Broad-spectrum Antiviral Agent Research Articles

Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects

Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice.

Zika Virus Replication Is Substantially Inhibited by Novel Favipiravir and Interferon Alpha Combination Regimens.

Zika virus (ZIKV) is a major public health concern due to its overwhelming spread into the Americas. Currently, there are neither licensed vaccines nor antiviral therapies available for the treatment of ZIKV. We aimed to identify and rationally optimize effective therapeutic regimens for ZIKV by evaluating the antiviral potentials of the approved broad-spectrum antiviral agents favipiravir (FAV), interferon alpha (IFN), and ribavirin (RBV) as single agents and in combinations. For these studies, Vero cells were infected with ZIKV in the presence of increasing concentrations of FAV, IFN, or/and RBV for 4 days. Supernatants were harvested daily, and the viral burden was quantified by a plaque assay on Vero cells. The time course of the viral burden during treatment in vitro was characterized by a novel translational, mechanism-based model, which was subsequently used to rationally optimize combination dosage regimens. The combination regimen of FAV plus IFN provided the greatest extent of viral inhibition without cytotoxicity, reducing the viral burden by 4.4 log10 PFU/ml at concentrations of 250 μM FAV and 100 IU/ml IFN. Importantly, these concentrations are achievable in humans. The translational, mechanism-based model yielded unbiased and reasonably precise curve fits. Simulations with the model predicted that clinically relevant regimens of FAV plus IFN would markedly reduce viral burdens in humans, resulting in at least a 10,000-fold reduction in the amount of the virus during the first 4 days of treatment. These findings highlight the substantial promise of rationally optimized combination dosage regimens of FAV plus IFN, which should be further investigated to combat ZIKV.

Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo

Targeting host functions essential for viral replication has been considered as a broad spectrum and resistance-refractory antiviral approach. However, only a few host functions have, thus far, been validated as broad-spectrum antiviral targets in vivo. ER α-glucosidases I and II have been demonstrated to be essential for the morphogenesis of many enveloped viruses, including members from four families of viruses causing hemorrhagic fever. In vivo antiviral efficacy of various iminosugar-based ER α-glucosidase inhibitors has been reported in animals infected with Dengue, Japanese encephalitis, Ebola, Marburg and influenza viruses. Herein, we established Huh7.5-derived cell lines with ER α-glucosidase I or II knockout using CRISPR/Cas9 and demonstrated that the replication of Dengue, Yellow fever and Zika viruses was reduced by only 1–2 logs in the knockout cell lines. The results clearly indicate that only a partial suppression of viral replication can possibly be achieved with a complete inhibition of ER-α-glucosidases I or II by their inhibitors. We therefore explore to improve the antiviral efficacy of a lead iminosugar IHVR-19029 through combination with another broad-spectrum antiviral agent, favipiravir (T-705). Indeed, combination of IHVR-19029 and T-705 synergistically inhibited the replication of Yellow fever and Ebola viruses in cultured cells. Moreover, in a mouse model of Ebola virus infection, combination of sub-optimal doses of IHVR-19029 and T-705 significantly increased the survival rate of infected animals. We have thus proved the concept of combinational therapeutic strategy for the treatment of viral hemorrhagic fevers with broad spectrum host- and viral- targeting antiviral agents.

Broad-spectrum antiviral agents: secreted phospholipase A2 targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane

Hepatitis C virus (HCV), dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the family Flaviviridae. Their viral particles have the envelope composed of viral proteins and a lipid bilayer acquired from budding through the endoplasmic reticulum (ER). The phospholipid content of the ER membrane differs from that of the plasma membrane (PM). The phospholipase A2 (PLA2) superfamily consists of a large number of members that specifically catalyse the hydrolysis of phospholipids at a particular position. Here we show that the CM-II isoform of secreted PLA2 obtained from Naja mossambica mossambica snake venom (CM-II-sPLA2) possesses potent virucidal (neutralising) activity against HCV, DENV and JEV, with 50% inhibitory concentrations (IC50) of 0.036, 0.31 and 1.34 ng/ml, respectively. In contrast, the IC50 values of CM-II-sPLA2 against viruses that bud through the PM (Sindbis virus, influenza virus and Sendai virus) or trans-Golgi network (TGN) (herpes simplex virus) were >10,000 ng/ml. Moreover, the 50% cytotoxic (CC50) and haemolytic (HC50) concentrations of CM-II-sPLA2 were >10,000 ng/ml, implying that CM-II-sPLA2 did not significantly damage the PM. These results suggest that CM-II-sPLA2 and its derivatives are good candidates for the development of broad-spectrum antiviral drugs that target viral envelope lipid bilayers derived from the ER membrane.

Identification of Novel 5,6-Dimethoxyindan-1-one Derivatives as Antiviral Agents.

Discovery of novel antiviral agents is essential because viral infection continues to threaten human life globally. Various heterocyclic small molecules have been developed as antiviral agents. The 5,6-dimethoxyindan-1-on nucleus is of considerable interest as this ring is the key constituent in a range of bioactive compounds, both naturally occurring and synthetic, and often of considerable complexity. The main purpose of this research was to discover and develop small molecule heterocycles as broad-spectrum of antiviral agents. A focused small set of 5,6-dimethoxyindan-1-one analogs (6-8) along with a thiopene derivative (9) was screened for selected viruses (Vaccinia virus - VACA, Human papillomavirus - HPV, Zika virus - ZIKV, Dengue virus - DENV, Measles virus - MV, Poliovirus 3 - PV, Rift Valley fever virus - RVFV, Tacaribe virus - TCRV, Venezuelan equine encephalitis virus - VEEV, Herpes simplex virus 1 -HSV-1 and Human cytomegalovirus - HCMV) using the National Institute of Allergy and Infectious Diseases (NIAID)'s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. These molecules demonstrated moderate to excellent antiviral activity towards variety of viruses. The 5,6-dimethoxyindan-1-one analog (7) demonstrated high efficacy towards vaccinia virus (EC50: <0.05 µM) and was nearly 232 times more potent than the standard drug Cidofovir (EC50: 11.59 µM) in primary assay whereas it demonstrated moderate activity (EC50: >30.00 µM) in secondary plaque reduction assay. The thiophene analog (9) has shown very good viral inhibition towards several viruses such as Human papillomavirus, Measles virus, Rift Valley fever virus, Tacaribe virus and Herpes simplex virus 1. Our research identified a novel 5,6-dimethoxyindan-1-one analog (compound 7), as a potent antiviral agent for vaccinia virus, and heterocyclic chalcone analog (compound 9) as a broad spectrum antiviral agent.

A screen for novel hepatitis C virus RdRp inhibitor identifies a broad-spectrum antiviral compound

Hepatitis C virus (HCV) is a global pathogen and infects more than 185 million individuals worldwide. Although recent development of direct acting antivirals (DAA) has shown promise in HCV therapy, there is an urgent need for the development of more affordable treatment options. We initiated this study to identify novel inhibitors of HCV through screening of compounds from the National Cancer Institute (NCI) diversity dataset. Using cell-based assays, we identified NSC-320218 as a potent inhibitor against HCV with an EC50 of 2.5 μM and CC50 of 75 μM. The compound inhibited RNA dependent RNA polymerase (RdRp) activity of all six major HCV genotypes indicating a pan-genotypic effect. Limited structure-function analysis suggested that the entire molecule is necessary for the observed antiviral activity. However, the compound failed to inhibit HCV NS5B activity in vitro, suggesting that it may not be directly acting on the NS5B protein but could be interacting with a host protein. Importantly, the antiviral compound also inhibited dengue virus and hepatitis E virus replication in hepatocytes. Thus, our study has identified a broad-spectrum antiviral therapeutic agent against multiple viral infections.

Understanding the Mechanism of the Broad-Spectrum Antiviral Activity of Favipiravir (T-705): Key Role of the F1 Motif of the Viral Polymerase.

Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.

Genotoxicity and acute and subchronic toxicity studies of a bioactive polyoxometalate in Wistar rats

BackgroundCs2K4Na [SiW9Nb3O40] (POM93) is a novel broad-spectrum antiviral agent with high activity, high stability, and low toxicity in vitro. Most toxicity studies for POM93 have been performed in cultured cell lines rather than in animals. Like other POMs, there is a lack of evidence for in vivo toxicity limits, oral bioavailability, and therapeutic applications.MethodsThe toxic properties of POM93 were evaluated comprehensively in vivo, including the acute and subchronic oral toxicity studies and genotoxicity tests.ResultsThe acute toxicity study showed no abnormal changes or mortality in rats treated with POM93 even at the single high dose of 5000 mg/kg body weight. In the subchronic toxicity study, regardless of the body weight, the organ weight, and the hematological parameters, similar results were observed between the control group and the experimental groups. POM93 produced mild changes in rare hematological parameters in the liver and kidneys, but did not induce the clinical symptoms of liver or kidneys injury in rats as confirmed by histopathological analysis. Moreover, neither mutagenicity nor clastogenicity was caused by POM93 treatment in vitro and in vivo.ConclusionsThe present study demonstrates that the oral administration of POM93 is presumed safe and poses a low risk of potential health risks.

Favipiravir can evoke lethal mutagenesis and extinction of foot-and-mouth disease virus

Antiviral agents are increasingly considered an option for veterinary medicine. An understanding of their mechanism of activity is important to plan their administration either as monotherapy or in combination with other agents. Previous studies have shown that the broad spectrum antiviral agent favipiravir (T-705) and its derivatives T-1105 and T-1106 are efficient inhibitors of foot-and-mouth disease virus (FMDV) replication in cell culture and in vivo. However, no mechanism for their activity against FMDV has been proposed. In the present study we show that favipiravir (T-705) can act as a lethal mutagen for FMDV in cell culture. Evidence includes virus extinction associated with increase in mutation frequency in the mutant spectrum of 860 residues of the 3D (polymerase)-coding region, and a decrease of specific infectivity while the consensus nucleotide sequence of the region analyzed remained invariant. The mutational spectrum evoked by favipiravir differs from that observed with other viruses in that no predominant transition type is observed, indicating that a movement towards A,U- or G,C-rich regions of sequence space is not a prerequisite for virus extinction. We discuss prospects for the use of favipiravir to assist in the control of FMDV, and its possible broader use in veterinary medicine as an extension of its current status as antiviral agent for human influenza virus.

Cellular protein GLTSCR2: A valuable target for the development of broad-spectrum antivirals

Viral infection induces translocation of the nucleolar protein GLTSCR2 from the nucleus to the cytoplasm, resulting in attenuation of the type I interferon IFN-β. Addressing the role of GLTSCR2 in viral replication, we detect that knocking down GLTSCR2 by shRNAs results in significant suppression of viral replication in mammalian and chicken cells. Injection of chicken embryo with the GLTSCR2-specific shRNA-1370 simultaneously or 24 h prior to infection with Newcastle disease virus (NDV) substantially reduces viral replication in chicken embryo fibroblasts. Injection of shRNA-1370 into chicken embryo also reduces the replication of avian influenza virus (AIV). In contrast, GLTSCR2-derived protein G4-T, forming α-helical dimers, increases replication of seven various DNA and RNA viruses in cells. Our studies reveal that alteration of the function of cellular GLTSCR2 plays a role in supporting viral replication. GLTSCR2 should be seriously considered as a therapeutic target for developing broad spectrum antiviral agents to effectively control viral infection.

Ribavirin attenuates the respiratory immune responses to influenza viral infection in mice.

Ribavirin is a broad-spectrum antiviral agent that is used against RNA and DNA viruses and has been reported to inhibit infection by influenza A and B virus in vitro and in vivo. Studies have shown that ribavirin can lower convalescent antibody titers in young children hospitalized with influenza. Here, we report that ribavirin administration in juvenile mice significantly attenuated respiratory immune responses, production of total IgA and hemagglutinin (HA)-specific secretory IgA responses on the mucosal surface. In contrast, systemic IgG and IgA responses were not affected. Ribavirin significantly suppressed toll-like receptor 2 and 4 expression in the lung and decreased the level of IL-1β, IL-6, TNF-α, and IFN-γ in lung tissues of mice infected with influenza virus. Our findings suggest ribavirin appears to be able to inhibit viral replication and, as a result, TLR and cytokine expression are not up-regulated, attenuating inflammation as well as the respiratory tract's immune response.

Rational Design of Novel Highly Potent and Selective Phosphatidylinositol 4-Kinase IIIβ (PI4KB) Inhibitors as Broad-Spectrum Antiviral Agents and Tools for Chemical Biology.

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.

In vitro antiviral activity of ribavirin against severe fever with thrombocytopenia syndrome virus.

Background/AimsSevere fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus. As yet, there is no effective antiviral therapy for SFTS. Ribavirin is a broad-spectrum antiviral agent, which has been tried for treatment of SFTS. In this study, antiviral activity of ribavirin against SFTSV has been investigated.MethodsVero cell-grown SFTSV strain Gangwon/Korea/2012 was treated with ribavirin at various concentrations. Antiviral activity of ribavirin was evaluated by inhibition of the SFTSV cytopathic effect in Vero cells and quantification of viral RNA load in culture supernatant using one-step real-time reverse transcription polymerase chain reaction. Cytotoxicity of ribavirin was determined by a tetrazolium-based colorimetric method.ResultsRibavirin reduced SFTSV titers in a dose-dependent manner, with a half-maximal inhibitory concentration ranged from 3.69 to 8.72 μg/mL. Cytopathic effects were reduced as ribavirin concentration increased. No significant cytotoxicity was detected at ribavirin concentrations of ≤ 31.3 μg/mL.ConclusionsRibavirin exhibited inhibitory activity against SFTSV replication in vitro, which suggests that ribavirin can be used as a potential antiviral agent for SFTS.

Inhibitory effects of bee venom and its components against viruses in vitro and in vivo.

Bee venom (BV) from honey bee (Apis Melifera L.) contains at least 18 pharmacologically active components including melittin (MLT), phospholipase A2 (PLA2), and apamin etc. BV is safe for human treatments dose dependently and proven to possess different healing properties including antibacterial and antiparasitidal properties. Nevertheless, antiviral properties of BV have not well investigated. Hence, we identified the potential antiviral properties of BV and its component against a broad panel of viruses. Co-incubation of non-cytotoxic amounts of BV and MLT, the main component of BV, significantly inhibited the replication of enveloped viruses such as Influenza A virus (PR8), Vesicular Stomatitis Virus (VSV), Respiratory Syncytial Virus (RSV), and Herpes Simplex Virus (HSV). Additionally, BV and MLT also inhibited the replication of non-enveloped viruses such as Enterovirus-71 (EV-71) and Coxsackie Virus (H3). Such antiviral properties were mainly explained by virucidal mechanism. Moreover, MLT protected mice which were challenged with lethal doses of pathogenic influenza A H1N1 viruses. Therefore, these results provides the evidence that BV and MLT could be a potential source as a promising antiviral agent, especially to develop as a broad spectrum antiviral agent.

Macromolecular Prodrugs of Ribavirin: Structure-Function Correlation as Inhibitors of Influenza Infectivity.

The requirement for new antiviral therapeutics is an ever present need. Particularly lacking are broad spectrum antivirals that have low toxicity. We develop such agents based on macromolecular prodrugs whereby both the polymer chain and the drug released from the polymer upon cell entry have antiviral effects. Specifically, macromolecular prodrugs were designed herein based on poly(methacrylic acid) and ribavirin. Structure-function parameter space was analyzed via the synthesis of 10 polymer compositions varied by molar mass and drug content. Antiviral activity was tested in cell culture against both low and high pathogenic strains of influenza. Lead compounds were successfully used to counter infectivity of influenza in chicken embryos. The lead composition with the highest activity against influenza was also active against another respiratory pathogen, respiratory syncytial virus, providing opportunity to potentially treat infection by the two pathogens with one antiviral agent. In contrast, structure-function activity against the herpes simplex virus was drastically different, revealing limitations of the broad spectrum antiviral agents based on macromolecular prodrugs.

Synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs

Thiazolides are polypharmacology agents with at least three mechanisms of action against a broad spectrum of parasites, bacteria and viruses. In respiratory viruses they inhibit the replication of orthomyxoviridae and paramyxoviridae at a post-translational level. Nitazoxanide 1a, the prototype thiazolide, was originally developed as an antiparasitic agent and later repurposed for the treatment of viral respiratory infections. The second generation thiazolides following nitazoxanide, such as the 5-chloro analogue RM-5038 2a, are also broad-spectrum antiviral agents as we have reported. Both 1a and its effective circulating metabolite, tizoxanide 1b, are 5-nitrothiazole derivatives, while RM-5038 2a and its de-acetyl derivative RM-4848 2b are the corresponding 5-chloro derivatives. Recently 1a has completed phase II-III clinical trials in the United States, Canada, Australia and New Zealand in a total of 2865 adults and adolescents of at least 12 months of age with viral acute respiratory illness. Since its biodisposition is primarily seen in the gastro-intestinal tract, its efficacy in systemic viral diseases requires relatively high oral doses. The chemical synthesis of new derivatives with a better systemic absorption was therefore urgently needed. In order to improve their systemic absorption, new amino-ester prodrug derivatives of 1b and RM4848 2b were prepared and tested for their animal pharmacology, pharmacokinetics and toxicology. RM-5061 8a in rats showed 7-fold higher blood concentration compared to 1a: absolute bioavailability increased from 3 to 20%, with a good safety profile in animal safety pharmacology and toxicology.

Antivirals for Respiratory Viral Infections: Problems and Prospects.

In the past two decades, several newly emerging and reemerging viral respiratory pathogens including several influenza viruses (avian influenza and pandemic influenza), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV), have continued to challenge medical and public health systems. Thereafter, the development of cost-effective, broad-spectrum antiviral agents is the urgent mission of both virologists and pharmacologists. Current antiviral developments have focused targets on viral entry, replication, release, and intercellular pathways essential for viral life cycle. Here, we review the current literature on challenges and prospects in the development of these antivirals.

Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors

Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CLpro of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R1 or R4 destabilizes the oxyanion hole in the 3CLpro. Interestingly, 3f, 3g and 3m could inhibit both NA and 3CLpro and serve as a starting point to develop broad-spectrum antiviral agents.

RIG-ging the host innate immune response for vaccine adjuvant and antiviral therapy

Abstract Retinoic acid inducible gene I (RIG-I) is essential for directing and priming the host immune response against many RNA viruses. A diverse small molecule library was used in a cell-based screening approach to identify drug-like compounds that could target the RIG-I signaling pathway and induce downstream IRF3 to drive innate antiviral immunity. We identified small molecule compounds that activate IRF3 to induce discrete subsets of innate immune and antiviral genes. We identified a class of hydroxyquinoline compounds that induce IRF3 activation and innate immunity of cultured cells to decrease viral RNA load and infectious virus production, thereby controlling infection by viruses of the families Flaviviridae, Filoviridae, Orthomyxoviridae, Arenaviridae and Paramyxoviridae. In addition, we identified a second class of benzo-bisthiazole compounds that directly engage and activate RIG-I to promote downstream IRF3 signaling and the expression of innate immune genes, cytokines and chemokines. Importantly, these compounds serve potent immune adjuvant functions to promote the maturation of dendritic cells to enhance antigen presentation and T cell activation. When administered to mice in combination with influenza A virus or West Nile virus vaccine, this compound conferred adjuvant activity to facilitate vaccine-mediated protection of mice from lethal virus challenge under conditions of otherwise suboptimal vaccine dose. Thus, we have identified novel small molecule RIG-I agonists that activate the innate immune response. Our studies reveal the strong potential of developing small molecule RIG-I agonists as broad-spectrum antiviral agents to control RNA virus infection and as adjuvants to potentiate vaccine immunity.