NS5B RNA-dependent RNA Polymerase Research Articles

Liver-Targeted Prodrugs of 2‘-C-Methyladenosine for Therapy of Hepatitis C Virus Infection

2'-C-Methyladenosine exhibits impressive inhibitory activity in the cell-based hepatitis C virus (HCV) subgenomic replicon assay, by virtue of intracellular conversion to the corresponding nucleoside triphosphate (NTP) and inhibition of NS5B RNA-dependent RNA polymerase (RdRp). However, rapid degradation by adenosine deaminase (ADA) limits its overall therapeutic potential. To reduce ADA-mediated deamination, we prepared cyclic 1-aryl-1,3-propanyl prodrugs of the corresponding nucleoside monophosphate (NMP), anticipating cytochrome P450 3A-mediated oxidative cleavage to the NMP in hepatocytes. Lead compounds identified in a primary rat hepatocyte screen were shown to result in liver levels of NTP predictive of efficacy after intravenous dosing to rats. The oral bioavailability of the initial lead was below 5%; therefore, additional analogues were synthesized and screened for liver NTP levels after oral administration to rats. Addition of a 2',3'-carbonate prodrug moiety proved to be a successful strategy, and the 1-(4-pyridyl)-1,3-propanyl prodrug containing a 2',3'-carbonate moiety displayed oral bioavailability of 39%.

Development of carboxylic acid replacements in indole- N-acetamide inhibitors of hepatitis C virus NS5B polymerase

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole- N-acetamides, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme with reduced potential for formation of glucuronide conjugates. Preliminary optimization of these series furnished compounds that are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.

Recent Progress in the Development of Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase

The global prevalence of hepatitis C virus (HCV) infection and the serious consequences associated with the chronic state of the disease have become a worldwide health problem. A combination therapy comprising Interferon-alpha and Ribavirin represents the current standard treatment for chronic HCV infection, although it has demonstrated limited success and causes serious side effects. Promising alternative approaches toward the control of HCV infection include the development of small molecule inhibitors of viral enzymes interfering with the essential steps in the life cycle of the virus. In this review we will focus on inhibitors of the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) which is essential for viral replication and has been recognized as a prime target for therapeutic intervention.

Further studies on hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors toward improved replicon cell activities: Benzimidazole and structurally related compounds bearing the 2-morpholinophenyl moiety

Following the discovery of JTK-109 ( 1) as a potent inhibitor of hepatitis C virus NS5B RNA-dependent RNA polymerase, [(a) Hirashima, S.; Suzuki, T.; Ishida, T.; Noji, S.; Yata, S.; Ando, I.; Komatsu, M.; Ikeda, S.; Hashimoto, H. J. Med. Chem. 2006, 49, 4721. (b) Hashimoto, H.; Mizutani, K.; Yoshida, A. Int. Patent Appl. WO 01/47883, 2001.] further studies toward the improvement of the cellular potency have been performed. A greater than 40-fold improvement was achieved through replacing the biphenyl moiety with a 2-morpholinophenyl group and the benzimidazole ring with the tetracyclic scaffold to afford compound 7 with an excellent replicon potency (EC 50 = 7.6 nM).

Specific targeted antiviral therapy for hepatitis C

Since the discovery of the hepatitis C virus (HCV) as the major cause of non-A, non-B hepatitis in 1989, the search for specific targeted antiviral therapy for HCV (STAT-C) has been underway. Recently, major advances in the understanding of HCV biology and the development of an in vitro system of HCV replication have contributed to the selection of multiple candidate drugs for the treatment of hepatitis C. In 2006, five such candidate drugs have entered phase II clinical trials in patients chronically infected with hepatitis C, including small molecule inhibitors of the HCV NS3 serine protease and NS5B RNA-dependent RNA polymerase. This review focuses on hepatitis C protease and polymerase inhibitors that have progressed to phase II clinical development, foreshadowing the era of STAT-Cs.

Structure–activity relationship (SAR) studies of quinoxalines as novel HCV NS5B RNA-dependent RNA polymerase inhibitors

From chemical compound library screening using an HCV NS5B RNA-dependent RNA polymerase enzymatic assay, we identified a substituted quinoxaline hit with an IC 50 of 5.5 μM. A series of substituted quinoxaline amide derivatives were synthesized based on the hit’s pharmacophore, and a good structure–activity relationship was observed. Computer modeling analysis was employed to help comprehend the SAR.

Topochemical models for predicting the activity of α,γ-diketo acids as inhibitors of the hepatitis c virus NS5B RNA-dependent RNA polymerase

A relationship between the topochemical indices of α,γ-diketo acids and their inhibitory activity with respect to the hepatitis C virus NS5b RNA-dependent RNA polymerase has been studied. The values of Wiener’s topochemical index W c (a distance based descriptor), the Zagreb topochemical index M 2 c (an adjacency based descriptor), and the topochemical eccentric connectivity index ξ c c (an adjacency-cum-distance based descriptor) were calculated for a set of 30 compounds using an in-house computer program. The resulting data array was analyzed and the models of activity were developed after determination of the active ranges of parameters. Subsequently, a biological activity was assigned using these models to each compound included in the data bank, and the assignment was compared with data reported on the inhibitory activity. A high accuracy of prediction was observed for the proposed models. These models possess vast potential in providing basis structures for the development of diketo acids capable of effectively inhibiting the hepatitis C virus NS5b RNA-dependent RNA polymerase.

Recent Patents on Nucleoside and Nucleotide Inhibitors for HCV

Hepatitis C virus (HCV) infection is a leading cause of liver diseases such as cirrhosis and hepatocellular carcinoma. There are estimated 170 million people worldwide chronically infected with the virus. The lack of highly effective and safe therapeutics for HCV infection has spurred intensive efforts to develop anti-HCV drugs as evidenced by the large number of new patent applications filed each year. Nucleoside and nucleotide inhibitors are the analogues of DNA or RNA substrates, and they inhibit viral polymerases by acting as chain terminators, viral mutagens, or simple competitive inhibitors. The successful development of various nucleoside and nucleotide inhibitors for the treatment of HIV and HBV infections has prompted the drug industry to seek similar strategies for HCV. This review summarizes recently issued or published patents covering nucleoside and nucleotide inhibitors for HCV. The claimed chemical structures and available biological activities, mechanism of action, and drug resistance profiles are discussed. The development status of several promising nucleoside inhibitors is also described.

General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2′-Modified Nucleotide Analogues

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2'-C-methyl CTP (2'-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2'-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2'-modified nucleotide analogues. 2'-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2'-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2'-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

Benzimidazole Inhibitors of Hepatitis C Virus NS5B Polymerase: Identification of 2‐[(4‐Diarylmethoxy)phenyl]‐benzimidazole.

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Benzimidazole Derivatives Bearing Substituted Biphenyls as Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase Inhibitors: Structure−Activity Relationship Studies and Identification of a Potent and Highly Selective Inhibitor JTK-109

Following the discovery of a new series of benzimidazole derivatives bearing a diarylmethyl group as inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase (HCV NS5B RdRp),1,2 we extended the structure-activity relationship (SAR) study to analogues bearing a substituted biphenyl group and succeeded in a significant advancement of activity. Starting from compound 1, optimization of the A, B, and C rings afforded potent inhibitors with low nanomolar potency against genotype 1b NS5B. The compounds, which have a substituent with a carbonyl function at the 4-position of the B-ring, efficiently blocked subgenomic viral RNA replication in the replicon cell assay at low submicromolar concentrations. Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.

Recent development of therapeutics for chronic HCV infection

The global prevalence of hepatitis C virus (HCV) infection and serious health consequences associated with chronic state of the disease have become a significant health problem worldwide. Currently, there is no vaccine to prevent the disease and no specific antiviral drug directed against HCV infection. The current standard of care, interferon-based therapies, both alone or in combination with ribavirin, has demonstrated limited success and is associated with undesirable side effects. Thus, the treatment of the chronic HCV infection represents an unmet medical need. With advances in the understanding of HCV replication and the crystal structures of the virally encoded enzymes, the HCV NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase have emerged as ideal targets toward the control of the disease and the development of new anti-HCV agents. In this review, we will summarize the current treatment options, and outline the approaches toward discovery of small molecule antivirals against the virally encoded enzymes. The current clinical studies of promising lead compounds are also reviewed.

Identification of thieno[3,2- b]pyrroles as allosteric inhibitors of hepatitis C virus NS5B polymerase

Thieno[3,2- b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently blocks subgenomic HCV RNA replication in HUH-7 cells with an EC 50 of 2.9 μM.

Discovery of Proline Sulfonamides as Potent and Selective Hepatitis C Virus NS5b Polymerase Inhibitors. Evidence for a New NS5b Polymerase Binding Site

Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor. Optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity. The crystal structure of 6 and NS5b polymerase complex confirmed the binding near the active site region. The optimization approach and SAR are discussed in detail.

Allosteric Inhibition of the Hepatitis C Virus NS5B RNA Dependent RNA Polymerase

The human and monetary costs of chronic hepatitis C and the complications arising from this disease emphasize the urgency to find a treatment for Hepatitis C Virus (HCV) infected patients. The current standard of treatment for patients chronically infected with HCV is combination therapy with pegylated interferon plus ribavirin. Recently, viral enzymes have become the target of efforts to develop small molecule inhibitors interfering with the essential steps in the life cycle of the virus. Amongst these enzymes the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) is essential for viral replication and has been recognized as a prime target for therapeutic intervention. Several distinct classes of inhibitors of NS5B RdRp have been disclosed in the literature, including active site inhibitors such as nucleosides and pyrophosphate mimetics, as well as non-nucleoside inhibitors. The latter, based on the success of allosteric inhibitors in the treatment of HIV infection, have been developed into compounds which show activity in the subgenomic cell-culture assay of HCV replication. This review provides an account of the recent developments in this field.

Biochemical and Pre-Steady-State Kinetic Characterization of the Hepatitis C Virus RNA Polymerase (NS5BΔ21, HC-J4)

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding, nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K(d)) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-)(1) s(-)(1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-)(1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Single-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-)(1) and a K(d) of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 microM. Using dynamic laser light scattering, it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore, nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.

Design and synthesis of 2,3,4,9-tetrahydro-1 H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[ b]indole derivatives as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase

A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 2,3,4,9-tetrahydro-1 H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[ b]indole scaffolds were designed and synthesized. Optimization of the aromatic region showed preference for 5,8-disubstitution pattern in both the scaffolds examined while favoring the n-propyl moiety for the C-1 position. 1,2,3,4-tetrahydro-cyclopenta[ b]indole scaffold was slightly more potent than the corresponding 2,3,4,9-tetrahydro-1 H-carbazole and analogue 36 displayed an IC 50 of 550 nM against HCV NS5B enzyme.

Benzimidazole inhibitors of hepatitis C virus NS5B polymerase: Identification of 2-[(4-diarylmethoxy)phenyl]-benzimidazole

A series of 1-cycloalkyl-2-phenyl-1 H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp). A SAR study was performed and led to identify the 2-[(4-diarylmethoxy)phenyl]-benzimidazoles as potent inhibitors. They inhibit subgenomic HCV RNA replication in the replicon cells at low micromolar concentrations (EC 50 as low as 1.1 μM). They are selective against DNA polymerases (IC 50 > 10 μM) and exhibit low cytotoxicity.

Novel Inhibitors of Hepatitis C Virus RNA-dependent RNA Polymerases

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide-and is the main cause of adult liver transplants in developed nations. We have identified a class of novel and specific inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) activity in vitro. Characterization of two such inhibitors, COMPOUND1 (5-(4-chlorophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine) and COMPOUND2 (5-(4-bromophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine), is reported here. With IC(50) values of 0.54muM and 0.44muM, respectively, they are reversible and non-competitive with nucleotides. Biochemical and structural studies have suggested that these compounds can inhibit the initiation of the RdRp reaction. Interestingly, these inhibitors appear to form a reversible covalent bond with the NS5B cysteine 366, a residue that is not only conserved among all HCV genotypes and a large family of viruses but also required for full NS5B RdRp activity. This may reduce the potential resistance of the viruses to this class of inhibitors.

Parallel synthesis of 5-cyano-6-aryl-2-thiouracil derivatives as inhibitors for hepatitis C viral NS5B RNA-dependent RNA polymerase

From random screening of our compound libraries, we identified a hit compound with an IC 50 of 27 μM against hepatitis C viral NS5B RNA-dependent RNA polymerase. By using a parallel synthetic strategy, a series of its derivatives were synthesized. From their anti-HCV activity screening, compounds with single digital 3.8 micromolar activity were obtained.