Inhibition of dengue virus by targeting viral NS4B protein.

We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 µM; and 50% cytotoxic concentration [CC(50)], >40 µM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis-P104L-abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.

Anti-infectives: can cellular screening deliver?

In an era of emerging and reemerging infectious diseases, and increasing multidrug resistance, the need to identify novel therapy is imperative. Unfortunately, the recent shift of the drug discovery paradigm from cellular screening to target-based approaches has not delivered the anticipated benefits. A recent renaissance of the traditional cell-based approach, on the other hand, has yielded several clinical candidates. Three successful examples are illustrated in this review, namely spiroindolone, thiazolidinone, and diarylquinoline for the treatment of malaria, hepatitis C virus, and tuberculosis, respectively. We describe in detail their identification, mechanism of action (MoA), and common features in the chemical structures. The challenges of the cell-based approach for anti-infective drug discovery are also discussed. We propose a shift from standard libraries to synthetic natural-product-like compound collections to improve the success of phenotypic lead finding and to facilitate the validation of hits.

Development and characterization of a stable luciferase dengue virus for high-throughput screening.

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z' factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.

Inhibition of dengue virus through suppression of host pyrimidine biosynthesis.

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC(50)) of 2.4 nM and a 50% cytotoxic concentration (CC(50)) of >5 µM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC(90)s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.