Neerja Kaushik-Basu

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.

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.

Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC50] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.

Structure-Based Discovery of Pyrazolobenzothiazine Derivatives As Inhibitors of Hepatitis C Virus Replication

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 μM, EC90 = 25.6 μM, and CC50 > 180 μM in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

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.

2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-nucleoside inhibitors of HCV NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as inhibitors of HCV NS5B polymerase. Eleven compounds tested were found to inhibit HCV NS5B with IC₅₀ values ranging between 19.8 and 64.9 μM. Compound 24 was the most active of this series with an IC₅₀ of 5.6 μM. A number of these derivatives further exhibited strong inhibition against HCV 1b and 2a genotypes in cell based antiviral assays. Molecular docking analysis predicted that the thiazolidinone derivatives bind to the NS5B thumb pocket-II (TP-II). Our results suggest that further optimization of the thiazolidinone scaffold may be possible to yield new derivatives with improved enzyme- and cell-based activity.

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.

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 α-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.

New Pyrazolobenzothiazine Derivatives as Hepatitis C Virus NS5B Polymerase Palm Site I Inhibitors

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 μM against NS5B polymerase and antiviral effect (EC50 = 8.1 μM; EC90 = 23.3 μM) coupled with the absence of any antimetabolic effect (CC50 > 224 μM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.