Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.

Interferon regulatory factor 5 (IRF5) suppresses hepatitis C virus (HCV) replication and HCV-associated hepatocellular carcinoma.

Hepatitis C virus (HCV) infection is a major risk factor for the development of chronic liver disease. The disease typically progresses from chronic HCV to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and death. Chronic inflammation associated with HCV infection is implicated in cirrhosis and HCC, but the molecular players and signaling pathways contributing to these processes remain largely unknown. Interferon regulatory factor 5 (IRF5) is a molecule of interest in HCV-associated HCC because it has critical roles in virus-, Toll-like receptor (TLR)-, and IFN-induced signaling pathways. IRF5 is also a tumor suppressor, and its expression is dysregulated in several human cancers. Here, we present first evidence that IRF5 expression and signaling are modulated during HCV infection. Using HCV infection of human hepatocytes and cells with autonomously replicating HCV RNA, we found that levels of IRF5 mRNA and protein expression were down-regulated. Of note, reporter assays indicated that IRF5 re-expression inhibited HCV protein translation and RNA replication. Gene expression analysis revealed significant differences in the expression of cancer pathway mediators and autophagy proteins rather than in cytokines between IRF5- and empty vector-transfected HCV replicon cells. IRF5 re-expression induced apoptosis via loss in mitochondrial membrane potential, down-regulated autophagy, and inhibited hepatocyte cell migration/invasion. Analysis of clinical HCC specimens supports a pathologic role for IRF5 in HCV-induced HCC, as IRF5 expression was down-regulated in livers from HCV-positive versus HCV-negative HCC patients or healthy donor livers. These results identify IRF5 as an important suppressor of HCV replication and HCC pathogenesis.

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION: HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

Discovery of conjugated thiazolidinone-thiadiazole scaffold as anti-dengue virus polymerase inhibitors.

Dengue virus (DENV) infection is a significant health threat to the global population with no therapeutic option. DENV NS5 RNA-dependent RNA polymerase (RdRp) is the key replicating protein of the virus and thus an attractive target for drug development. Herein, we report on the synthesis and biological evaluation of a series of hybrid thiazolidinone-thiadiazole derivatives as a new class of DENV-2 NS5 RdRp inhibitors. This yielded compounds 12 and 21 with IC50 values of 2.3 µM and 2.1 µM, respectively, as promising leads. Limited SAR analysis indicated 3-fluorobenzylidene as the optimal substituent at C5-position of the thiazolidinone core, whereas both 2-chlorophenyl and 3-fluorophenyl substituents were equally effective at C5-position of the 1,3,4-thiadiazole core. Biophysical characterization and molecular docking studies conferred the binding site of this scaffold on DENV NS5 polymerase. Binding mode of compound 21 in Thumb pocket-II of DENV-2 NS5 polymerase will form the basis for future structure-activity relationship optimization.

Anti-cancer and anti-hepatitis C virus NS5B polymerase activity of etodolac 1,2,4-triazoles.

Arachidonic acid is an unsaturated fatty acid liberated from phospholipids of cell membranes. NSAIDs are known as targets of cyclooxygenase enzyme (COX-1, COX-2 and COX-3) in arachidonic acid metabolism. This mechanism of COX-2 in carcinogenesis causes cancer. In addition, COX-2 plays a role in the early stages of hepatocarcinogenesis. Hepatitis C virus (HCV) infection is cause of liver cirrhosis and hepatocellular carcinoma (HCC). The aim of our study was to improve effective agents against HCV. A novel series of new etodolac 1,2,4-triazoles derivatives (4a-h) have been synthesized and investigated for their activity against HCV NS5B polymerase. Compound 4a was found to be the most active with IC(50) value of 14.8 µM. In accordance with these results, compound 4a was screened for anti-cancer activity on liver cancer cell lines (Huh7, Mahlavu, HepG2, FOCUS). Compound 4a showed anti-cancer activity against Huh7 human hepatoma cell line with IC(50) value of 4.29 µM. Therefore, compound 4a could be considered as a new anti-cancer and anti-HCV lead compound.

Novel 4-thiazolidinones as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase.

In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29-42, together with their synthetic precursors 22-28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC50 values between 25.3 and 54.1 µM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.

Design of novel rho kinase inhibitors using energy based pharmacophore modeling, shape-based screening, in silico virtual screening, and biological evaluation.

Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound 8 exhibited an IC50 value of 1.5 µM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI50 value of 0.27 µM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound 8 with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 µM CC50 (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor.

Multiple e-pharmacophore modeling, 3D-QSAR, and high-throughput virtual screening of hepatitis C virus NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRP) is a crucial and unique component of the HCV RNA replication machinery and a validated target for drug discovery. Multiple crystal structures of NS5B inhibitor complexes have facilitated the identification of novel compound scaffolds through in silico analysis. With the goal of discovering new NS5B inhibitor leads, HCV NS5B crystal structures bound with inhibitors in the palm and thumb allosteric pockets in combination with ligands with known inhibitory potential were explored for a comparative pharmacophore analyses. The energy-based and 3D-QSAR-based pharmacophore models were validated using enrichment analysis, and the six models thus developed were employed for high-throughput virtual screening and docking to identify nonpeptidic leads. The hits derived at each stage were analyzed for diversity based on the six pharmacophore models, followed by molecular docking and filtering based on their interaction with amino acids in the NS5B allosteric pocket and 3D-QSAR predictions. The resulting 10 hits displaying diverse scaffold were then screened employing biochemical and cell-based NS5B and anti-HCV inhibition assays. Of these, two molecules H-5 and H-6 were the most promising, exhibiting IC50 values of 28.8 and 47.3 µM against NS5B polymerase and anti-HCV inhibition of 96% and 86% at 50 µM, respectively. The identified leads comprised of benzimidazole (H-5) and pyridine (H-6) scaffolds thus constitute prototypical molecules for further optimization and development as NS5B inhibitors.

Inhibitors for the hepatitis C virus RNA polymerase explored by SAR with advanced machine learning methods.

Hepatitis C virus (HCV) is a global health challenge, affecting approximately 200 million people worldwide. In this study we developed SAR models with advanced machine learning classifiers Random Forest and k Nearest Neighbor Simulated Annealing for 679 small molecules with measured inhibition activity for NS5B genotype 1b. The activity was expressed as a binary value (active/inactive), where actives were considered molecules with IC50 ≤0.95 µM. We applied our SAR models to various drug-like databases and identified novel chemical scaffolds for NS5B inhibitors. Subsequent in vitro antiviral assays suggested a new activity for an existing prodrug, Candesartan cilexetil, which is currently used to treat hypertension and heart failure but has not been previously tested for anti-HCV activity. We also identified NS5B inhibitors with two novel non-nucleoside chemical motifs.

Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50=7.7 µM) and 2 (IC50=10.6 µM) as represented by hybrid compound 27 (IC50=6.7 µM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC50 of 2.6 µM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the D-isomers 41 (IC50=19.3 µM) and 45 (IC50=5.4 µM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.

Some hydrazones of 2-aroylamino-3-methylbutanohydrazide: synthesis, molecular modeling studies, and identification as stereoselective inhibitors of HIV-1.

In accordance with our antiviral drug development attempt, acylhydrazone derivatives bearing amino acid side chains were synthesized for the evaluation of their antiviral activity against various types of viruses. Among these compounds, 8(S) , 11(S) , and 12(S) showed anti-HIV-1 activity with a 50% inhibitory concentration (IC(50)) =123.8 µM (selectivity index, SI>3), IC(50) =12.1 µM (SI>29), IC(50) =17.4 µM (SI>19), respectively. Enantiomers 8(R) , 11(R) , and 12(R) were inactive against the HIV-1 strain III(B) . Hydrazones 8(S) , 11(S) , and 12(S) which were active against HIV-1 wild type showed no inhibition against a double mutant NNRTI-resistant strain (K103N;Y181C). Molecular docking calculations of R- and S-enantiomers of 8, 11, and 12 were performed using the hydrazone-bound novel site of HIV-1 RT.

Synthesis and characterization of celecoxib derivatives as possible anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV agents.

A series of novel N-(3-substituted aryl/alkyl-4-oxo-1,3-thiazolidin-2-ylidene)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamides 2a-e were synthesized by the addition of ethyl a-bromoacetate and anhydrous sodium acetate in dry ethanol to N-(substituted aryl/alkylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoro-methyl)-1H-pyrazol-1-yl]benzene sulfonamides 1a-e, which were synthesized by the reaction of alkyl/aryl isothiocyanates with celecoxib. The structures of the isolated products were determined by spectral methods and their anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV NS5B RNA-dependent RNA polymerase (RdRp) activities evaluated. The compounds were also tested for gastric toxicity and selected compound 1a was screened for its anticancer activity against 60 human tumor cell lines. These investigations revealed that compound 1a exhibited anti-inflammatory and analgesic activities and further did not cause tissue damage in liver, kidney, colon and brain compared to untreated controls or celecoxib. Compounds 1c and 1d displayed modest inhibition of HCV NS5B RdRp activity. In conclusion, N-(ethylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (1a) may have the potential to be developed into a therapeutic agent.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

The glutamine side chain at position 91 on the ß5a-ß5b loop of human immunodeficiency virus type 1 reverse transcriptase is required for stabilizing the dNTP binding pocket.

Earlier, we postulated that Gln91 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) stabilizes the side chain of Tyr183 via hydrogen bonding interaction between O(H) of Tyr183 and CO of Q91 [Harris, D., et al. (1998) Biochemistry 37, 9630-9640]. To test this hypothesis, we generated mutant derivatives of Gln91 and analyzed their biochemical properties. The efficiency of reverse transcription was severely impaired by nonconservative substitution of Gln with Ala, while conservative substitution of Gln with Asn resulted in an approximately 70% loss of activity, a value similar to that observed with the Y183F mutation. The loss of polymerase activity from both Q91A and Q91N was significantly improved by a Met to Val substitution at position 184. Curiously, the Q91N mutant exhibited stringency in discriminating between correct and incorrect nucleotides, suggesting its possible interaction with residues influencing the flexibility of the dNTP binding pocket. In contrast, both double mutants, Q91A/M184V and Q91N/M184V, are found to be as error prone as the wild-type enzyme. We propose a model that suggests that subtle structural changes in the region due to mutation at position 91 may influence the stability of the side chain of Tyr183 in the catalytic YMDD motif of the enzyme, thus altering the active site geometry that may interfere in substrate recognition.

Combining 3-D quantitative structure-activity relationship with ligand based and structure based alignment procedures for in silico screening of new hepatitis C virus NS5B polymerase inhibitors.

The viral NS5B RNA-dependent RNA-polymerase (RdRp) is one of the best-studied and promising targets for the development of novel therapeutics against hepatitis C virus (HCV). Allosteric inhibition of this enzyme has emerged as a viable strategy toward blocking replication of viral RNA in cell based systems. Herein, we describe how the combination of a complete computational procedure together with biological studies led to the identification of novel molecular scaffolds, hitherto untested toward NS5B polymerase. Structure based 3-D quantitative structure-activity relationship (QSAR) models were generated employing NS5B non-nucleoside inhibitors (NNIs), whose bound conformations were readily available from the protein database (PDB). These were grouped into two training sets of structurally diverse NS5B NNIs, based on their binding to the enzyme thumb (15 NNIs) or palm (10 NNIs) domains. Ligand based (LB) and structure based (SB) alignments were rigorously investigated to assess the reliability on the correct molecular alignment for unknown binding mode modeled compounds. Both Surflex and Autodock programs were able to reproduce with minimal errors the experimental binding conformations of 24 experimental NS5B allosteric inhibitors. Eighty-one (thumb) and 223 (palm) modeled compounds taken from literature were LB and SB aligned and used as external validation sets for the development of 3-D QSAR models. Low error of prediction proved the 3-D QSARs to be useful scoring functions for the in silico screening procedure. Finally, the virtual screening of the NCI Diversity Set led to the selection for enzymatic assays of 20 top-scoring molecules for each final model. Among the 40 selected molecules, preliminary data yielded four derivatives exhibiting IC(50) values ranging between 45 and 75 microM. Binding mode analysis of hit compounds within the NS5B polymerase thumb domain showed that one of them, NSC 123526, exhibited a docked conformation which was in good agreement with the thumb training set most active compound (6).

Structure-based virtual screening, synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening, synthesis and structure-activity relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1), sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for productive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC50 values ranging from 7.7 to 68.0 µM were developed. Docking analysis of lead compound 28 within the tetracyclic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed topological similarities between these two pockets. Compound 28, a novel rhodanine analog with NS5B inhibitory potency in the low micromolar level range may be a promising lead for future development of more potent NS5B inhibitors.

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.

4-Thiazolidinones: a novel class of hepatitis C virus NS5B polymerase inhibitors.

In a quest to identify novel compounds targeting HCV viral replicase, we evaluated a new series of 4-thiazolidinone derivatives (18 compounds). Our in vitro NS5B RdRp inhibition analysis with a series of 2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid (2-(5-nitro-2-furyl/substituted phenyl)-4-thiazolidinone-3-yl) amides (1-7) yielded IC50 values ranging between 45-75 microM. Of these, lead compound 6: 2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid(2-(2-fluorophenyl)-4-thiazolidinone-3-yl)amide exhibited an IC50 value of 48 microM and inhibited NS5B non-competitively with respect to UTP and exhibited a mixed mode of inhibition with respect to RNA. Molecular docking of thiazolidinone derivatives within the allosteric site of NS5B yielded significant correlation between their calculated binding affinity and IC50 values. Taken together, these data suggest that the 4-thiazolidinone scaffold may be optimized for generating new analogues with improved anti-NS5B potency.

Peptide inhibition of HIV-1: current status and future potential.

More than 2 decades of intensive research has focused on defining replication mechanisms of HIV type 1 (HIV-1), the etiologic agent of AIDS. The delineation of strategies for combating this viral infection has yielded many innovative approaches toward this end. HIV-1 is a lentivirus in the family retroviridae that is relatively small with regard to both structure and genome size, having a diploid RNA genome of approximately 9 kb, with only three major genes and several gene products resulting from alternate splicing and translational frameshifting. Most marketed drugs for treating AIDS are inhibitors of HIV-1 reverse transcriptase or protease enzymes, but new targets include the integrase enzyme, cell surface interactions that facilitate viral entry, and also virus particle maturation and assembly. The emergence of drug-resistant variants of HIV-1 has been the main impediment to successful treatment of AIDS. Thus, there is a pressing need to develop novel treatment strategies targeting multiple stages of the virus life-cycle. Research efforts aimed at developing successful means for combating HIV-1 infection have included development of peptide inhibitors of HIV-1. This article summarizes past and current endeavors in the development of peptides that inhibit replication of HIV-1 and the role of peptide inhibitors in the search for new anti-HIV drugs.

Non-nucleoside inhibitors of the hepatitis C virus NS5B RNA-dependant RNA polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives.

Hepatitis C virus (HCV) NS5B RNA polymerase is crucial for replicating the HCV RNA genome and is an attractive target for developing anti-HCV drugs. A novel series of 2,3-diaryl-1,3-thiazolidin-4-one derivatives were evaluated for their ability to inhibit HCV NS5B. Of this series, compounds 4c, 5b, 5c and 6 emerged as more potent, displaying over 95% inhibition of NS5B RNA polymerase activity in vitro. The two most active compounds 4c and 5c exhibited an IC(50) of 31.9 microM and 32.2 microM, respectively, against HCV NS5B.