Nucleotide Prodrugs for the Treatment of HCV Infection

f The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2=-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2=-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively. H epatitis C virus (HCV) has a single-stranded RNA genome of positive polarity and belongs to the Flaviviridae family (1). Chronic HCV infection is associated with severe liver disease, including cirrhosis, and an increased risk of hepatocellular carcinoma (2). If adequately treated, HCV can be cured. Previously, treatment options for HCV infected persons were limited to combination therapy with pegylated alpha interferon (IFN-␣) and ribavirin (3). Ribavirin is a nucleoside analog, with a guanine-like base moiety, that can be incorporated by the HCV RNA-dependent RNA polymerase (RdRp) opposite cytosine or uracil, although the detailed mechanism of action remains elusive (4). In 2011, the first direct-acting antivirals (DAAs) targeting the viral protease, or nonstructural protein 3 (NS3), were approved as a component of IFN-based therapies (5); however, the clinical use of the narrow-spectrum protease inhibitors boceprevir and telaprevir is limited by a narrow coverage of HCV genotypes and a low barrier to the selection of resistance (6). Nucleoside or nucleotide inhibitors (NIs) that target the HCV RdRp, or nonstructural protein 5B (NS5B), address these weaknesses (7). Sofosbuvir, a nucleotide prodrug of 2=-deoxy-2=-␣-fluoro-␤-C-methyluridine, was approved by the U.S. Food and Drug Administration (FDA) in Dece...

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confidence: 99%

A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin

Kulkarni

Damha

Schinazi

et al. 2016

“…Once inside the cell, host kinases must first phosphorylate rNAIs into the active ribonucleoside triphosphate (rNTP) form. Therefore, sufficient intracellular levels of triphosphorylated ribonucleoside analog (rNAI-TP) metabolites are critical for antiviral activity (14)(15)(16). The presence of a member of the phosphoramidate prodrug group on rNAI, such as sofosbuvir, allows the first, often rate-limiting, phosphorylation step to be bypassed, leading to increased intracellular rNAI-TP generation.…”

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confidence: 99%

Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094

Ehteshami

Tao

Öztürk

et al. 2016

bRibonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ␤-D-2=-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2=-C-methyl-GTP, is a wellcharacterized inhibitor of NS5B polymerase, whereas the second metabolite, 2=-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo. Finally, we found that although both 2=-C-methyl-GTP and 2=-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2=-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2=-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites. Chronic infection with hepatitis C virus (HCV) represents a significant disease burden on global health. It is estimated that 3% of the human population carries a chronic HCV infection, which translates to roughly 170 million infections worldwide and 3.2 million infections in the United States (1). Although up to 25% of acutely infected individuals clear the virus spontaneously, chronic HCV infection develops in the remaining 75% of those infected and can lead to the development of liver cirrhosis and hepatocellular carcinoma (2-4).HCV contains a 9.6-kb positive-strand RNA genome that codes for three structural and seven nonstructural proteins. The nonstructural protein 5B (NS5B) is the viral RNA-dependent RNA polymerase and is responsible for genome replication. Because of the high mutation rate of this enzyme, as well as the high virus replication rate, HCV propagation results in the generation of thousands of viral quasispecies (5, 6). Therefore, drug combination therapy is required in order to overcome development of resistance and to achieve successful viral clearance. HCV treatment comprised of ribavirin and pegylated interferon alpha, with or without protease inhibitors, can achieve sustained virologic responses in 40 to 80% of treated patients (7-9). However, curative outcomes historically have been suboptimal due to host polymorphisms, viral genotypic variability, and onset ...

“…Chain-terminating nucleotide analog inhibitors are promising drugs against HCV because they target the conserved active site and exhibit pan-genotype activity and a high barrier of resistance (8). One such compound, sofosbuvir (GS-7977) ( Fig.…”

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confidence: 99%

Molecular and Structural Basis for the Roles of Hepatitis C Virus Polymerase NS5B Amino Acids 15, 223, and 321 in Viral Replication and Drug Resistance

Lam¹,

Edwards

Mosley³

et al. 2014

Self Cite

cResistance to the 2=-F-2=-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2=-F-2=-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes. Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus and the cause of hepatitis C. Nonstructural protein 5B (NS5B) is the RNA-dependent RNA polymerase (RdRp) responsible for replication of the viral genome (1) and adopts a right-hand structure with palm, finger, and thumb domains common to nucleotide polymerases (2-4). In addition, HCV NS5B contains an extension of the finger region called the fingertips, as well as a ␤-hairpin loop or ␤-flap insertion into the thumb domain that protrudes deep into the active site and appears to be a hallmark of Flaviviridae RdRps (5). It has been suggested that a significant structural rearrangement of the thumb domain, including relocation of the ␤-flap and a C-terminal-membrane-anchoring linker from the active-site cavity, must take place to accommodate a growing primer-template RNA chain (3,5,6). Indeed, such a rearrangement was observed in an elongationphase binary complex of an NS5B ␤-flap deletion mutant with primer-template RNA (7). A crystal structure of an HCV NS5B ternary assembly is not yet available, and thus, insights into nucleoside triphosphate (NTP) binding and incorporation have been derived from biochemical studies and homology modeling.NS5B is critical for viral replication, exhibits important differences from cellular polymerases, and has become a major target for antiviral drug development. Chain-terminating nucleotide analog inhibitors are promising drugs against HCV because they target the conserved active site and exhibit pan-genotype activity and a high barrier of resistance (8). One such compound, sofosbuvir...