3-Hydroxyisoquinolines as inhibitors of HCV NS5b RNA-dependent RNA polymerase

Isoquinoline-based non-nucleoside inhibitors of HCV NS5b RNA-dependent RNA-polymerase are described. The synthesis and structure–activity relationships are detailed, along with enzyme and cellular activity.     

Expression and purification of untagged full-length HCV NS5B RNA-dependent RNA polymerase

The NS5B encoded by the hepatitis C virus genome is a RNA-dependent RNA polymerase essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. Reported here is the molecular cloning and expression of the full-length NS5B polymerase (NS5B-FL) in bacterial cells as a non-fusion protein. The non-tagged NS5B-FL was purified to homogeneity using sequential chromatographic columns and its identity was confirmed using anti-NS5B peptide antibodies and amino acid sequencing. Purified NS5B-FL demonstrated RNA-dependent RNA polymerase activity and was able to replicate a HCV RNA genome fragment through both copy-back and de novo mechanisms. Its biochemical properties were further characterized in comparison with a truncated form of NS5B polymerase with a deletion of 51 residues from its C-terminus.     

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 microM. 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.     

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.     

Quinolones as HCV NS5B polymerase inhibitors

Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle. A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allosteric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.     

1,5-Benzodiazepine inhibitors of HCV NS5B polymerase

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC₅₀ = 400 nM and 270 nM, respectively) and selective (CC₅₀ > 20 μM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.     

Discovery of novel tricyclic indole derived inhibitors of HCV NS5B RNA dependent RNA polymerase

The characterization of HCV genome has identified various vital functional proteins involved in the life cycle of hepatitis C virus. This has resulted in many novel enzymatic targets that are potential for development of therapeutic agents. The HCV RNA dependent RNA polymerase (HCV NS5B) is one such essential enzyme for HCV replication that has been well characterized and studied by various groups to develop novel therapies for hepatitis C. In this paper, we describe our efforts towards the identification and structure–activity relationship (SAR) of novel tricyclic indole derivatives that bind close to the palm site of the NS5B polymerase. X-ray crystal structure of an inhibitor bound to the polymerase is also described.     

Isothiazoles as active-site inhibitors of HCV NS5B polymerase

Isothiazole analogs were discovered as a novel class of active-site inhibitors of HCV NS5B polymerase. The best compound has an IC(50) of 200 nM and EC(50) of 100 nM, which is a significant improvement over the starting inhibitor (1). The X-ray complex structure of 1 with HCV NS5B was obtained at a resolution of 2.2A, revealing that the inhibitor is covalently linked with Cys 366 of the 'primer-grip'. Furthermore, it makes considerable contacts with the C-terminus, beta-loop, and more importantly, to the active-site of the enzyme. The uniqueness of this binding mode offers a new insight for the rational design of novel inhibitors for HCV NS5B polymerase.     

3-heterocyclyl quinolone inhibitors of the HCV NS5B polymerase

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.     

The monoethyl ester of meconic acid is an active site inhibitor of HCV NS5B RNA-dependent RNA polymerase

Screening of the in-house sample collection for compounds with HCV NS5B RNA dependent RNA polymerase inhibition led to the identification of a new lead. Afterwards, we discovered that the screening lead, rather than containing the expected structure 1, was comprised of roughly a 1:1 mixture of meconic acid 2 and its monoethyl ester 3, with all inhibitory potency residing with 3. We propose that this compound shares critical common features for activity with alpha,gamma-diketoacids inhibitors previously discovered by our group. SAR around this molecule will be presented to provide an improved basis for structure-based ligand design.     

Parallel synthesis of pteridine derivatives as potent inhibitors for hepatitis C virus NS5B RNA-dependent RNA polymerase

From compound library screening using an HCV NS5B RNA-dependent RNA polymerase enzymatic assay, we identified a pteridine hit compound with an IC(50) of 15 microM. Our SAR studies were focused on the different groups at the 6- and 7-positions, substitutions at the 4-position, and replacement of N(1) or N(3) with carbon in the pteridine ring. We found that NH or OH at 4-position is critical for the inhibitory activity. Furthermore, a hydrophobic substituent at the 4-position may help compounds permeate through the cell membrane.     

Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.     

Synthesis of thiazolone-based sulfonamides as inhibitors of HCV NS5B polymerase

Several thiazolone-based sulfonamides were prepared, utilizing various hetero-aryl sulfonyl chlorides and different aldehydes, as inhibitors of NS5B polymerase, to target HCV. The best compound showed 0.6 microM [corrected] of IC50 inhibitory activity.     

Non-nucleoside inhibitors of HCV NS5B polymerase. Part 1: Synthetic and computational exploration of the binding modes of benzothiadiazine and 1,4-benzothiazine HCV NS5b polymerase inhibitors

The importance of internal hydrogen bonding in a series of benzothiadiazine and 1,4-benzothiazine NS5b inhibitors has been explored. Computational analysis has been used to compare the protonated vs. anionic forms of each series and we demonstrate that activity against HCV NS5b polymerase is best explained using the anionic forms. The syntheses and structure–activity relationships for a variety of new analogs are also discussed.     

5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC₅₀ (1b) < 10 nM; IC₅₀ (1a) < 25 nM, EC₅₀ (1b) = 16 nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F = 24%).     

Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides

The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in Huh-7 cells.     

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.     

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3′ terminus at the enzyme active site.     

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 IC50 of 27 microM 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.     

Identification of [(naphthalene-1-carbonyl)-amino]-acetic acid derivatives as nonnucleoside inhibitors of HCV NS5B RNA dependent RNA polymerase

A novel series of HCV NS5B RNA dependent RNA polymerase inhibitors containing a naphthalene carboxamide scaffold were identified by high throughput screening. Optimization of substituents by parallel synthesis and the iterative design towards understanding structure-activity relationship to improve potency are described. Tetra substituted naphthalene 31 displayed potent activity with IC(50) of 120 nM against HCV NS5B enzyme and was selective over a panel of polymerases.