) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K m of 81 M and a k cat of 0.007 s ؊1 , but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K i ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 M. Similar results were obtained with 2-C-methyladenosine triphosphate (K i ‫؍‬ 1.5 M) and 2-C-methylcytidine triphosphate (K i ‫؍‬ 1.6 M). NS5B with the S282T mutation, which is known to confer resistance to 2-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5,6,21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3Ј-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine (2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the...

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 μM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Entecavir (ETV) is a potent antiviral nucleoside analogue that is used to treat hepatitis B virus (HBV) infection. Recent clinical studies have demonstrated that ETV is also active against the human immunodeficiency virus type 1 (HIV-1).Unlike all approved nucleoside analogue reverse transcriptase RT) inhibitors (NRTIs), ETV contains a 3-hydroxyl group that allows further nucleotide incorporation events to occur. Thus, the mechanism of inhibition probably differs from classic chain termination. Here, we show that the incorporated ETV-monophosphate (MP) can interfere with three distinct stages of DNA synthesis. First, incorporation of the next nucleotide at position n ؉ 1 following ETV-MP is compromised, although DNA synthesis eventually continues. Second, strong pausing at position n ؉ 3 suggests a long range effect, referred to as "delayed chain-termination." Third, the incorporated ETV-MP can also act as a "base pair confounder" during synthesis of the second DNA strand, when the RT enzyme needs to pass the inhibitor in the template. Enzyme kinetics revealed that delayed chain termination is the dominant mechanism of action. High resolution footprinting experiments suggest that the incorporated ETV-MP "repels" the 3-end of the primer from the active site of HIV-1 RT, which, in turn, diminishes incorporation of the natural nucleotide substrate at position n ؉ 4. Most importantly, delayed chain termination protects ETV-MP from phosphorolytic excision, which represents a major resistance mechanism for approved NRTIs. Collectively, these findings provide a rationale and important tools for the development of novel, more potent delayed chain terminators as anti-HIV agents.

Nucleoside reverse transcriptase inhibitors (NRTI) require intracellular phosphorylation, which involves multiple enzymatic steps to inhibit the human immunodeficiency virus type 1 (HIV-1). NRTI-triphosphates (NRTI-TP) compete with endogenous 2′-deoxyribonucleosides-5′-triphosphates (dNTP) for incorporation by the HIV-1 reverse transcriptase (RT). Thus, a highly sensitive analytical methodology capable of quantifying at the low femtomoles/106 cells level was necessary to understand the intracellular metabolism and antiviral activity of NRTIs in human peripheral blood mononuclear (PBM) cells and in macrophages. A novel, rapid, and a reproducible ion-pair chromatography–tandem mass spectrometry (MS/MS) method was developed to simultaneously quantify the intracellular phosphorylated metabolites of abacavir, emtricitabine, tenofovir disoproxil fumarate, amdoxovir, and zidovudine, as well as four natural endogenous dNTP. Positive or negative electrospray ionization was chosen with specific MS/MS transitions for improved selectivity on all the compounds studied. The sample preparation, the ion-pair reagent concentration, and buffer composition were optimized, resulting in the simultaneous quantification of 13 different nucleotides in a total run time of 30 min. This novel method demonstrated optimal sensitivity (limit of detection 1–10 nM for various analytes), specificity, and reproducibility to successfully measure NRTI-TP and dNTP in human PBM cells and macrophages.

It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.