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Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1 log PFU/ml reduction of the virus infectious titre. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is the only causative agent of CHIKV fever with persistent arthralgia, and in some cases may lead to neurological complications which can be highly fatal, therefore it poses severe health issues in many parts of the world. CHIKV transmission can be mediated via the Aedes albopictus mosquito; however, very little is currently known about the involvement of mosquito cellular factors during CHIKV-infection within the mosquito cells. Unravelling the neglected aspects of mosquito proteome changes in CHIKV-infected mosquito cells may increase our understanding on the differences in the host factors between arthropod and mammalian cells for successful replication of CHIKV. In this study, the CHIKV-infected C6/36 cells with differential cellular proteins expression were profiled using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2DE analysis on CHIKV-infected C6/36 cells has shown 23 mosquito cellular proteins that are differentially regulated, and which are involved diverse biological pathways, such as protein folding and metabolic processes. Among those identified mosquito proteins, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD have been found to regulate CHIKV infection. Furthermore, siRNA-mediated gene knockdown of these proteins has demonstrated the biological importance of these host proteins that mediate CHIKV infection. These findings have provided an insight to the importance of mosquito host factors in the replication of CHIKV, thus providing a potential channel for developing novel antiviral strategies against CHIKV transmission. Author Summary: Chikungunya virus (CHIKV), a re-emerging mosquito-borne virus, is the main cause of CHIKV fever, persistent arthralgia and serious neurological complications which can be highly fatal; therefore, it poses serious health threats worldwide. Unraveling the underappreciated aspects of mosquito cellular factors that contribute to the replication processes of CHIKV was performed using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The 2DE proteomics profiling of CHIKV-infected mosquito C6/36 cells has revealed twenty-three proteins that were differentially regulated upon CHIKV infection. These proteins are shown to be involved in diverse biological pathways and cellular processes. Notably, upon selected genes knockdown, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD are found to be important during the replication processes of CHIKV. This study illustrates the importance of mosquito cellular factors in association with CHIKV infection in mosquito cells and reveals an interesting portal for developing novel antiviral strategies in CHIKV studies. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is the only causative agent of CHIKV fever with persistent arthralgia, and in some cases may lead to neurological complications which can be highly fatal, therefore it poses severe health issues in many parts of the world. CHIKV transmission can be mediated via the Aedes albopictus mosquito; however, very little is currently known about the involvement of mosquito cellular factors during CHIKV-infection within the mosquito cells. Unravelling the neglected aspects of mosquito proteome changes in CHIKV-infected mosquito cells may increase our understanding on the differences in the host factors between arthropod and mammalian cells for successful replication of CHIKV. In this study, the CHIKV-infected C6/36 cells with differential cellular proteins expression were profiled using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2DE analysis on CHIKV-infected C6/36 cells has shown 23 mosquito cellular proteins that are differentially regulated, and which are involved diverse biological pathways, such as protein folding and metabolic processes. Among those identified mosquito proteins, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD have been found to regulate CHIKV infection. Furthermore, siRNA-mediated gene knockdown of these proteins has demonstrated the biological importance of these host proteins that mediate CHIKV infection. These findings have provided an insight to the importance of mosquito host factors in the replication of CHIKV, thus providing a potential channel for developing novel antiviral strategies against CHIKV transmission. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) infection, a febrile illness caused by a mosquito-transmitted alphavirus, has afflicted millions of people worldwide. There is currently no approved effective antiviral treatment for CHIKV infection. In this study, we report a new class of small-molecule CHIKV inhibitors, the oxindole-labdanes, that potently block the replication of CHIKV with good selectivity. Andrographolide, a previously reported inhibitor of CHIKV infection, was used as the lead compound for our initial structure-activity relationship (SAR) study. From a focused library of 72 andrographolide analogues, we identified the lead compound (E) - 2 with improved antiviral activities. Further optimization of (E) - 2 led to the discovery of the normal -labdane 7-chloro-oxindole (E) - 42 as potent inhibitor against two low-passage CHIKV isolates from human patients with an EC 50 of 1.55 μM against CHIKV-122508 and 0.14 μM against CHIKV-6708. Compound (E) - 42 displayed minimal cytotoxic liability (CC 50 > 100 μM), thus furnishing good selectivity relative to the host cells. Mechanistically, (E) - 42 does not inactivate the viral particles but rather acts on the host cells to interfere with the viral replication, demonstrating both prophylactic and therapeutic effects. Our findings open a new avenue for the development of oxindole-labdane compounds as promising antiviral drugs against CHIKV infection. [Display omitted] • A series of novel oxindole-labdane hybrids were developed as small-molecule inhibitors against Chikungunya virus infection. • Compound (E) - 42 displays potent inhibitory effects against two CHIKV variants isolated from infected patients. • (E) - 42 acts on the host cells to interfere with the viral replication, demonstrating prophylactic and therapeutic effects. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne virus that has garnered a lot of attention in recent years, due to the explosive epidemic from 2014 to 2016. Since its introduction in the Americas in late 2014, ZIKV has spread at an unprecedented rate and scale throughout the world and infected millions of people. Its infection has also been associated with severe neurological disorders like Guillain-Barré syndrome and microcephaly in fetuses. Despite these, there is currently no approved antiviral against ZIKV. In this study, an immunofluorescence-based high throughput screen was conducted on a library of 483 flavonoid derivatives to identify potential anti-ZIKV compounds. Flavonoids, which are natural polyphenolic compounds found in plants, represent an attractive source of antivirals due to their abundance in food and expected low toxicity. From the primary screen, three hits were selected for validation by cell viability and viral plaque reduction assays. Pinocembrin, a flavanone found in honey, tea and red wine, was chosen for downstream studies as it exhibited the strongest inhibition of ZIKV infection in human placental JEG-3 cells (IC 50 = 17.4 μM). Time-course studies revealed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. Furthermore, pinocembrin inhibits viral RNA production and envelope protein synthesis based on quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses. This study has demonstrated for the first time the in vitro anti-ZIKV activity of pinocembrin. Image 1 • 483 flavonoid derivatives were screened for anti-Zika virus (ZIKV) activity in human placental JEG-3 cells. • Three hits were validated using dose response and cell viability assays. • Pinocembrin, a flavanone found in honey and tea, exhibited the strongest inhibition of ZIKV replication (IC 50 = 17.4 μM). • Mechanism studies showed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. • Pinocembrin was also shown to have broad-spectrum antiviral effects against DENV2 and CHIKV. [ABSTRACT FROM AUTHOR]

Hand Foot Mouth Disease (HFMD), resulting from human enterovirus A71 (HEVA71) infection can cause severe neurological complications leading to fatality in young children. Currently, there is no approved antiviral for therapeutic treatment against HEVA71 infection. In this study, a 500-compound flavonoid library was screened to identify potential inhibitors of HEVA71 using high-throughput immunofluorescence-based phenotypic screening method. Two lead flavonoid compounds, ST077124 and ST024734 at the non-cytotoxic concentration of 50 μM were found to be effective antivirals that inhibited replication of HEVA71, reducing infectious viral titers by 3.5 log 10 PFU/ml and 2.5 log 10 PFU/ml respectively. Our study revealed that ST077124 is a specific antiviral compound that inhibits human enteroviruses while ST024734 exhibited antiviral activity against human enteroviruses as well as dengue virus type-2. We also identified that both compounds affected the viral RNA transcription and translation machinery of HEVA71 but did not interfere with the viral internal ribosomal entry site (IRES) activity. Hence, our findings strongly suggest that ST077124 and ST024734 are effective antiviral compounds of minimal cytotoxicity and could serve as promising therapeutic agents against HEVA71 infection. [ABSTRACT FROM AUTHOR]

[Display omitted] Human enterovirus A71 (EV-A71) is a major etiological agent of hand-foot-and-mouth disease (HFMD) and there is presently no internationally approved antiviral against EV-A71. In this study, it is disclosed that 14 S -(2'-chloro-4'-nitrophenoxy)-8 R / S ,17-epoxy andrographolide (2) was discovered to be an effective inhibitor against EV-A71 infection showing significant reduction of viral titre. In addition to EV-A71, compound 2 exerts broad-spectrum antiviral effects against other enteroviruses. It is revealed that compound 2 inhibits the post-entry stages of EV-A71 viral replication cycle and significantly reduces viral protein expression of structural proteins such as VP0 and VP2 via inhibiting EV-A71 RNA replication. Moreover, the inhibitory property of compound 2 is specific to viral RNA replication. Furthermore, compound 2 is more likely to target a host factor in EV-A71 RNA replication. As a result, introduction of epoxide at positions 8 and 17 of andrographolide is effective for anti-EV-A71 infection and is a potential anti-EV-A71 strategy. Further work to discover more potent andrographolide derivatives and elucidate comprehensive SAR is under way. [ABSTRACT FROM AUTHOR]