Attenuation of neurovirulence of chikungunya virus by a single amino acid mutation in viral E2 envelope protein.

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. METHODS: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. RESULTS: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. CONCLUSIONS: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.

Flavivirus genome recoding by codon optimisation confers genetically stable in vivo attenuation in both mice and mosquitoes.

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

Chelerythrine chloride inhibits Zika virus infection by targeting the viral NS4B protein.

Zika virus (ZIKV) is a mosquito-borne virus that has re-emerged as a significant threat to global health in the recent decade. Whilst infections are primarily asymptomatic, the virus has been associated with the manifestation of severe neurological complications. At present, there is still a lack of approved antivirals for ZIKV infections. In this study, chelerythrine chloride, a benzophenanthridine alkaloid, was identified from a mid-throughput screen conducted on a 502-compound natural products library to be a novel and potent inhibitor of ZIKV infection in both in-vitro and in-vivo assays. Subsequent downstream studies demonstrated that the compound inhibits a post-entry step of the viral replication cycle and is capable of disrupting viral RNA synthesis and protein expression. The successful generation and sequencing of a ZIKV resistant mutant revealed that a single S61T mutation on the viral NS4B allowed ZIKV to overcome chelerythrine chloride inhibition. Further investigation revealed that chelerythrine chloride could directly inhibit ZIKV protein synthesis, and that the NS4B-S61T mutation confers resistance to this inhibition. This study has established chelerythrine chloride as a potential candidate for further development as a therapeutic agent against ZIKV infection.

AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.

BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.

Antiviral Activity of Catechin against Dengue Virus Infection.

Dengue virus (DENV) is the cause of dengue fever, infecting 390 million people worldwide per year. It is transmitted to humans through the bites of mosquitoes and could potentially develop severe symptoms. In spite of the rising social and economic impact inflicted by the disease on the global population, a conspicuous lack of efficacious therapeutics against DENV still persists. In this study, catechin, a natural polyphenol compound, was evaluated as a DENV infection inhibitor in vitro. Through time-course studies, catechin was shown to inhibit a post-entry stage of the DENV replication cycle. Further investigation revealed its role in affecting viral protein translation. Catechin inhibited the replication of all four DENV serotypes and chikungunya virus (CHIKV). Together, these results demonstrate the ability of catechin to inhibit DENV replication, hinting at its potential to be used as a starting scaffold for further development of antivirals against DENV infection.

The host-targeting compound peruvoside has a broad-spectrum antiviral activity against positive-sense RNA viruses.

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.

Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants.

The COVID-19 pandemic has brought about unprecedented medical and healthcare challenges worldwide. With the continual emergence and spread of new COVID-19 variants, four drug compound libraries were interrogated for their antiviral activities against SARS-CoV-2. Here, we show that the drug screen has resulted in 121 promising anti-SARS-CoV-2 compounds, of which seven were further shortlisted for hit validation: citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate. In particular, the active form of vitamin D, calcitriol, exhibits strong potency against SARS-CoV-2 on cell-based assays and is shown to work by modulating the vitamin D receptor pathway to increase antimicrobial peptide cathelicidin expression. However, the weight, survival rate, physiological conditions, histological scoring, and virus titre between SARS-CoV-2 infected K18-hACE2 mice pre-treated or post-treated with calcitriol were negligible, indicating that the differential effects of calcitriol may be due to differences in vitamin D metabolism in mice and warrants future investigation using other animal models.

Discovery of Novel Andrographolide Derivatives as Antiviral Inhibitors against Human Enterovirus A71.

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

Discovery and development of labdane-oxindole hybrids as small-molecule inhibitors against chikungunya virus infection.

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 EC50 of 1.55 µM against CHIKV-122508 and 0.14 µM against CHIKV-6708. Compound (E)-42 displayed minimal cytotoxic liability (CC50 > 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.

Discovery of 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide as EV-A71 infection inhibitor.

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 14S-(2'-chloro-4'-nitrophenoxy)-8R/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.

A single-dose live attenuated chimeric vaccine candidate against Zika virus.

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.

Betulinic acid exhibits antiviral effects against dengue virus infection.

Dengue virus (DENV) is an arthropod-borne virus that has developed into a prominent global health threat in recent decades. The main causative agent of dengue fever, the virus infects an estimated 390 million individuals across the globe each year. Despite the sharply increasing social and economic burden on global society caused by the disease, there is still a glaring lack of effective therapeutics against DENV. In this study, betulinic acid, a naturally occurring pentacyclic triterpenoid was established as an inhibitor of DENV infection in vitro. Time-course studies revealed that betulinic acid inhibits a post-entry stage of the DENV replication cycle and subsequent analyses also showed that the compound is able to inhibit viral RNA synthesis and protein production. Betulinic acid also demonstrated antiviral efficacy against other serotypes of DENV, as well as against other mosquito-borne RNA viruses such as Zika virus and Chikungunya virus, which are commonly found co-circulating together with DENV. As such, betulinic acid may serve as a valuable starting point for the development of antivirals to combat potential DENV outbreaks, particularly in tropical and subtropical regions which make up a large majority of documented infections.

Drug repurposing of pyrimidine analogs as potent antiviral compounds against human enterovirus A71 infection with potential clinical applications.

Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine's synergistic effects with interferon-ß at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.

Antiviral activity of the FDA-approved drug candesartan cilexetil against Zika virus infection.

Zika virus (ZIKV) is a mosquito-borne virus that has risen to prominence as a significant threat to public health in the recent decade. Since its re-emergence in 2007, ZIKV has spread at an alarming rate and has since become endemic to multiple regions around the world. Infections are primarily asymptomatic, however the virus has become associated with the development of severe neurological complications such as Guillain-Barré syndrome (GBS) and congenital microcephaly. At present, there are currently no approved antivirals for ZIKV infections. In this study, we utilised a phenotype-based screening platform to perform a high-throughput screen on a 1172-compound US FDA-approved drug library to identify potential novel inhibitors against ZIKV. Candesartan cilexetil, an angiotensin II receptor inhibitor, displayed potent inhibition effects against ZIKV and subsequent downstream time-course studies revealed that it targets a post-entry stage(s) of the ZIKV replication cycle. Moreover, candesartan cilexetil also inhibited viral RNA production and viral protein synthesis. Candesartan cilexetil also exhibited antiviral effects against Dengue virus serotype-2 (DENV2), Kunjin virus (KUNV) and Chikungunya virus (CHIKV), indicating that its antiviral properties may not be restricted to ZIKV. Our study has demonstrated for the first time the potential application of candesartan cilexetil as an antiviral.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.

Antiviral activity of ST081006 against the dengue virus.

Dengue virus, the causative agent for the dengue fever, infects approximately 50-100 million people worldwide per year. The high incidence of dengue fever, along with its potential to develop into a severe, life-threatening form, resulted in great interest in the discovery of an antiviral against it. In this study, we constructed a DENV2-EGFP infectious clone, established a fluorescence-based, high-throughput screening platform, and conducted a screen for anti-DENV compounds on a flavonoid-derivative library, Amongst the hits identified, ST081006 was found to be a strong inhibitor of the DENV replication. Time-course studies suggest that the presence of ST081006 is necessary to inhibit successive rounds of virus replication. Further investigations demonstrated that ST081006 affects the synthesis of both viral protein and viral RNA, and one anti-DENV mechanism is the direct inhibition of viral protein synthesis. The replication of all dengue serotypes, along with that of the enterovirus EV-A71, was shown to be affected by ST081006. Attempts to generate ST081006-resistant DENV were unsuccessful, and thus hints at host factors as potential drug target. Together, these results suggest that ST081006 affect DENV replication, likely by acting on a target involved in the viral protein and/or RNA synthesis pathway.

Zika virus alters DNA methylation status of genes involved in Hippo signaling pathway in human neural progenitor cells.

Aim: This study was aimed to understand if Zika virus (ZIKV) alters the DNA methylome of human neural progenitor cells (hNPCs). Materials & methods: Whole genome DNA methylation profiling was performed using human methylationEPIC array in control and ZIKV infected hNPCs. Results & conclusion: ZIKV infection altered the DNA methylation of several genes such as WWTR1 (TAZ) and RASSF1 of Hippo signaling pathway which regulates organ size during brain development, and decreased the expression of several centrosomal-related microcephaly genes, and genes involved in stemness and differentiation in human neural progenitor cells. Overall, ZIKV downregulated the Hippo signaling pathway genes which perturb the stemness and differentiation process in hNPCs, which could form the basis for ZIKV-induced microcephaly.

Antiviral activity of pinocembrin against Zika virus replication.

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 (IC50 = 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.

A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication.

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 log10 PFU/ml and 2.5 log10 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.

Discovery of selective dengue virus inhibitors using combination of molecular fingerprint-based virtual screening protocols, structure-based pharmacophore model development, molecular dynamics simulations and in vitro studies.

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.