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Flaviviruses are important human pathogens and include dengue (DENV), West Nile (WNV), Yellow fever virus (YFV), Japanese encephalitis (JEV) and Zika virus (ZIKV). DENV, transmitted by mosquitoes, causes diseases ranging in severity from mild dengue fever with non-specific flu-like symptoms to fatal dengue hemorrhagic fever and dengue shock syndrome (Clyde et al., 2006). DENV infections are caused by four serotypes, DENV1-4, which interact differently with antibodies in blood serum. The incidence of DENV infection has increased dramatically in recent decades and the CDC estimates 400 million dengue infections occur each year, resulting in ∼25,000 deaths mostly among children and elderly people. Similarly, ZIKV infections are caused by infected mosquito bites to humans, can be transmitted sexually and through blood transfusions. If a pregnant woman is infected, the virus can cross the placental barrier and can spread to her fetus, causing severe brain malformations in the child including microcephaly and other birth defects. It is noteworthy that the neurological manifestations of ZIKV were also observed in DENV endemic regions, suggesting that pre-existing antibody response to DENV could augment ZIKV infection. WNV, previously unknown in the US (and known to cause only mild disease in Middle East), first arrived in New York city in 1999 (NY99) and spread throughout the US and Canada by Culex mosquitoes and birds. WNV is now endemic in North America. Thus, emerging and re-emerging flaviviruses are significant threat to human health. However, vaccines are available for only a limited number of flaviviruses, and antiviral therapies are not available for any flavivirus. Hence, there is an urgent need to develop therapeutics that interfere with essential enzymatic steps, such as protease in the flavivirus lifecycle as these viruses possess significant threat to future pandemics. In this review, we focus on our E. coli expression of NS2B hydrophilic domain (NS2BH) covalently linked to NS3 protease domain (NS3Pro) in their natural context which is processed by the combined action of both subunits of the NS2B-NS3Pro precursor. Biochemical activities of the viral protease such as solubility and autoproteolysis of NS2BH-NS3Pro linkage depended on the C-terminal portion of NS2BH linked to the NS3Pro domain. Since 2008, we also focus on the use of the recombinant protease in high throughput screens and characterization of small molecular compounds identified in these screens.

In a previous study, twelve antimalarial compounds, amodiaquine (AQ) and derivatives, were shown to have potent anti-dengue viral (DENV) activity by using the stable DENV2 Renilla luciferase reporter replicon expressing BHK-21 cells, infectivity (plaque), and the qRT-PCR assays. In this study, we performed molecular modeling on these compounds to determine their stereo-electronic properties required for optimal antiviral activity. Based on the similarity of calculated stereo-electronic profiles, specifically the electrostatic potential profiles of the compounds, and in silico screening of related compounds from literature, we identified three additional compounds, Quinacrine (QC), Mefloquine (MQ), and GSK369796. Analysis of their antiviral activities indicated that all three compounds have high anti-DENV activity in the DENV2 replicon expressing cells with EC50 values of 5.30 ± 1.31 μM (QC), 3.22 ± 0.37 μM (MQ), and 5.06 ± 0.86 μM (GSK369796). The infectivity assays revealed the EC50 values of 7.09 ± 1.67 μM (QC), 4.36 ± 0.31 μM (MQ) and 3.03 ± 0.35 μM (GSK369796). The mode of action of these compounds is through inhibition of autophagy, thereby affecting DENV2 replication. Moreover, these compounds also showed antiviral activity against the rapidly emerging Zika virus (ZIKV) with EC50 values of 2.27 ± 0.14 μM (QC), 3.95 ± 0.21 μM (MQ), and 2.57 ± 0.09 μM (GSK369796).

The mosquito-borne dengue virus serotypes 1–4 (DENV1-4) and West Nile virus (WNV) cause serious illnesses worldwide associated with considerable morbidity and mortality. According to the World Health Organization (WHO) estimates, there are about 390 million infections every year leading to ∼500,000 dengue haemorrhagic fever (DHF) cases and ∼25,000 deaths, mostly among children. Antiviral therapies could reduce the morbidity and mortality associated with flaviviral infections, but currently there are no drugs available for treatment. In this study, a high-throughput screening assay for the Dengue protease was employed to screen ∼120,000 small molecule compounds for identification of inhibitors. Eight of these inhibitors have been extensively analyzed for inhibition of the viral protease in vitro and cell-based viral replication using Renilla luciferase reporter replicon, infectivity (plaque) and cytotoxicity assays. Three of these compounds were identified as potent inhibitors of DENV and WNV proteases, and viral replication of DENV2 replicon and infectious RNA. Fluorescence quenching, kinetic analysis and molecular modeling of these inhibitors into the structure of NS2B-NS3 protease suggest a mode of inhibition for three compounds that they bind to the substrate binding pocket.

The dengue virus is considered to be a globally important human pathogen prevalent in tropical and subtropical regions of the world. According to a recent estimate, the disease burden due to DENV infections is ∼390 million infections per year globally in ∼100 countries including the southern US, Puerto Rico and Hawaii, resulting in nearly ∼25,000 deaths mostly among children. Despite the significant morbidity and mortality that results from DENV infections, there is currently no effective chemotherapeutic treatment for DENV infections. We identified curcumin as an inhibitor of DENV2 NS2B/NS3protease in a previous high-throughput screening (HTS) campaign. We synthesized four analogues of curcumin (curcuminoids) and tested the in vitro protease inhibition activity and inhibition of replication by cell-based assays. The results revealed that curcumin is a weak inhibitor of the viral protease. However, the analogues exhibited more potent inhibition of DENV infectivity in plaque assays suggesting that the cellular pathway(s) required for viral replication and/or assembly are targeted by these compounds. Further analysis shows that inhibition of genes involved in lipid biosynthesis, and of actin polymerization by curcuminoids, are likely to be involved as their mode of action in DENV2-infected cells. Three of the curcumin derivatives possess good selectivity indices (SI) (>10) when compared to the parent curcumin.

Dengue virus serotypes 1–4 (DENV1–4) are transmitted by mosquitoes which cause most frequent arboviral infections in the world resulting in ∼390 million cases with ∼25,000 deaths annually. There is no vaccine or antiviral drug currently available for human use. Compounds containing quinoline scaffold were shown to inhibit flavivirus NS2B–NS3 protease (NS2B–NS3pro) with good potencies. In this study, we screened quinoline derivatives, which are known antimalarial drugs for inhibition of DENV2 and West Nile virus (WNV) replication using the corresponding replicon expressing cell-based assays. Amodiaquine (AQ), one of the 4-aminoquinoline drugs, inhibited DENV2 infectivity measured by plaque assays, with EC50 and EC90 values of 1.08 ± 0.09 μM and 2.69 ± 0.47 μM, respectively, and DENV2 RNA replication measured by Renilla luciferase reporter assay, with EC50 value of 7.41 ± 1.09 μM in the replicon expressing cells. Cytotoxic concentration (CC50) in BHK-21 cells was 52.09 ± 4.25 μM. The replication inhibition was confirmed by plaque assay of the extracellular virions as well as by qRT-PCR of the intracellular and extracellular viral RNA levels. AQ was stable for at least 96 h and had minor inhibitory effect on entry, translation, and post-replication stages in the viral life cycle. DENV protease, 5′-methyltransferase, and RNA-dependent RNA polymerase do not seem to be targets of AQ. Both p-hydroxyanilino and diethylaminomethyl moieties are important for AQ to inhibit DENV2 replication and infectivity. Our results support AQ as a promising candidate for anti-flaviviral therapy.

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.

Four serotypes of dengue virus (DENV1–4), mosquito-borne members of Flaviviridae family cause frequent epidemics causing considerable morbidity and mortality in humans throughout tropical regions of the world. There is no vaccine or antiviral therapeutics available for human use. In a previous study, we reported that compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of West Nile virus serine protease. In this study, we analyzed potencies of some compounds with (8-HQ)-aminobenzothiazole derivatives for inhibition of DENV2 protease in vitro. We identified analogs 1–4 with 2-aminothiazole or 2-aminobenzothiazole scaffold with sub-micromolar potencies (IC50) in the in vitro protease assays. The kinetic constant (Ki) for the most potent 8-HQ-aminobenzothiazole inhibitor (compound 1) with an IC50 value of 0.91 ± 0.05 μM was determined to be 2.36 ± 0.13 μM. This compound inhibits the DENV2 NS2B/NS3pro by a competitive mode of inhibition.

West Nile virus (WNV) is a mosquito-borne member of flaviviruses that causes significant morbidity and mortality especially among children. There is currently no approved vaccine or antiviral therapeutic for human use. In a previous study, we described compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of WNV serine protease (NS2B/NS3pro) in a high throughput screen (HTS) using the purified WNV NS2B/NS3pro as the target. In this study, we analyzed potencies of some commercially available as well as chemically synthesized derivatives of 8-HQ by biochemical assays. An insight into the contribution of various substitutions of 8-HQ moiety for inhibition of the protease activity was revealed. Most importantly, the substitution of the N1 of the 8-HQ ring by –CH– in compound 26 significantly reduced the inhibition of the viral protease by this naphthalen-1-ol derivative. The kinetic constant ( K i ) for the most potent 8-HQ inhibitor (compound 14) with an IC 50 value of 2.01 ± 0.08 μM using the tetra -peptide substrate was determined to be 5.8 μM. This compound inhibits the WNV NS2B/NS3pro by a competitive mode of inhibition which is supported by molecular modeling.