Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through Structure-Based Virtual Screening.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

Homology modeling of DFG-in FMS-like tyrosine kinase 3 (FLT3) and structure-based virtual screening for inhibitor identification.

The inhibition of FMS-like tyrosine kinase 3 (FLT3) activity using small-molecule inhibitors has emerged as a target-based alternative to traditional chemotherapy for the treatment of acute myeloid leukemia (AML). In this study, we report the use of structure-based virtual screening (SBVS), a computer-aided drug design technique for the identification of new chemotypes for FLT3 inhibition. For this purpose, homology modeling (HM) of the DFG-in FLT3 structure was carried using two template structures, including PDB ID: 1RJB (DFG-out FLT3 kinase domain) and PDB ID: 3LCD (DFG-in CSF-1 kinase domain). The modeled structure was able to correctly identify known DFG-in (SU11248, CEP-701, and PKC-412) and DFG-out (sorafenib, ABT-869 and AC220) FLT3 inhibitors, in docking studies. The modeled structure was then used to carry out SBVS of an HTS library of 125,000 compounds. The top scoring 97 compounds were tested for FLT3 kinase inhibition, and two hits (BPR056, IC50 = 2.3 and BPR080, IC50 = 10.7 µM) were identified. Molecular dynamics simulation and density functional theory calculation suggest that BPR056 (MW: 325.32; cLogP: 2.48) interacted with FLT3 in a stable manner and could be chemically optimized to realize a drug-like lead in the future.

Identification of ligand efficient, fragment-like hits from an HTS library: structure-based virtual screening and docking investigations of 2H- and 3H-pyrazolo tautomers for Aurora kinase A selectivity.

Furanopyrimidine 1 (IC50 = 273 nM, LE = 0.36, LELP = 10.28) was recently identified by high-throughput screening (HTS) of an in-house library (125,000 compounds) as an Aurora kinase inhibitor. Structure-based hit optimization resulted in lead molecules with in vivo efficacy in a mouse tumour xenograft model, but no oral bioavailability. This is attributed to "molecular obesity", a common problem during hit to lead evolution during which degradation of important molecular properties such as molecular weight (MW) and lipophilicity occurs. This could be effectively tackled by the right choice of hit compounds for optimization. In this regard, ligand efficiency (LE) and ligand efficiency dependent lipophilicity (LELP) indices are more often used to choose fragment-like hits for optimization. To identify hits with appropriate LE, we used a MW cut-off <250, and pyrazole structure to filter HTS library. Next, structure-based virtual screening using software (Libdock and Glide) in the Aurora A crystal structure (PDB ID: 3E5A) was carried out, and the top scoring 18 compounds tested for Aurora A enzyme inhibition. This resulted in the identification of a novel tetrahydro-pyrazolo-isoquinoline hit 7 (IC50 = 852 nM, LE = 0.44, LELP = 8.36) with fragment-like properties suitable for further hit optimization. Moreover, hit 7 was found to be selective for Aurora A (Aurora B IC50 = 35,150 nM) and the possible reasons for selectivity investigated by docking two tautomeric forms (2H- and 3H-pyrazole) of 7 in Auroras A and B (PDB ID: 4AF3) crystal structures. This docking study shows that the major 3H-pyrazole tautomer of 7 binds in Aurora A stronger than in Aurora B.

Ligand efficiency based approach for efficient virtual screening of compound libraries.

Here we report for the first time the use of fit quality (FQ), a ligand efficiency (LE) based measure for virtual screening (VS) of compound libraries. The LE based VS protocol was used to screen an in-house database of 125,000 compounds to identify aurora kinase A inhibitors. First, 20 known aurora kinase inhibitors were docked to aurora kinase A crystal structure (PDB ID: 2W1C); and the conformations of docked ligand were used to create a pharmacophore (PH) model. The PH model was used to screen the database compounds, and rank (PH rank) them based on the predicted IC50 values. Next, LE_Scale, a weight-dependant LE function, was derived from 294 known aurora kinase inhibitors. Using the fit quality (FQ = LE/LE_Scale) score derived from the LE_Scale function, the database compounds were reranked (PH_FQ rank) and the top 151 (0.12% of database) compounds were assessed for aurora kinase A inhibition biochemically. This VS protocol led to the identification of 7 novel hits, with compound 5 showing aurora kinase A IC50 = 1.29 µM. Furthermore, testing of 5 against a panel of 31 kinase reveals that it is selective toward aurora kinase A & B, with <50% inhibition for other kinases at 10 µM concentrations and is a suitable candidate for further development. Incorporation of FQ score in the VS protocol not only helped identify a novel aurora kinase inhibitor, 5, but also increased the hit rate of the VS protocol by improving the enrichment factor (EF) for FQ based screening (EF = 828), compared to PH based screening (EF = 237) alone. The LE based VS protocol disclosed here could be applied to other targets for hit identification in an efficient manner.

Development of an anti-influenza drug screening assay targeting nucleoproteins with tryptophan fluorescence quenching.

Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV.

Diosgenin, a plant-derived sapogenin, exhibits antiviral activity in vitro against hepatitis C virus.

Diosgenin (3ß-hydroxy-5-spirostene, 1), a plant-derived sapogenin, is used as a dietary supplement. However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 µM. No cellular toxicity was observed at this concentration. Diosgenin (1) also significantly reduced the levels of viral RNA and viral proteins as evaluated by quantitative real-time reverse transcriptase PCR and Western blot analysis, respectively. In addition, in an alternative HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 µM. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1 and interferon-α exerted an additive effect on the resultant anti-HCV activity.

BPR2-D2 targeting viral ribonucleoprotein complex-associated function inhibits oseltamivir-resistant influenza viruses.

OBJECTIVES: The emergence of oseltamivir-resistant viruses raised the global threat with regard to influenza virus infection. To develop alternative antiviral agents against influenza virus infection is significant and urgent. METHODS: A neutralization test was applied as a screening assay and a plaque reduction assay was used for confirmation. Expression plasmids for viral ribonucleoproteins (RNPs) and a plasmid that allowed expression of a pseudoviral reporter RNA were transfected into cells to investigate the effects of a novel antiviral compound on viral RNA synthesis. RESULTS: BPR2-D2 was identified as a novel inhibitor against influenza virus from a hit obtained from high throughput screening of 20 000 or more compounds. BPR2-D2 exhibited an excellent antiviral efficacy for the oseltamivir-resistant virus (EC(50) ranging from 0.021 to 0.040 microM). No resistant virus was produced throughout 20 passages in the presence of BPR2-D2, whereas oseltamivir-resistant virus was generated at passage 8 using the same experimental system. A molecular target other than neuraminidase (NA) was found because BPR2-D2 inhibited the synthesis of viral RNA that was driven by influenza viral RNP in a transfection assay. BPR2-D2 also exhibited a broad antiviral spectrum against various strains of influenza A and influenza B viruses. CONCLUSIONS: BPR2-D2 was identified as a novel inhibitor of influenza virus. It may target viral RNPs that are responsible for viral RNA synthesis. Targeting different molecules compared with NA allows BPR2-D2 to inhibit oseltamivir-resistant viruses.

Anti-HSV activity of digitoxin and its possible mechanisms.

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC(50)) of 0.05 microM. The 50% cytotoxicity concentration (CC(50)) of digitoxin is 10.66 microM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors.

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.

High-throughput cell-based screening for hepatitis C virus NS3/4A protease inhibitors.

Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.

Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate (TF3).

SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C-like protease (3CL(Pro)) has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CL(Pro). Two compounds in the library were found to be inhibitive: tannic acid (IC(50) = 3 microM) and 3-isotheaflavin-3-gallate (TF2B) (IC(50) = 7 microM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CL(Pro)-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CL(Pro). Several other known compositions in teas were also evaluated for their activities in inhibiting 3CL(Pro). We found that caffeine, (-)-epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CL(Pro) activity. Only theaflavin-3,3'-digallate (TF3) was found to be a 3CL(Pro) inhibitor. This study has resulted in the identification of new compounds that are effective 3CL(Pro) inhibitors.

Inhibition of hepatitis C virus replication by arsenic trioxide.

Hepatitis C virus (HCV) is a serious global problem, and present therapeutics are inadequate to cure HCV infection. In the present study, various antiviral assays show that As2O3 at submicromolar concentrations is capable of inhibiting HCV replication. The 50% effective concentration (EC50) of As2O3 required to inhibit HCV replication was 0.35 microM when it was determined by a reporter-based HCV replication assay, and the EC50 was below 0.2 microM when it was determined by quantitative reverse transcription-PCR analysis. As2O3 did not cause cellular toxicity at this concentration, as revealed by an MTS [3-(4,5-dimethylthiozol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. A combination of As2O3 and alpha interferon exerted synergistic effects against HCV, as revealed by a multiple linear logistic model and isobologram analysis. Furthermore, in an alternative HCV antiviral system that may recapitulate additional steps involved in HCV infection and replication, As2O3 at 0.3 microM totally abolished the HCV signal, whereas alpha interferon at a high dose (5,000 IU/ml) only partially suppressed the HCV signal. The study highlights the indications for use of a novel class of anti-HCV agent. Further elucidation of the exact antiviral mechanism of As2O3 may lead to the development of agents with potent activities against HCV or related viruses.

Anti-HCV activities of selective polyunsaturated fatty acids.

HCV infection can lead to chronic infectious hepatitis disease with serious sequelae. Interferon-alpha, or its PEGylated form, plus ribavirin is the only treatment option to combat HCV. Alternative and more effective therapy is needed due to the severe side effects and unsatisfactory curing rate of the current therapy. In this study, we found that several polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are able to exert anti-HCV activities using an HCV subgenomic RNA replicon system. The EC(50) (50% effective concentration to inhibit HCV replication) of AA was 4microM that falls in the range of physiologically relevant concentration. At 100microM, alpha-linolenic acid, gamma-linolenic, and linoleic acid only reduced HCV RNA levels slightly and saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid had no inhibitory activities toward HCV replication. When AA was combined with IFN-alpha, strong synergistic anti-HCV effect was observed as revealed by an isobologram analysis. It will be important to determine whether PUFAs can provide synergistic antiviral effects when given as food supplements during IFN-based anti-HCV therapy. Further elucidation of the exact anti-HCV mechanism caused by AA, DHA, and EPA may lead to the development of agents with potent activity against HCV or related viruses.

A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.