2′-Deoxy-2′-α-fluoro-2′-β-C-methyl 3′,5′-cyclic phosphate nucleotide prodrug analogs as inhibitors of HCV NS5B polymerase: Discovery of PSI-352938

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2=F-2=C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2=F-2=C-Me-UTP was efficiently incorporated by HCV polymerase with apparent K d (equilibrium constant) and k pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ؎ 28 M and 0.67 ؎ 0.05 s ؊1 , respectively, giving an overall substrate efficiency (k pol /K d ) of 0.0059 ؎ 0.0015 M ؊1 s ؊1 . We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2= position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH 2 > F-C-Me > C-Me > N 3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2=F-2=C-Me-, 2=C-Me-, and 2=ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2=-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2=-modified NTP analogs against HCV polymerase. . The primary mode of transmission for HCV is via exposure to infected blood, including transfusions from infected donors, and through intravenous use of illicit drugs. Although a minority of all HCV infections will spontaneously resolve without any clinical outcome, an estimated 80% of cases will progress into chronic hepatitis, leading to a significant proportion of cirrhosis and cases of hepatocellular carcinoma (2). This makes HCV the leading cause of liver transplantation in the United States.Hepatitis C virus is a member of the Flaviviridae family (3). It contains a single, positive-strand RNA genome of about 9.5 kb. The viral genome encodes only one open reading frame translated to a polyprotein of approximately 3,000 amino acids. The NS5B protein is composed of 591 amino acids that are cleaved at the C-terminal end of the polyprotein. NS5B acts as the RNA-dependent RNA polymerase (RdRp), with critical functions in RNA replication and transcription. Similar to other known RdRps, NS5B contains six conserved motifs, designated A through F. The amino acids involved in the catalytic activity of NS5B are located within motif A (aspartate at position 220) and in the catalytic triad GDD at positions 318 to ...

supporting

confidence: 88%

“…Several nucleotide analogs inhibiting HCV NS5B have been reported (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). These inhibitors must enter liver cells as non-…”

mentioning

confidence: 99%

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Fung¹,

Zhang²,

Dyatkina³

et al. 2014

“…PSI-7977 (prodrug of 2=-F-2=-C-methyluridine monophosphate) (38), PSI-352938 (prodrug of 2=-F-2=-C-methylguanosine monophosphate) (34), PSI-6130 (2=-F-2=-C-methylcytidine) (40), R1479 (4=-azidocytidine) (13), IDX-184 and INX-189 (prodrugs of 2=-C-meth-ylguanosine monophosphate) (43,48), NS5B nonnucleoside inhibitors NNI-1 (an indole analog), NNI-2 (a thiophene analog), NNI-3 (a benzothiadiazine analog), and HCV-796 (a benzofuran analog or NNI-4) (39), NS3 protease inhibitors telaprevir and RG7227 (ITMN-191) (21,22), NS3/4a inhibitor ACH-806 (46), and NS5A inhibitor BMS-790052 (8) were synthesized at Pharmasset and shown to be Ͼ95 to 99% pure by proton nuclear magnetic resonance, mass spectroscopy, and highperformance liquid chromatography analysis. Ribavirin was obtained from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

Lam¹,

Espiritu²,

Bansal³

et al. 2012

Self Cite

222

186

PSI-7977, a prodrug of 2=-F-2=-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC 50 ) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons. Hepatitis C virus (HCV) currently infects more than 170 million people worldwide and is the leading cause of chronic liver disease and liver transplantation in the United States. HCV is a single plus-strand RNA virus about 9.6 kb in genome size and contains one open reading frame that encodes 10 structural and nonstructural (NS) proteins. The structural proteins (core, E1, and E2), which constitute the viral capsid and envelope proteins, are located at the amino terminus, followed by p7, which oligomerizes to form an ion channel critical for viral assembly, and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which are responsible for viral replication (35). Recently, two antiviral agents have been approved, in combination with pegylated alpha interferon and ribavirin (Peg-IFN/RBV), to treat patients infected with genotype (GT) 1 HCV. Both of these compounds, boceprevir (Victrelis) and telaprevir (Incivek), target the NS3/4A protease and inhibit HCV replication by blocking processing of NS3, NS4A/B, and NS5A/B (25, 33). These treatments showed improvement over Peg-IFN/RBV; however, patients may develop additional side effects corresponding to each of these antiviral compounds (4). Furthermore, viral resistance against these compounds has emerged both in vitro and in vivo as a result of the high genetic diversity of HCV and error-prone nature of the HCV NS5B RNA-dependent RNA polymerase (12). Therefore, research...

“…PSI-352938 was previously reported not to be cytotoxic (50% cytotoxic concentrations [CC 50 s] Ͼ 100 M) or toxic to mitochondria (CC 50 s Ͼ 100 M) in various cell types (47). Because bone marrow toxicity has been associated with a number of nucleoside antiviral analogs (49), we evaluated the effect of PSI-352938 on the proliferation of human bone marrow progenitor cells.…”

Section: Antiviral Activity Of Psi-352938 and Activity Of Psi-352666mentioning

confidence: 99%

“…PSI-352938 was synthesized as a nucleotide prodrug to bypass the ratelimiting first phosphorylation step in the metabolism of the parent nucleoside to the active triphosphate, ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylguanosine-5Ј-triphosphate (PSI-352666) (47). Structure-activity relationship (SAR) studies showed that PSI-352938 was about 60-fold more active than its corresponding nucleoside analog, with no significant cellular and mitochondrial toxicity, and produced high levels of triphosphate in both primary human hepatocytes and the liver of rats administered a single dose of PSI-352938 of 50 mg/kg of body weight (47). Herein we report on the activity of PSI-352938 and its triphosphate metabolite in a panel of cell-based HCV and NS5B polymerase assays demonstrating that PSI-352938 has pan-genotype anti-HCV activity.…”

mentioning

confidence: 99%

Inhibition of Hepatitis C Virus Replicon RNA Synthesis by PSI-352938, a Cyclic Phosphate Prodrug of β-d-2′-Deoxy-2′-α-Fluoro-2′-β-C-Methylguanosine

Lam¹,

Espiritu²,

Murakami³

et al. 2011

Self Cite

PSI-352938 is a novel cyclic phosphate prodrug of ␤-D-2-deoxy-2-␣-fluoro-2-␤-C-methylguanosine 5-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 M, respectively. The active 5-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. Hepatitis C virus (HCV), an important member of the Flaviviridae family of viruses, is a major health problem affecting approximately 170 million people worldwide. In the United States, where it is the leading cause of liver transplantation, nearly 2% of the U.S. population are HCV carriers (2). The current standard of care (SOC), which consists of pegylated alpha interferon (IFN-␣) and ribavirin, results in only about a 50% sustained virological response in patients infected with genotype 1 HCV, the predominant genotype in the United States and Europe (13, 18). The limited cure rate and potential side effects associated with interferon and ribavirin (10, 11) prompted the development of direct-acting antiviral agents (DAAs) in order to improve clinical efficacy and tolerability.Among the DAAs currently in clinical development, nucleoside/nucleotide analogs as inhibitors of HCV replication have the advantage of broad genotype coverage and a high barrier to resistance (14). The active 5Ј-triphosphates of nucleoside/ nucleotide analogs target the HCV NS5B RNA-dependent RNA polymerase (Pol) by serving as alternative substrate inhibitors during RNA synthesis (6). Both of the two resistance mutations identified so far, S96T and S282T, are highly conserved residues and unfit for HCV replicon replication (33). Among the nucleoside/nucleotide analogs, RG7128 (the prodrug of PSI-6130, ␤-D-2Ј-fluoro-2Ј-C-methylcytidine) is the most advanced anti-HCV nucleoside and is currently in phase IIb clinical studies. So far, results from the clinical studies showed that RG7128 is generally safe and effective in reducing the viral loads for genotype 1, 2, 3, and 4 HCV-infected patients when it is combined with SOC, with no resistance-related breakthrough (16,21,27,37).More recently, we reported results of in vitro studies characterizing PSI-7851 and PSI-7977 (the single Sp isomer of PSI-7851), phosphoramidate prodrugs of ␤-D-2Ј-fluoro-2Ј-Cmethyluridine 5Ј-monophosphate (30,45,48). While RG7128 was designed to improve the pharmacokinetic profile of PSI-6130, PSI-7851 and PSI-7977 were synthesized to bypass the nonproductive first phosphorylation step, as the corresponding nucleoside analog was incapable of being converted to the monophosphate form. In a 3-day phase I monotherapy study, a 1.95-log vira...