μM potency displayed by certain ProTides of AZU; a nucleoside analogue, which was itself inactive in the assay. In some cases, we were able to separate, and separately evaluate the phosphate stereoisomers generated in the synthesis; in a few cases the absolute phosphate stereochemistry was solved (Fig. 2). The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy