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Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment. ispartof: JOURNAL OF MEDICAL VIROLOGY vol:94 issue:7 pages:3101-3111 ispartof: location:United States status: published

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran−1,3-imidazolidin-4-one)s and bis(benzofuran−1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC50 of 15.3. The results are valuable for the development of novel antiviral drug leads against emerging diseases. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:20 ispartof: location:Switzerland status: published

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran-1,3-imidazolidin-4-one)s and bis(benzofuran-1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC

Chikungunya virus (CHIKV) has repeatedly spread via the bite of an infected mosquito and affected more than 100 countries. The disease poses threats to public health and the economy in the infected locations. Many efforts have been devoted to identifying compounds that could inhibit CHIKV. Unfortunately, successful clinical candidates have not been found yet. Computations through the simulating recognition process were performed on complexation of the nsP3 protein of CHIKV with the structures of triply conjugated drug lead candidates. The outcomes provided the aid on rational design of functionalized quinazoline-(α-substituted coumarin)-arylsulfonate compounds to inhibit CHIKV in Vero cells. The molecular docking studies showed a void space around the β carbon atom of coumarin when a substituent was attached at the α position. The formed vacancy offered a good chance for a Michael addition to take place owing to steric and electronic effects. The best conjugate containing a quinazolinone moiety exhibited potency with EC50 = 6.46 μM, low toxicity with CC50 = 59.7 μM, and the selective index (SI) = 9.24. Furthermore, the corresponding 4-anilinoquinazoline derivative improved the anti-CHIKV potency to EC50 = 3.84 μM, CC50 = 72.3 μM, and SI = 18.8. The conjugate with 4-anilinoquinazoline exhibited stronger binding affinity towards the macro domain than that with quinazolinone via hydrophobic and hydrogen bond interactions. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:14 ispartof: location:Switzerland status: published

We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved. We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved. ispartof: Antiviral Research vol:198 ispartof: location:Netherlands status: published

BACKGROUND: Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV-2. First efficacy data have been recently reported in COVID-19 patients. METHODS: We here report on the combined antiviral effect of both drugs in a SARS-CoV-2 Syrian hamster infection model. The infected hamsters were treated twice daily with the vehicle (the control group) or a suboptimal dose of each compound or a combination of both compounds. FINDINGS: When animals were treated with a combination of suboptimal doses of Molnupiravir and Favipiravir at the time of infection, a marked combined potency at endpoint is observed. Infectious virus titers in the lungs of animals treated with the combination are reduced by ∼5 log10 and infectious virus are no longer detected in the lungs of >60% of treated animals. When start of treatment was delayed with one day a reduction of titers in the lungs of 2.4 log10 was achieved. Moreover, treatment of infected animals nearly completely prevented transmission to co-housed untreated sentinels. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs of treated animals. In the combo-treated hamsters, an increased frequency of C-to-T mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. INTERPRETATION: Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir/Favipiravir in the treatment of COVID-19. FUNDING: stated in the acknowledgment. ispartof: EBIOMEDICINE vol:72 ispartof: location:Netherlands status: published

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10 , as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15). ispartof: JOURNAL OF MEDICAL VIROLOGY vol:93 issue:7 pages:4454-4460 ispartof: location:United States status: published

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.

Vapendavir is a rhino/enterovirus inhibitor that targets a hydrophobic pocket in the viral capsid preventing the virus from entering the cell. We set out to study and compare the molecular mechanisms of resistance to vapendavir among clinically relevant Picornavirus species. To this end in vitro resistance selection of drug-resistant isolates was applied in rhinovirus 2 and 14, enterovirus-D68 and Poliovirus 1 Sabin. Mutations in the drug-binding pocket in VP1 (C199R/Y in hRV14; I194F in PV1; M252L and A156T in EV-D68), typical for this class of compounds, were identified. Interestingly, we also observed mutations located outside the pocket (K167E in EV-D68 and G149C in hRV2) that contribute to the resistant phenotype. Remarkably, the G149C substitution rendered the replication of human rhinovirus 2 dependent on the presence of vapendavir. Our data suggest that the binding of vapendavir to the capsid of the G149C isolate may be required to stabilize the viral particle and to allow efficient dissemination of the virus. We observed the dependency of the G149C isolate on other compounds of this class, suggesting that this phenotype is common for capsid binders. In addition the VP1 region containing the G149C substitution has not been associated with antiviral resistance before. Our results demonstrate that the phenotype and genotype of clinically relevant vapendavir-resistant picornavirus species is more complex than generally believed. ispartof: ANTIVIRAL RESEARCH vol:195 ispartof: location:Netherlands status: published

The modern world has no available drugs for the treatment of enteroviruses (EV), which affect millions of people worldwide each year. The EV71 is a major causative disease for hand, foot, and mouth disease, sometimes it is associated with severe central nervous system diseases. Treatment for enteroviral infection is mainly supportive, treatment for aseptic meningitis caused by enteroviruses is also generally symptomatic. Upon the urgent request of new anti-enterovirus drugs, a series of hinged aromatic compounds with polynulei were synthesized through two different chemical pathways. Among these morpholine&ndash, furan/thiophene/pyrrole&ndash, benzene&ndash, pyrazole conjugates, three new agents exhibited inhibitory activity with EC50 = 2.29&ndash, 6.16 &mu, M toward EV71 strain BrCr in RD cells. Their selectivity index values were reached as high as 33.4. Their structure&ndash, activity relationship was deduced that a thiophene derivative with morpholine and trifluorobenzene rings showed the greatest antiviral activity, with EC50 = 2.29 &mu, M.

International audience; Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve similar to 18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.

Chikungunya and yellow fever virus cause vector-borne viral diseases in humans. There is currently no specific antiviral drug for either of these diseases. Banana plants are used in traditional medicine for treating viral diseases such as measles and chickenpox. Therefore, we tested selected banana cultivars for their antiviral but also cytotoxic properties. Different parts such as leaf, pseudostem and corm, collected separately and extracted with four different solvents (hexane, acetone, ethanol, and water), were tested for in vitro antiviral activity against Chikungunya virus (CHIKV), enterovirus 71 (EV71), and yellow fever virus (YFV). Extracts prepared with acetone and ethanol from leaf parts of several cultivars exhibited strong (EC50 around 10 &mu, g/mL) anti-CHIKV activity. Interestingly, none of the banana plant extracts (concentration 1&ndash, 100 &micro, g/mL) were active against EV71. Activity against YFV was restricted to two cultivars: Namwa Khom&ndash, Pseudostem&ndash, Ethanol (5.9 &plusmn, 5.4), Namwa Khom&ndash, Corm&ndash, Ethanol (0.79 &plusmn, 0.1) and Fougamou&ndash, Acetone (2.5 &plusmn, 1.5). In most cases, the cytotoxic activity of the extracts was generally 5- to 10-fold lower than the antiviral activity, suggesting a reasonable therapeutic window.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:63 issue:9 pages:4562-4578 ispartof: location:United States status: published

Vector-borne diseases cause more than 1 million deaths annually. The research into new medicines is urgent, especially as there is currently no specific treatment. In this study, the authors have selected 64 endemic plants from the Mascarene Islands based on their endemism, their medicinal use and their registration in the French Pharmacopeia to evaluate the antiplasmodial, anti-chikungunya and antioxidant activities. The list of these 64 plants including their local name, population, data of collection and voucher number are available in the Supporting Information. Forty active extracts were identified from the 38 species: 22 responded positively to the antiplasmodial activity, 8 to the anti-chikungunya activity and 8 to the antioxidant activity. Six plants demonstrated high antiplasmodial activity (concentration inhibiting 50% of parasitic growth (IC50) 2000 µM of Trolox equivalent per mg/mL of extract: Bertiera borbonica, Erythroxylon laurifolium, Erythroxylon sideroxyloides, I. ammoxylum, P. borbonica, Scolopia heterophylla, Sophora denudata, and Terminalia bentzoe. Some data obtained tend to corroborate the reported traditional use of the plant, such as Z. heterophyllum (antiplasmodial), A. theiformis (anti-chikungunya), and E. laurifolium (antioxidant). ispartof: INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS vol:52 issue:5 pages:622-628 ispartof: location:Netherlands status: published

In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral activity against a panel of alpha- and beta-coronaviruses, including the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were the most potent with antiviral EC50 values in the range of 0.12–12 μM. Chloroquine displayed the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with amodiaquine, ferroquine and mefloquine, although this was associated with substantial cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked coronavirus replication at higher concentrations, while piperaquine completely lacked antiviral and cytotoxic effects. A time-of-addition experiment in HCoV-229E-infected HEL cells revealed that chloroquine interferes with viral entry at a post-attachment stage. Using confocal microscopy, no viral RNA synthesis could be detected upon treatment of SARS-CoV-2-infected cells with chloroquine. The inhibition of SARS-CoV-2 replication by chloroquine and hydroxychloroquine coincided with an inhibitory effect on the autophagy pathway as visualized by a dose-dependent increase in LC3-positive puncta. The latter effect was less pronounced or even absent with the other quinolines. In summary, we showed that several quinoline analogues, including chloroquine, hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus activity in vitro.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells., LS, KL, LD, PL, CM and JN received funding of Belgian Interuniversity Attraction Poles (IAP) Phase VII–P7/45 (BELVIR) and the EU FP7 Industry-Academia Partnerships and Pathways Project AIROPICO. LS received funding of China Scholarship Council (CSC) (Grant 201403250056). HL, CB and SH received a grant with the Pennsylvania Department of Health using Tobacco CURE Funds. ERB, BMG and ASF received the grant “Ministerio de Economia, Industriay Competitividad, Gobierno de España” and “European Regional Development Funds” projects number SAF2015-64629-C2-1-R (MINECO/ FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range., This work has been supported by the Spanish MINECO (Project SAF2012-39760-C02-01, cofinanced by the FEDER program; Plan Nacional de Cooperación Público-Privada; and Subprograma INNPACTO IPT-2012-0213-060000, cofinanced by the FEDER program) and the Comunidad de Madrid (BIPEDD2-CM-S2010/BMD-2457). This work was also funded by EU FP7 (FP7/2007-2013) Project EUVIRNA under Grant408 Agreement 264286 by EU FP7 SILVER (Contract HEALTH-F3-2010- 260644), a grant from the Belgian Interuniversity Attraction Poles (IAP) Phase VII–P7/45 (BELVIR), and the EU FP7 Industry-Academia Partnerships and Pathways Project AIROPICO. The Spanish MEC/MINECO is also acknowledged for a grant to E.R.-B. L.S. was funded by China Scholarship Council (CSC) Grant 201403250056. We also acknowledge Charlotte Vanderheydt for help with the processing of the antiviral data.

Dengue virus (DV) is an important mosquito-borne flavivirus threatening almost half of the world's population. Prophylaxis and potent anti-DV drugs are urgently needed. Here, we developed a high content imaging-based (HCI) assay with DV type 2 expressing the fluorescent protein mCherry (DV2/mCherry) to improve the efficiency and robustness of the drug discovery process. For the construction of the reporter virus, the mCherry gene followed by the ribosome-skipping 2A sequence of the Thosea asigna virus (T2A) was placed upstream of the full DV2 open reading frame. The biological characteristics including mCherry expression, virus replication rate, and plaque phenotype was examined and validated in BHK-21, Vero and C6/36 cells. A robust image-based antiviral assay combined with an automated robotic system was then developed, with a Z' factor of 0.73. To validate the image-based antiviral assay, a panel of reference compounds with different molecular mechanisms of anti-DV activity was assessed: (i) the glycosylation inhibitor, Celgosivir, (ii) two NS4b-targeting compounds: a 3-Acyl-indole derivative and NITD618, and (iii) two nucleoside viral polymerase inhibitors, 2'CMC and 7DMA. The inhibition profiles were quantified and obtained by means of HCI and RT-qPCR. Both methods resulted in very comparable inhibition profiles. In conclusion, a powerful and robust assay was developed with a fully automated data generation and processing pipeline. It makes the new reporter virus assay amenable to high-throughput screening of large libraries of small molecules. ispartof: ANTIVIRAL RESEARCH vol:183 ispartof: location:Netherlands status: published

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 μM), calpain inhibitors II and XII (IC50 ~20–25 μM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human β-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2., Highlights • A novel label-free SARS-CoV-2 3CLpro enzymatic assay was developed using mass spectrometry (SAMDI-MS). • Protease inhibitors active in the SAMDI-MS assay under reducing conditions also have antiviral effects in cells. • Shikonin, disulfiram, and ebselen do not inhibit SARS-CoV-2 3CLpro in the SAMDI-MS assay. • Shikonin, disulfiram, and ebselen do not directly inhibit SARS-CoV-2 but instead are toxic to cells.

Chikungunya virus is a re-emerging arbovirus transmitted to humans by Aedes mosquitoes, responsible for an acute febrile illness associated with painful and debilitating arthralgia, which can persist for several months or become chronic. Over the past few years, infection with this virus has spread worldwide with a previously unknown virulence. No specific antiviral treatments nor vaccines are currently available against this important pathogen. Starting from the structure of a class of selective anti-CHIKV agents previously identified in our research group, different modifications to this scaffold were rationally designed, and 69 novel small-molecule derivatives were synthesised and evaluated for their inhibition of Chikungunya virus replication in Vero cells. Further structure-activity relationships associated with this class of antiviral agents were elucidated for the original scaffolds, and novel antiviral compounds with EC50 values in the low micromolar range were identified. This work provides the foundation for further investigation of these new structures as antivirals against Chikungunya virus.

The re-emergence of chikungunya virus (CHIKV) is a serious global health threat. CHIKV is an alphavirus that is transmitted to humans by Aedes mosquitoes; therefore, their wide distribution significantly contributes to the globalization of the disease. Unfortunately, no effective antiviral drugs are available. We have identified a series of 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication. New series of compounds have now been synthesized with the aim to improve their physicochemical properties and to potentiate the inhibitory activity against different CHIKV strains. Among these newly synthesized compounds modified at position 3 of the aryl ring, tetrahydropyranyl and N-t-butylpiperidine carboxamide derivatives have shown to elicit potent antiviral activity against different clinically relevant CHIKV isolates with 50% effective concentration (EC) values ranging from 0.30 to 4.5 μM in Vero cells, as well as anti-CHIKV activity in human skin fibroblasts (EC = 0.1 μM), a clinically relevant cell system for CHIKV infection., We thank Caroline Collard and Nick Verstraeten for their excellent technical assistance in the acquisition of the antiviral data. This work has been supported by grants from MINECO/ FEDER SAF2015-64629-C2-1-R and by European Union FP7 Program under SILVER grant agreement no. 260644.

OBJECTIVES: Many positive-stranded RNA viruses, including HCV, drastically remodel intracellular membranes to generate specialized environments for RNA replication. Phosphatidylinositol 4-kinase III (PI4KIII)α plays an essential role in the formation of HCV replication complexes and has therefore been explored as a potential drug target. Here, we characterized the anti-HCV activity of the PI4KIII inhibitors enviroxime and BF738735 and elucidated their mechanism of action. METHODS: Antiviral assays were performed using HCV subgenomic replicons and infectious HCV. Enviroxime- and BF738735-resistant HCV replicons were generated by long-term culture with increasing compound concentrations. Intracellular localization of phosphatidylinositol 4-phosphate (PI4P) lipids was analysed by confocal microscopy. RESULTS: HCV subgenomic replicons resistant to either enviroxime or BF738735 proved cross-resistant and carried mutations in the NS3, NS4B and NS5A genes. Knockdown of PI4KIIIβ by small interfering RNA (siRNA) did not affect the replication of the HCV subgenomic replicon in this study. Furthermore, the compounds did not affect PI4P lipid levels at the replication complexes nor the phosphorylation status of NS5A, activities attributed to PI4KIIIα. Interestingly, the broad-spectrum phosphoinositide 3-kinase (PI3K) inhibitor LY294002 proved to be 10-fold less effective against the resistant replicons. In addition, enviroxime and BF738735 inhibited several PI3Ks in enzymatic assays. CONCLUSIONS: Contrary to assumptions, our data indicate that PI4KIIIα and PI4KIIIβ are not the main targets for the anti-HCV activity of enviroxime and BF738735. Instead, we demonstrated that both molecules impede HCV replication at least partially by an inhibitory effect on PI3Ks. Moreover, HCV is able to bypass PI3K inhibition by acquiring mutations in its genome. ispartof: Journal Of Antimicrobial Chemotherapy vol:73 issue:12 pages:3375-3384 ispartof: location:England status: published

Bioassay-guided fractionation of an EtOAc extract of the trunk bark of Sandwithia guyanensis, using a chikungunya virus (CHIKV)-cell-based assay, afforded 17 new diterpenoids 1-17 and the known jatrointelones A and C (18 and 19). The new compounds included two tetranorditerpenoids 1 and 2, a trinorditerpenoid 3, euphoractines P-W (4-11), and euphactine G (13) possessing the rare 5/6/7/3 (4-7), 5/6/6/4 (8-11), and 5/6/8 (13) fused ring skeletons, sikkimenoid E (12), and jatrointelones J-M (14-17) possessing jatropholane and lathyrane carbon skeletons, respectively. Jatrointelones J (14) and M (17) represent the first naturally occurring examples of C-15 nonoxidized lathyrane-type diterpenoids. The structures of the new compounds were elucidated by NMR spectroscopic data analysis. The relative configuration of compound 16 and the absolute configurations of compounds 3-6 and 14 were determined by single-crystal X-ray diffraction analysis. In addition, jatrointelone K (15) was chemically transformed to euphoractine T (8) supporting the biosynthetic relationships between the two types of diterpenoids. Only compound 15 showed a moderate anti-CHIKV activity with an EC

Chikungunya virus is a re-emerging arbovirus transmitted to humans by mosquitoes, responsible for an acute flu-like illness associated with debilitating arthralgia, which can persist for several months or become chronic. In recent years, this viral infection has spread worldwide with a previously unknown virulence. To date, no specific antivirals treatments nor vaccines are available against this important pathogen. Starting from the structures of two antiviral hits previously identified in our research group with in silico techniques, this work describes the design and preparation of 31 novel structural analogues, with which different pharmacophoric features of the two hits have been explored and correlated with the inhibition of Chikungunya virus replication in cells. Structure-activity relationships were elucidated for the original scaffolds, and different novel antiviral compounds with EC50 values in the low micromolar range were identified. This work provides the foundation for further investigation of these promising novel structures as antiviral agents against Chikungunya virus.

The novel enterovirus protease inhibitor (PI) SG85 effectively inhibits the in vitro replication of 14 rhinoviruses representative of species A and B (median 50% effective concentration, 0.04 μM). A low-level SG85-resistant variant was selected that carried amino acid substitutions S127G and T143A in the 3C protease. Both substitutions are required for low-level resistance to SG85, as demonstrated by reverse genetics. Interestingly, there is no cross-resistance to SG85 and rupintrivir (another PI); a structural explanation is provided for this observation.

International audience; In an effort to identify new potent and selective inhibitors of chikungunya virus and HIV-1 and HIV-2 virus replication, the endemic Mascarene species Stillingia lineata was investigated. LC/MS and bioassay-guided purification of the EtOAc leaf extract using a chikungunya virus-cell-based assay led to the isolation of six new (4–9) and three known (1–3) tonantzitlolones possessing the rare C20-flexibilane skeleton, along with tonantzitloic acid (10), a new linear diterpenoid, and three new (11, 13, and 15) and two known (12 and 14) tigliane-type diterpenoids. The planar structures of the new compounds and their relative configurations were determined by spectroscopic analysis, and their absolute configurations were determined through comparison with literature data and from biogenetic considerations. These compounds were investigated for selective antiviral activity against chikungunya virus (CHIKV), Semliki Forest virus, Sindbis virus, and, for compounds 11–15, the HIV-1 and HIV-2 viruses. Compounds 12–15 were found to be the most potent and are selective inhibitors of CHIKV, HIV-1, and HIV-2 replication. In particular, compound 14 inhibited CHIKV replication with an EC50 value of 1.2 μM on CHIKV and a selectivity index of >240, while compound 15 inhibited HIV-1 and HIV-2 with EC50 values of 0.043 and 0.018 μM, respectively. It was demonstrated further that potency and selectivity are sensitive to the substitution pattern on the tigliane skeleton. The cytotoxic activities of compounds 1–10 were evaluated against the HCT-116, MCF-7, and PC3 cancer cell lines.

Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. publisher: Elsevier articletitle: Itraconazole Inhibits Enterovirus Replication by Targeting the Oxysterol-Binding Protein journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2014.12.054 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Inc. ispartof: Cell Reports vol:10 issue:4 pages:600-615 ispartof: location:United States status: published

In this study, we demonstrate that N(6)-isopentenyladenosine, which essentially is a plant cytokinin-like compound, exerts a potent and selective antiviral effect on the replication of human enterovirus 71 with an EC50 of 1.0 ± 0.2 μM and a selectivity index (SI) of 5.7. The synthesis of analogs with modification of the N(6)-position did not result in a lower EC50 value. However, in particular with the synthesis of N(6)-(5-hexene-2-yne-1-yl)adenosine (EC50 = 4.3 ± 1.5 μM), the selectivity index was significantly increased: because of a reduction in the adverse effect of this compound on the host cells, an SI > 101 could be calculated. With this study, we for the first time provide proof that a compound class that is based on the plant cytokinin skeleton offers an interesting starting point for the development of novel antivirals against mammalian viruses, in the present context in particular against enterovirus 71. publisher: Elsevier articletitle: Chemical modification of the plant isoprenoid cytokinin N6-isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication journaltitle: European Journal of Medicinal Chemistry articlelink: http://dx.doi.org/10.1016/j.ejmech.2014.11.048 content_type: article copyright: Copyright © 2014 Elsevier Masson SAS. All rights reserved. ispartof: European Journal of Medicinal Chemistry vol:90 pages:406-413 ispartof: location:France status: published

We report on the synthesis of novel conformationally locked nucleoside and nucleotide derivatives, which are structurally closely related to clinically used antivirals such as didanosine and abacavir. As a suitable conformationally rigid substitute of the sugar/pseudosugar ring allowing a permanent stabilization of the nucleoside in North conformation we employed bicyclo[2.2.1]heptane (norbornane) substituted in the bridgehead position with a hydroxymethyl group and in the C-3 position with a nucleobase. Prepared nucleoside derivatives were also converted into appropriate phosphoramidate prodrugs (ProTides) in order to increase delivery of the compounds in the cells. All target compounds were evaluated in a broad antiviral and cytostatic assay panel.

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16–18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC50 = 0.45 μM), while compound 15 proved to be a potent and selective inhibitor of H...

Six new premyrsinol esters (1–6) and one new myrsinol ester (8) were isolated from an aerial parts extract of Euphorbia pithyusa, together with a known premyrsinol (7) and two known dideoxyphorbol esters (9 and 10), following a bioactivity-guided purification procedure using a chikungunya virus (CHIKV) cell-based assay. The structures of the new diterpene esters (1–6 and 8) were elucidated by MS and NMR spectroscopic data interpretation. Compounds 1–10 were evaluated against CHIKV replication, and results showed that the 4β-dideoxyphorbol ester 10 was the most active compound, with an EC50 value of 4.0 ± 0.3 μM and a selectivity index of 10.6. To gain more insight into the structural diversity of diterpenoids produced by E. pithyusa, the initial extract and chromatographic fractions were analyzed by LC-MS/MS. The generated data were annotated using a molecular networking procedure and revealed that dozens of unknown premyrsinane, myrsinane, and tigliane analogues were present.

Recently, we demonstrated that the natural cytokinin nucleosides N6-isopentenyladenosine (iPR) and N6-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N6-trifluoromethylbenzyladenosine derivatives (9–11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N6-(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development.

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.

A ligand-based approach was applied to screen in silico a library of commercially available compounds, with the aim to find novel inhibitors of the HCV replication starting from the study of the viral NS3 helicase. Six structures were selected for evaluation in the HCV subgenomic replicon assay and one hit was found to inhibit the HCV replicon replication in the low micromolar range. A small series of new pyrrolone compounds was designed and synthesised, and novel structures were identified with improved antiviral activity.

A structure-based virtual screening of commercial compounds was carried out on the HCV NS3 helicase structure, with the aim to identify novel inhibitors of HCV replication. Among a selection of 13 commercial structures, one compound was found to inhibit the subgenomic HCV replicon in the low micromolar range. Different series of new piperazine-based analogues were designed and synthesised, and among them, several novel structures exhibited antiviral activity in the HCV replicon assay. Some of the new compounds were also found to inhibit HCV NS3 helicase function in vitro, and one directly bound NS3 with a dissociation constant of 570 ± 270 nM.

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2016.12.010., We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship studies of this family to identify key features that might be significant for the antiviral activity. With this aim, novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a “decarboxylated” analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71 clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the divergent approach previously reported., This work has been supported by the Spanish MINECO [projects SAF2012-39760-C02 and SAF2015-64629-C2-1-R (MINECO/ FEDER)], “The Centers of Excellence” of the KU Leuven (EF-05/15 and PF-10/18), EU FP7 (FP7/2007e2013) Project EUVIRNA (Grant 408 Agreement 264286), EU FP7 SILVER (Contract HEALTH-F3- 2010-260644), a grant from the Belgian Interuniversity Attraction Poles (IAP) Phase VIIeP7/45 (BELVIR) and the EU FP7 Industry- Academia Partnerships and Pathways Project AIROPICO. The Spanish MEC/MINECO is also acknowledged for a grant to B.M.G and E.R. and the China Scholarship Council (CSC) (Grant 201403250056) for a grant to L.S. We also thank Charlotte Vanderheydt and Evelyne Van Kerckhove for help with the processing of the antiviral data.

Chikungunya virus (CHIKV), a mosquito-borne arthrogenic Alphavirus, causes an acute febrile illness in humans, that is, accompanied by severe joint pains. In many cases, the infection leads to persistent arthralgia, which may last for weeks to several years. The re-emergence of this infection in the early 2000s was exemplified by numerous outbreaks in the eastern hemisphere. Since then, the virus is rapidly spreading. Currently, no drugs have been approved or are in development for the treatment of CHIKV, which makes this viral infection particularly interesting for academic medicinal chemistry efforts. Several molecules have already been identified that inhibit CHIKV replication in phenotypic virus-cell-based assays. One of these is arbidol, a molecule that already has been licensed for the treatment of influenza A and B virus infections. For structural optimization, a dedicated libraries of 43 indole-based derivatives were evaluated leading to more potent analogues (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities higher than those of the other derivatives, including the lead compound, and with a selective index of inhibition 13.2 and 14.6, respectively, higher than that of ARB (4.6).

International audience; A bioassay-guided purification of an EtOAc extract of the leaves of Croton mauritianus using a chikungunya virus-cell-based assay led to the isolation of 12-O-decanoylphorbol-13-acetate (1) and the new 12-O-decanoyl-7-hydroperoxy-phorbol-5-ene-13-acetate (2), along with loliolide, vomifoliol, dehydrovomifoliol, annuionone D and bluemol C. The planar structure and the relative configuration of compound 2 were elucidated based on spectroscopic analysis, including 1D- and 2D-NMR experiments, mass spectrometry, and comparison with literature data. Compounds 1 and 2 inhibited chikungunya virus-induced cell death in cell culture with EC50s of 2.4±0.3 and 4.0±0.8 μM, respectively.

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl] quinolone derivative 8 proved to be the best compound of this series, exhibiting IC50 value of 0.069 µM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 µM, EC90 =13.5 µM) coupled with the absence of any cytostatic effect (CC50 >163 µM, SI >54) in Huh-9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.

The dengue fever virus (DENV) and the yellow fever virus (YFV) are members of the genus flavivirus in the family Flaviviridae. An estimated 50-100 million cases of DENV infections occur each year and approximately half a million patients require hospitalization. There is no vaccine or effective antiviral treatment available. There is an urgent need for potent and safe inhibitors of DENV replication; ideally such compounds should have broad-spectrum activity against flaviviruses. We here report on the in vitro activity of 3',5'di-O-trityluridine on flavivirus replication. The compound results in a dose-dependent inhibition of (i) DENV- and YFV-induced cytopathic effect (CPE) (EC50 values in the low micromolar range for the 4 DENV serotypes), (ii) RNA replication (DENV-2 EC50=1.5μM; YFV-17D EC50=0.83μM) and (iii) viral antigen production. Antiviral activity was also demonstrated in DENV subgenomic replicons (which do not encode the structural viral proteins) (EC50=2.3μM), indicating that the compound inhibits intracellular events of the viral replication cycle. Preliminary data indicate that the molecule may inhibit the viral RNA-dependent RNA polymerase. publisher: Elsevier articletitle: 3′,5′Di-O-trityluridine inhibits in vitro flavivirus replication journaltitle: Antiviral Research articlelink: http://dx.doi.org/10.1016/j.antiviral.2013.01.011 content_type: article copyright: Copyright © 2013 Elsevier B.V. All rights reserved. ispartof: Antiviral Research vol:98 issue:2 pages:242-247 ispartof: location:Netherlands status: published

We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3C pro ). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3C pro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3C pro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic α,β-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3C pro , which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1′; the most potent inhibitors comprise P4 to P1′. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC 50 ] = 0.5 μM for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC 50 s of ≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus–cell-based assay. Even the shorter SG75, spanning only P3 to P1′, displayed significant activity (EC 50 = 2 to 5 μM) against various rhinoviruses.

International audience; An efficient process was used to isolate six new jatrophane esters, euphodendroidins J (3), K (5), L (6), M, (8), N (10), and O (11), along with seven known diterpenoid esters, namely, euphodendroidins A (4), B (9), E (1), and F (2), jatrophane ester (7), and 3α-hydroxyterracinolides G and B (12 and 13), and terracinolides J and C (14 and 15) from the latex of Euphorbia dendroides. Their 2D structures and relative configurations were established by extensive NMR spectroscopic analysis. The absolute configurations of compounds 1, 11, and 15 were determined by X-ray diffraction analysis. Euphodendroidin F (2) was obtained in 18% yield from the diterpenoid ester-enriched extract after two consecutive flash chromatography steps, making it an interesting starting material for chemical synthesis. Euphodendroidins K and L (5 and 6) showed an unprecedented NMR spectroscopic behavior, which was investigated by variable-temperature NMR experiments and molecular modeling. The structure–conformation relationships study of compounds 1, 5, and 6, using DFT-NMR calculations, indicated the prominent role of the acylation pattern in governing the conformational behavior of these jatrophane esters. The antiviral activity of compounds 1–15 was evaluated against Chikungunya virus (CHIKV) replication.

Previously, we reported that salicylate-based analogs of bryostatin protect cells from chikungunya virus (CHIKV)-induced cell death. Interestingly, 'capping' the hydroxyl group at C26 of a lead bryostatin analog, a position known to be crucial for binding to and modulation of protein kinase C (PKC), did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a pathway independent of PKC. The work detailed in this study demonstrates that salicylate-derived analog 1 and two capped analogs (2 and 3) are not merely cytoprotective compounds, but act as selective and specific inhibitors of CHIKV replication. Further, a detailed comparative analysis of the effect of the non-capped versus the two capped analogs revealed that compound 1 acts both at early and late stages in the chikungunya virus replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with the PKC inhibitors sotrastaurin and Gö6976 counteracts the antiviral activity of compound 1 without affecting that of capped analogs 2 and 3, providing further evidence that the latter elicit their anti-CHIKV activity independently of PKC. Remarkably, treatment of CHIKV-infected cells with a combination of compound 1 and a capped analog resulted in a pronounced synergistic antiviral effect. Thus, these salicylate-based bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway. publisher: Elsevier articletitle: Comparative analysis of the anti-chikungunya virus activity of novel bryostatin analogs confirms the existence of a PKC-independent mechanism journaltitle: Biochemical Pharmacology articlelink: http://dx.doi.org/10.1016/j.bcp.2016.09.020 content_type: article copyright: © 2016 Elsevier Inc. All rights reserved. ispartof: Biochemical Pharmacology vol:120 pages:15-21 ispartof: location:England status: published

The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive reemergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections., This work was supported by the European Union Seventh Framework Program (FP7/2007–2013) under SILVER grant agreement (grant number 260644), EUVIRNA (grant agreement number 264286), BELSPO (IUAP-BELVIR), and the Spanish CICYT (SAF2012-39760-C02-01). L.D. is supported by a fellowship of the Fund for Scientific Research, Flanders (FWO). N.S.G. has been supported by the Doctoral Research Training Program “Francisco Jose de Caldas 494-2009” from Colombia and an ERACOL scholarship. A.G. has been supported by a JAEpredoctoral fellowship financed by the CSIC and the FSE (Fondo Social Europeo).

International audience; Human rhinoviruses (HRV) are the predominant cause of common colds and flu-like illnesses, but are also responsible for virus-induced exacerbations of asthma and chronic obstructive pulmonary disease. However, to date, no drug has been approved yet for clinical use. In this study, we present the results of the structure-based lead optimization of a class of new small-molecule inhibitors that we previously reported to bind into the pocket beneath the canyon of the VP1 protein. A small series of analogues that we designed based on the available structure and interaction data were synthesized and evaluated for their potency to inhibit the replication of HRV serotype 14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (3v) was found to be a potent inhibitor exhibiting micromolar activity (EC50 = 3.4 ± 1.0 μM) with a toxicity for HeLa cells that was significantly lower than that of our previous hit (LPCRW_0005, CC50 = 104.0 ± 22.2 μM; 3v, CC50 > 263 μM).

The Global Polio Eradication Initiative, launched in 1988, had as its goal the eradication of polio worldwide by the year 2000 through large-scale vaccinations campaigns with the live attenuated oral PV vaccine (OPV) (Griffiths et al., Biologicals 34:73-74, 2006). Despite substantial progress, polio remains endemic in several countries and new imported cases are reported on a regular basis ( http://www.polioeradication.org/casecount.asp ).It was recognized by the poliovirus research community that developing antivirals against poliovirus would be invaluable in the post-OPV era. Here, we describe three methods essential for the identification of selective inhibitors of poliovirus replication and for determining their mode of action by time-of-drug-addition studies as well as by the isolation of compound-resistant poliovirus variants.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus showing a recent resurgence and rapid spread worldwide. While vaccines are under development, there are currently no therapies to treat this disease, except for over-the-counter (OTC) analgesics, which alleviate the devastating arthritic and arthralgic symptoms. To identify novel inhibitors of the virus, analogues of the natural product bryostatin 1, a clinical lead for the treatment of cancer, Alzheimer's disease, and HIV eradication, were investigated for in vitro antiviral activity and were found to be among the most potent inhibitors of CHIKV replication reported to date. Bryostatin-based therapeutic efforts and even recent anti-CHIKV strategies have centered on modulation of protein kinase C (PKC). Intriguingly, while the C ring of bryostatin primarily drives interactions with PKC, A- and B-ring functionality in these analogues has a significant effect on the observed cell-protective activity. Significantly, bryostatin 1 itself, a potent pan-PKC modulator, is inactive in these assays. These new findings indicate that the observed anti-CHIKV activity is not solely mediated by PKC modulation, suggesting possible as yet unidentified targets for CHIKV therapeutic intervention. The high potency and low toxicity of these bryologs make them promising new leads for the development of a CHIKV treatment. ispartof: Journal of Natural Products vol:79 issue:4 pages:675-9 ispartof: location:United States status: published

A series of new conjugated compounds with a -SCH₂- linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 μM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents. ispartof: Molecules vol:21 issue:2 ispartof: location:Switzerland status: published

We recently identified indole derivatives (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities at lower micro molar concentrations and a selective index of inhibition higher than the lead compound Arbidol. Here we highlight new structural information for the optimization of the previously identified lead compounds that contain the indole chemical core. Based on the structural data, a series of indole derivatives was synthesized and tested for their antiviral activity against chikungunya virus in Vero cell culture by a CPE reduction assay. Systematic optimization of the lead compounds resulted in tert-butyl-5-hydroxy-1-methyl-2-(2-trifluoromethysulfynyl)methyl)-indole-3-carboxylate derivative IIc with a 10-fold improved anti-CHIKV inhibitory activity (EC50=6.5±1μM) as compared to Arbidol demonstrating a potent, selective and specific inhibition of CHIKV replication with only a moderate cell protective effect against other related alphaviruses. The reported computational insights, together with the accessible synthetic procedure, pave the road towards the design of novel synthetic derivatives with enhanced anti-viral activities. publisher: Elsevier articletitle: Discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication journaltitle: Bioorganic & Medicinal Chemistry articlelink: http://dx.doi.org/10.1016/j.bmc.2016.10.037 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved. ispartof: Bioorganic & Medicinal Chemistry vol:25 issue:1 pages:327-337 ispartof: location:England status: published

A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of ∼450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.