Search

2-(Methylthio)- N -(4-(naphthalen-2-yl)thiazol-2-yl)nicotinamide 1 was identified as an inhibitor against Chikungunya virus (CHIKV) with good antiviral activity [EC 50 = 0.6 μM; EC 90 = 0.93 μM and viral titer reduction (VTR) of 6.9 logs at 10 μM concentration] with no observed cytotoxicity (CC 50 = 132 μM) in normal human dermal fibroblast (NHDF) cells. Structure-activity relationship (SAR) studies to further improve the potency, efficacy, and drug-like properties of 1 led to the discovery of a new potent inhibitor N -(4-(3-((4-cyanophenyl)amino)phenyl)thiazol-2-yl)-2-(methylthio)nicotinamide 26 , which showed a VTR of 8.7 logs at 10 μM against CHIKV and an EC 90 of 0.45 μM with considerably improved MLM stability ( t 1/2 = 74 min) as compared to 1 . Mechanism of action studies show that 26 inhibits alphavirus replication by blocking subgenomic viral RNA translation and structural protein synthesis. The in vivo efficacy studies of compound 26 on CHIKV infection in mice are reported.

A benzo[6]annulene, 4-( tert -butyl)- N -(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide ( 1a ), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC 90 = 1.45 μM and viral titer reduction (VTR) of 2.5 log at 10 μM with no observed cytotoxicity (CC 50 = 169 μM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q , which possessed excellent cellular antiviral activity (EC 90 = 270 nM and VTR of 4.5 log at 10 μM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a , compound 1c , tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions and cause significant morbidity and mortality worldwide. Although a partially effective vaccine is in use in several countries in which DENV are endemic, no antiviral therapeutics are approved for combating DENV-associated disease. Herein, we report the characterization of novel small molecule inhibitors of DENV replication, VGTI-A3 and VGTI-A3-03, as well as structure-activity relationship analysis of the molecules using a panel of chemical analogs. VGTI-A3 and VGTI-A3-03 are highly virus-specific, with greatest activity against DENV serotype 2. Further analysis revealed that treatment of infected cells with VGTI-A3-03 does not inhibit viral RNA replication or secretion of viral particles. Rather, the infectivity of secreted particles from A3-03 treated cells is significantly diminished compared to particles secreted from control cells. Elicitation of VGTI-A3-03-resistant mutants demonstrated a clear binding pocket in the capsid molecule at the dimerization interface. Additionally, we show that VGTI-A3-03 is incorporated into virus particles released from infected cells. In summary, these data provide detailed analysis of a potentially useful class of anti-DENV inhibitors and further identify a region of the viral capsid protein as a druggable target for other therapeutic approaches., (Copyright © 2018. Published by Elsevier B.V.)

The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy's potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3- c ]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity. IMPORTANCE The type I interferon system is part of the innate immune response that has evolved in vertebrates as a first line of broad-spectrum immunological defense against an unknowable diversity of microbial, especially viral, pathogens. Here, we characterize a novel small molecule that artificially activates this response and in so doing generates a cellular state antagonistic to growth of currently emerging viruses: Zika virus, Chikungunya virus, and dengue virus. We also show that this molecule is capable of eliciting cellular responses that are predictive of establishment of adaptive immunity. As such, this agent may represent a powerful and multipronged therapeutic tool to combat emerging and other viral diseases., (Copyright © 2017 Pryke et al.)

Unlabelled: Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds., Importance: The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.

Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.

The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5' cyclization sequence (5'CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an "in vivo-ready" version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.

Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration-approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC₅₀ potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.

We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 microM, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication.