Redox Regulation and Metabolic Dependency of Zika Virus Replication: Inhibition by Nrf2-Antioxidant Response and NAD(H) Antimetabolites.

Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

Metformin Inhibits Zika Virus Infection in Trophoblast Cell Line.

Zika virus (ZIKV) infections have been associated with severe clinical outcomes, which may include neurological manifestations, especially in newborns with intrauterine infection. However, licensed vaccines and specific antiviral agents are not yet available. Therefore, a safe and low-cost therapy is required, especially for pregnant women. In this regard, metformin, an FDA-approved drug used to treat gestational diabetes, has previously exhibited an anti-ZIKA effect in vitro in HUVEC cells by activating AMPK. In this study, we evaluated metformin treatment during ZIKV infection in vitro in a JEG3-permissive trophoblast cell line. Our results demonstrate that metformin affects viral replication and protein synthesis and reverses cytoskeletal changes promoted by ZIKV infection. In addition, it reduces lipid droplet formation, which is associated with lipogenic activation of infection. Taken together, our results indicate that metformin has potential as an antiviral agent against ZIKV infection in vitro in trophoblast cells.

Antiviral Potential of Fucoxanthin, an Edible Carotenoid Purified from Sargassum siliquastrum, against Zika Virus.

Considering the lack of antiviral drugs worldwide, we investigated the antiviral potential of fucoxanthin, an edible carotenoid purified from Sargassum siliquastrum, against zika virus (ZIKV) infection. The antiviral activity of fucoxanthin was assessed in ZIKV-infected Vero E6 cells, and the relevant structural characteristics were confirmed using molecular docking and molecular dynamics (MD) simulation. Fucoxanthin decreased the infectious viral particles and nonstructural protein (NS)1 mRNA expression levels at concentrations of 12.5, 25, and 50 µM in ZIKV-infected cells. Fucoxanthin also decreased the increased mRNA levels of interferon-induced proteins with tetratricopeptide repeat 1 and 2 in ZIKV-infected cells. Molecular docking simulations revealed that fucoxanthin binds to three main ZIKV proteins, including the envelope protein, NS3, and RNA-dependent RNA polymerase (RdRp), with binding energies of -151.449, -303.478, and -290.919 kcal/mol, respectively. The complex of fucoxanthin with RdRp was more stable than RdRp protein alone based on MD simulation. Further, fucoxanthin bonded to the three proteins via repeated formation and disappearance of hydrogen bonds. Overall, fucoxanthin exerts antiviral potential against ZIKV by affecting its three main proteins in a concentration-dependent manner. Thus, fucoxanthin isolated from S. siliquastrum is a potential candidate for treating zika virus infections.

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections.

Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection.

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.

A cathelicidin antimicrobial peptide from Hydrophis cyanocinctus inhibits Zika virus infection by downregulating expression of a viral entry factor.

Zika virus (ZIKV) is a re-emerging flavivirus that causes conditions such as microcephaly and testis damage. The spread of ZIKV has become a major public health concern. Recent studies indicated that antimicrobial peptides are an ideal source for screening antiviral candidates with broad-spectrum antiviral activities, including against ZIKV. We herein found that Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus in our previous work, conferred protection against ZIKV infection in host cells and showed preventative efficacy and therapeutic efficacy in C57BL/6J mice, Ifnar1-/- mice, and pregnant mice. Intriguingly, we revealed that Hc-CATH decreased the susceptibility of host cells to ZIKV by downregulating expression of AXL, a TAM (TYRO3, AXL and MERTK) family kinase receptor that mediates ZIKV infection, and subsequently reversed the negative regulation of AXL on host's type I interferon response. Furthermore, we showed that the cyclo-oxygenase-2/prostaglandin E2/adenylyl cyclase/protein kinase A pathway was involved in Hc-CATH-mediated AXL downregulation, and Hc-CATH in addition directly inactivated ZIKV particles by disrupting viral membrane. Finally, while we found Hc-CATH did not act on the late stage of ZIKV infection, structure-function relationship studies revealed that α-helix and phenylalanine residues are key structural requirements for its protective efficacy against initial ZIKV infection. In summary, we demonstrate that Hc-CATH provides prophylactic and therapeutic efficacy against ZIKV infection via downregulation of AXL, as well as inactivating the virion. Our findings reveal a novel mechanism of cathelicidin against viral infection and highlight the potential of Hc-CATH to prevent and treat ZIKV infection.

The role of glial cells in Zika virus-induced neurodegeneration.

Zika virus (ZIKV) is a strongly neurotropic flavivirus whose infection has been associated with microcephaly in neonates. However, clinical and experimental evidence indicate that ZIKV also affects the adult nervous system. In this regard, in vitro and in vivo studies have shown the ability of ZIKV to infect glial cells. In the central nervous system (CNS), glial cells are represented by astrocytes, microglia, and oligodendrocytes. In contrast, the peripheral nervous system (PNS) constitutes a highly heterogeneous group of cells (Schwann cells, satellite glial cells, and enteric glial cells) spread through the body. These cells are critical in both physiological and pathological conditions; as such, ZIKV-induced glial dysfunctions can be associated with the development and progression of neurological complications, including those related to the adult and aging brain. This review will address the effects of ZIKV infection on CNS and PNS glial cells, focusing on cellular and molecular mechanisms, including changes in the inflammatory response, oxidative stress, mitochondrial dysfunction, Ca2+ and glutamate homeostasis, neural metabolism, and neuron-glia communication. Of note, preventive and therapeutic strategies that focus on glial cells may emerge to delay and/or prevent the development of ZIKV-induced neurodegeneration and its consequences.

Drug repurposing toward the inhibition of RNA-dependent RNA polymerase of various flaviviruses through computational study.

Numerous pathogens affecting human is present in the flavivirus family namely west nile, dengue, yellow fever, and zika which involves in development of global burden and distressing the environment economically. Till date, no approved drugs are available for targeting these viruses. The threat which urged the identification of small molecules for the inhibition of these viruses is the spreading of serious viral diseases. The recent outbreak of zika and dengue infections postured a solemn risk to worldwide public well-being. RNA-dependent RNA polymerase (RdRp) is the supreme adaptable enzymes of all the RNA viruses which is responsible for the replication and transcription of genome among the structural and nonstructural proteins of flaviviruses. It is understood that the RdRp of the flaviviruses are similar stating that the japanese encephalitis and west nile shares 70% identity with zika whereas the dengue serotype 2 and 3 shares the identity of 76% and 81%, respectively. In this study, we investigated the binding site of four flaviviral RdRp and provided insights into various interaction of the molecules using the computational approach. Our study helps in recognizing the potent compounds that could inhibit the viral protein as a common inhibitor. Additionally, with the conformational stability analysis, we proposed the possible mechanism of inhibition of the identified common small molecule toward RdRp of flavivirus. Finally, this study could be an initiative for the identification of common inhibitors and can be explored further for understanding the mechanism of action through in vitro studies for the study on efficacy.

Pharmacological Elevation of Cellular Dihydrosphingomyelin Provides a Novel Antiviral Strategy against West Nile Virus Infection.

The flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our collection of polyphenols against West Nile virus (WNV), a representative medically relevant flavivirus, led to the identification of N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (half-maximal effective concentrations [EC50s] of 2.2 and 0.24 µM, respectively, in Vero cells; EC50s of 2.2 and 1.9 µM, respectively, in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely, Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1), promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles in membrane properties. The addition of exogenous dhSM or Des1 blockage by using the reference inhibitor GT-11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide} confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.

Heparin Protects Human Neural Progenitor Cells from Zika Virus-Induced Cell Death While Preserving Their Differentiation into Mature Neuroglial Cells.

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.

The in vitro and in vivo antiviral effects of aloperine against Zika virus infection.

Zika virus (ZIKV) infection poses a significant threat to global public health and is associated with microcephaly. There are no approved ZIKV-specific vaccines or drugs for the clinical treatment of the infection. Currently, there are no approved ZIKV-specific vaccines or drugs for the clinical treatment of the infection. In this study, we investigated the antiviral potential of aloperine, a quinolizidine alkaloid, against ZIKV infection in vivo and in vitro. Our results demonstrate that aloperine effectively inhibits ZIKV infection in vitro, with a low nanomolar half maximal effective concentration (EC50 ). Specifically, aloperine strongly protected cells from ZIKV multiplication, as indicated by decreased expression of viral proteins and virus titer. Our further investigations using the time-of-drug-addition assay, binding, entry, and replication assays, detection of ZIKV strand-specific RNA, the cellular thermal shift assay, and molecular docking revealed that aloperine significantly inhibits the replication stage of the ZIKV life cycle by targeting the domain RNA-dependent RNA polymerase (RDRP) of ZIKV NS5 protein. Additionally, aloperine reduced viremia in mice and effectively lowered the mortality rate in infected mice. These findings highlight the potency of aloperine and its ability to target ZIKV infection, suggesting its potential as a promising antiviral drug against ZIKV.

Placenta-derived interferon-stimulated gene 20 controls ZIKA virus infection.

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and has adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterize the IFNß-mediated anti-viral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication and determine whether components of the IFN-induced response can be used as a replacement therapy for ZIKA virus infection during pregnancy. We identify and characterize interferon-stimulated gene 20 (ISG20) as playing a central role in controlling Zika virus infection in trophoblast cells and successfully establish a recombinant ISG20-Fc protein that effectively decreases viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying potential immune modulatory functions. Recombinant ISG20-Fc has thus the potential to be further developed as an anti-viral treatment against ZIKA viral infection in high-risk populations, particularly in pregnant women.

Discovery and structural optimization of a new series of N-acyl-2-aminobenzothiazole as inhibitors of Zika virus.

Zika virus infection is associated to severe diseases such as congenital microcephaly and Zika fever causing serious harm to humans and special concern to health systems in low-income countries. Currently, there are no approved drugs against the virus, and the development of anti-Zika virus drugs is thus urgent. The present investigation describes the discovery and hit expansion of a N-acyl-2-aminobenzothiazole series of compounds against Zika virus replication. A structure-activity relationship study was obtained with the synthesis and evaluation of anti-Zika virus activity and cytotoxicity on Vero cells of nineteen derivatives. The three optimized compounds were 2.2-fold more potent than the initial hit and 20.9, 7.7 and 6.4-fold more selective. Subsequent phenotypic and biochemical assays were performed to evidence whether non-structural proteins, such as the complex NS2B-NS3pro, are related to the mechanism of action of the most active compounds.

Identification of an N-phenylsulfonyl-2-(piperazin-1-yl)methyl-benzonitrile derivative as Zika virus entry inhibitor.

Zika virus (ZIKV) infection could cause severe neurological complications such as neonatal microcephaly, Guillain-Barré syndrome, and myelitis in adults. No vaccine or therapeutic drug is available for prevention and control of ZIKV infection yet. Based on previously reported anti-ZIKV hit compound 1, a series of novel N-benzoyl or phenylsulfonyl substituted 2-(piperazin-1-yl)methyl-benzonitrile (PMBN) derivatives was designed, synthesized, and investigated for the antiviral activity against ZIKV replication in different cell-based phenotypic assays. The results indicated that N-phenylsulfonyl-PMBN derivative 24 displayed the comparable antiviral activity and higher oral availability than hit compound 1. Meanwhile, mechanism of action study confirmed that compound 24 acts on the early entry stage of ZIKV life cycle. The identification of this new ZIKV entry inhibitor chemotype provided a promising lead for further optimization to develop new drug for ZIKV infection.

sEVsRVG selectively delivers antiviral siRNA to fetus brain, inhibits ZIKV infection and mitigates ZIKV-induced microcephaly in mouse model.

Zika virus (ZIKV), a flavivirus associated with neurological disorders, constitutes a global health threat. During pregnancy, ZIKV traverses the placenta and causes congenital disease such as microcephaly and Guillain-Barré syndrome in newborns. To develop a specific antiviral therapy against ZIKV-induced microcephaly that could cross placental and blood-brain barriers, we designed targeted small extracellular vesicles (sEVs) encapsulating antiviral siRNA (small interfering RNA) to inhibit ZIKV. The neuro-specific targeting was achieved by engineering EVs membrane protein lamp2b fused with a neuron-specific rabies virus glycoprotein derived peptide (RVG). Intravenous administration of the RVG-engineered sEVs loaded with siRNA (ZIKV-specific siRNA) protected pregnant AG6 mice against vertical transmission of ZIKV. Particularly, sEVsRVG-siRNA traversed placental and blood-brain barriers and suppressed ZIKV infection in fetal brains. Moreover, sEVsRVG-siRNA alleviated the neuroinflammation and neurological damage caused by ZIKV in the fetal mouse model. In general, we developed a sEVs-based targeted system of antiviral therapy for brain and fetal brain infections.

Identification of potential inhibitors of Zika virus targeting NS3 helicase using molecular dynamics simulations and DFT studies.

Despite the various research efforts towards the drug discovery program for Zika virus treatment, no antiviral drugs or vaccines have yet been discovered. The spread of the mosquito vector and ZIKV infection exposure is expected to accelerate globally due to continuing global travel. The NS3-Hel is a non-structural protein part and involved in different functions such as polyprotein processing, genome replication, etc. It makes an NS3-Hel protein an attractive target for designing novel drugs for ZIKV treatment. This investigation identifies the novel, potent ZIKV inhibitors by virtual screening and elucidates the binding pattern using molecular docking and molecular dynamics simulation studies. The molecular dynamics simulation results indicate dynamic stability between protein and ligand complexes, and the structures keep significantly unchanged at the binding site during the simulation period. All inhibitors found within the acceptable range having drug-likeness properties. The synthetic feasibility score suggests that all screened inhibitors can be easily synthesizable. Therefore, possible inhibitors obtained from this study can be considered a potential inhibitor for NS3 Hel, and further, it could be provided as a lead for drug development.

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

A gossypol derivative effectively protects against Zika and dengue virus infection without toxicity.

BACKGROUND: Zika virus (ZIKV) and dengue virus (DENV) cause microcephaly and dengue hemorrhagic fever, respectively, leading to severe problems. No effective antiviral agents are approved against infections of these flaviviruses, calling for the need to develop potent therapeutics. We previously identified gossypol as an effective inhibitor against ZIKV and DENV infections, but this compound is toxic and not suitable for in vivo treatment. RESULTS: In this study, we showed that gossypol derivative ST087010 exhibited potent and broad-spectrum in vitro inhibitory activity against infections of at least ten ZIKV strains isolated from different hosts, time periods, and countries, as well as DENV-1-4 serotypes, and significantly reduced cytotoxicity compared to gossypol. It presented broad-spectrum in vivo protective efficacy, protecting ZIKV-infected Ifnar1-/- mice from lethal challenge, with increased survival and reduced weight loss. Ifnar1-/- mice treated with this gossypol derivative decreased viral titers in various tissues, including the brain and testis, after infection with ZIKV at different human isolates. Moreover, ST087010 potently blocked ZIKV vertical transmission in pregnant Ifnar1-/- mice, preventing ZIKV-caused fetal death, and it was safe for pregnant mice and their pups. It also protected DENV-2-challenged Ifnar1-/- mice against viral replication by reducing the viral titers in the brain, kidney, heart, and sera. CONCLUSIONS: Overall, our data indicate the potential for further development of this gossypol derivative as an effective and safe broad-spectrum therapeutic agent to treat ZIKV and DENV diseases.