NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility.

Dengue NS2A Protein Orchestrates Virus Assembly.

Dengue virus assembly requires cleavage of viral C-prM-E polyprotein into three structural proteins (capsid, premembrane, and envelope), packaging of viral RNA with C protein into nucleocapsid, and budding of prM and E proteins into virions. The molecular mechanisms underlying these assembly events are unclear. Here, we show that dengue nonstructural protein 2A (NS2A protein) recruits viral RNA, structural proteins, and protease to the site of virion assembly and coordinates nucleocapsid and virus formation.

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit.

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC<0.1μM.

Flavivirus Entry Inhibitors.

Many flaviviruses are significant human pathogens that are transmitted by mosquitoes and ticks. Although effective vaccines are available for yellow fever virus, Japanese encephalitic virus, and tick-borne encephalitis virus, these and other flaviviruses still cause thousands of human deaths and millions of illnesses each year. No clinically approved antiviral therapy is available for flavivirus treatment.

Secondary Structure and Membrane Topology of the Full-Length Dengue Virus NS4B in Micelles.

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments.

Secondary structure and membrane topology of dengue virus NS4B N-terminal 125 amino acids.

The transmembrane NS4B protein of dengue virus (DENV) is a validated antiviral target that plays important roles in viral replication and invasion of innate immune response. The first 125 amino acids of DENV NS4B are sufficient for inhibition of alpha/beta interferon signaling. Resistance mutations to NS4B inhibitors are all mapped to the first 125 amino acids.

Discovery of Dengue Virus NS4B Inhibitors.

The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo.

Lead optimization of spiropyrazolopyridones: a new and potent class of dengue virus inhibitors.

Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described.

Development of a stable Gaussia luciferase enterovirus 71 reporter virus.

We report a stable Gaussia luciferase enterovirus 71 (Gluc-EV71) reporter virus to facilitate drug discovery. The Gluc-EV71 reporter virus was generated by engineering the Gaussia luciferase (Gluc) gene between the 5' untranslated region and VP4 gene of the EV71 genome. We could recover Gluc-EV71 after transfection of Vero cells with the cDNA clone-derived RNA.

Targeting dengue virus NS4B protein for drug discovery.

The flavivirus nonstructural 4B protein (NS4B) has recently emerged as a valid antiviral target for drug discovery. Here we review (i) the current understanding of the structure and function of DENV NS4B, (ii) the approaches that have been taken to identify NS4B inhibitors, and (iii) the known inhibitors of flavivirus NS4B protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.

Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens.

Mapping the Interactions between the NS4B and NS3 proteins of dengue virus.

Unlabelled: Flavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive.

Characterization of dengue virus NS4A and NS4B protein interaction.

Unlabelled: Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins.

Anti-dengue-virus activity and structure-activity relationship studies of lycorine derivatives.

Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. No vaccines or specific therapeutics are currently available for dengue. Lycorine, which is a natural plant alkaloid, has been shown to possess antiviral activities against flaviviruses.

Generation and characterization of mouse monoclonal antibodies against NS4B protein of dengue virus.

Dengue virus (DENV) non-structural protein 4B (NS4B) has been demonstrated to be an attractive antiviral target. Due to its nature as an integral membrane protein, NS4B remains poorly characterized. In this study, we generated and characterized two monoclonal antibodies (mAb) that selectively bind to DENV NS4B protein.

Dimerization of flavivirus NS4B protein.

Unlabelled: Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood.

Ten years of dengue drug discovery: progress and prospects.

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV.

NMR analysis of a novel enzymatically active unlinked dengue NS2B-NS3 protease complex.

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target.

Genomic organization and promoter characterization of FcCTL, a C-type lectin-like protein in Fenneropenaeus chinensis.

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. C-type lectins may play an important role in shrimp immunity. The gene encoding FcCTL, a C-type lectin-like protein, was isolated from Fenneropenaeus chinensis and characterized.

Molecular cloning and differential expression of three GnRH genes during ovarian maturation of spotted halibut, Verasper variegatus.

In this study, the gonadotropin-releasing hormone (GnRH) genes in spotted halibut were cloned and sequenced by isolating their cDNAs. The species expressed three molecular forms of GnRH in the brain: chicken-type GnRH-II (cGnRH-II), seabream-type GnRH (sbGnRH), and salmon-type GnRH (sGnRH). Phylogenetic analysis divided the molecular forms of GnRHs into three branches: cGnRH-II branch, sGnRH branch, and fish-specific GnRH branch.

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).

A translation inhibitor that suppresses dengue virus in vitro and in vivo.

We describe a novel translation inhibitor that has anti-dengue virus (DENV) activity in vitro and in vivo. The inhibitor was identified through a high-throughput screening using a DENV infection assay. The compound contains a benzomorphan core structure.

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.

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.

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.