Optimization of a class of tryptophan dendrimers that inhibit HIV replication leads to a selective, specific, and low-nanomolar inhibitor of clinical isolates of enterovirus A71

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.Peer Reviewe

Design, synthesis and evaluation against Chikungunya virus of novel small-molecule antiviral agents

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

A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are capsid binders that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.Peer reviewe

The postbinding activity of scavenger receptor class B type I mediates initiation of hepatitis C virus infection and viral dissemination.

International audienceUNLABELLED: Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti-SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. CONCLUSION: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI-mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI

Antiviral Therapy for Hepatitis C Virus: Beyond the Standard of Care

Hepatitis C virus (HCV) represents a major health burden, with an estimated 180 million chronically infected individuals worldwide. These patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Infection with HCV is the leading cause of liver transplantation in the Western world. Currently, the standard of care (SoC) consists of pegylated interferon alpha (pegIFN-α) and ribavirin (RBV). However this therapy has a limited efficacy and is associated with serious side effects. Therefore more tolerable, highly potent inhibitors of HCV replication are urgently needed. Both Specifically Targeted Antiviral Therapy for HCV (STAT-C) and inhibitors that are believed to interfere with the host-viral interaction are discussed

Meeting report: 34th international conference on antiviral research

As a result of the multiple gathering and travels restrictions during the SARS-CoV-2 pandemic, the annual meeting of the International Society for Antiviral Research (ISAR), the International Conference on Antiviral Research (ICAR), could not be held in person in 2021. Nonetheless, ISAR successfully organized a remote conference, retaining the most critical aspects of all ICARs, a collegiate gathering of researchers in academia, industry, government and non-governmental institutions working to develop, identify, and evaluate effective antiviral therapy for the benefit of all human beings. This article highlights the 2021 remote meeting, which presented the advances and objectives of antiviral and vaccine discovery, research, and development. The meeting resulted in a dynamic and effective exchange of ideas and information, positively impacting the prompt progress towards new and effective prophylaxis and therapeutics

STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients. SARS-CoV-2 infection can result in severe lung inflammation and pathology, but host response remains incompletely understood. Here the authors show in Syrian hamsters that STAT2 signaling restricts systemic virus dissemination but also drives severe lung injury, playing a dual role in SARS-CoV-2 infection

Animal models for COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (frst detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the fndings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.info:eu-repo/semantics/acceptedVersio

Arbovirus infections: time to act, time to treat?

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Identification of novel targets for the inhibition of hepatitis c virus replication and strategies to prevent the development of drug resistance

An estimated 170 million people are chronically infected with the hepatitis C virus (HCV) worldwide and thus at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Moreover, late complications of HCV infection are the most important indication for liver transplantation in Western countries. The current standard therapy consists of the combination of pegylated interferon-alfa and ribavirin. The efficacy of standard therapy is however limited, particularly in genotype 1 infected patients, and therapy is associated with serious adverse effects. Therefore, more potent and better tolerated drugs are urgently needed to treat HCV infected patients. The last decade great progress has been made in the development of direct acting antivirals (DAA) against HCV. Very recently, the first DAA received market authorisation by the FDA, being protease inhibitors telaprevir (Incivek®) and boceprevir (Victrelis®). A major challenge for future HCV therapy will be the prevention of drug resistance development. Due to the poor fidelity of the HCV polymerase and the high magnitude of replication, drug resistant variants pre-exist as minor populations within the patients population of HCV quasispecies. As shown by in vitro and in vivo studies, these resistant variants will rapidly become dominant in the virus population when treated with one DAA. For several classes of HCV inhibitors, the development of drug resistance has been studied in vitro. However, since different resistance selection protocols are used in different studies, it is not possible to directly compare the genetic barrier to antiviral drug resistance of various (classes of) HCV drugs. In the first part of this thesis (chapter 2) we therefore present a comparative study of the genetic barrier to resistance of different classes of HCV inhibitors. We demonstrated that the choice of the resistance selection protocol can influence the emergence of resistance mutations. Furthermore, we confirmed that nucleoside polymerase inhibitors and host-targeting antivirals (DEB025) have a high genetic barrier to resistance as compared to protease inhibitors and non-nucleoside polymerase inhibitors (NNI). A mutation that is known to be involved in resistance to a benzofuran NNI, C445F (in NS5B), may compensate for the loss of replication fitness caused by unfit drug resistance mutations for other classes of NNIs. Our observations highlight the importance of in depth studies on the in vitro drug resistant genotype and phenotype of inhibitors when planning (combination) clinical studies.In the slipstream of the resistance studies that were performed for a number of HCV reference inhibitors, we identified a novel resistance mutation selected for by the non-nucleoside polymerase inhibitor benzimidazole JT-16, T389S/A. The characterization of this mutation is described in chapter 3. The signature resistance mutations for benzimidazole and indole derivatives are located at residues P495, P496 and V499 in thumb domain 1 of HCV NS5B. We here demonstrated that T389A and T389S resulted in moderate levels of resistance to JT-16. On the contrary, P495A resulted in higher levels of resistance. Although associated with a lower resistance level, mutation T389S was found to be the dominant resistance mutation in the JT-16res population. This is probably due to the good replicative capacity of this mutant, whereas the replication fitness of P495A proved to be markedly impaired when compared to wild-type. We thereby demonstrated that the resistance mutations emerging during resistance selection can be affected by structural modifications within a class of inhibitors with a similar scaffold. To address the major challenge of HCV therapy regarding resistance development, it will be necessary to combine antivirals that have a different mode of action or that target different positions on the same protein. In chapter 4, we determined the antiviral efficacy of various combinations of NNIs and the barrier towards resistance development of such combinations. Three NNIs with a different allosteric binding site were studied: benzimidazole JT-16 (thumb domain 1), thiophene carboxylic acid (thumb domain 2) and benzofuran HCV-796 (palm domain 2). The emergence of double resistant variants was efficiently prevented by short-term treatment with double combinations of either HCV-796, JT-16 or TCA whereas NNI monotherapy resulted readily in the emergence of resistant mutants. However, when a long-term, stepwise resistance selection protocol was used, double and triple NNI resistant replicons could be generated. The triple NNI resistant replicon proved resistant to all four classes of NNIs because of the partial overlap between the palm 1 and palm 2 binding pocket on NS5B. Furthermore, we demonstrated that combination therapy of NNIs did not significantly complicate the resistance profiles that are observed with monotherapy. Interestingly, different signature mutations emerged for some NNI depending on the other NNI in the particular combination. With these results we demonstrated that the design of a combination therapy solely based on the use of NNIs that target different allosteric pockets should be in theory possible. Another strategy to delay or prevent the development of HCV resistance to antivirals could be the use of host-targeting antivirals (HTA) in HCV therapy. A particular characteristic of HCV is the intimate link with the lipid metabolism of the host cell. Therefore, targeting enzymes of the host lipid metabolism could be a new approach for the inhibition of HCV replication. Statins, the cholesterol-lowering drugs, have been reported to inhibit HCV replication in vitro. In chapter 5, we characterized the in vitro anti-HCV activity of statins alone and in combination with HCV DAA or interferon-alfa. When combined with HCV DAA or interferon-alfa, statins resulted in an additive antiviral activity. Furthermore, statin-containing combinations exhibited a great potential to clear hepatoma cells from their HCV replicon. We also demonstrated that statins are able to delay or prevent the development of resistance towards DAAs. Our data therefore encourage the potential use of statins in combination with the standard therapy or HCV DAA.The mechanism by which statins inhibit HCV replication has not yet been elucidated. Since statins were found to inhibit viral RNA replication in subgenomic replicons that only encode for the non-structural HCV proteins, the intracellular replication machinery is expected to be the target. In chapter 6, we aimed to light a tip of the veil on the mechanism of the anti-HCV activity of statins. Recent studies suggest that the in vitro anti-HCV activity of statins is due to the inhibition of geranylgeranylation of cellular proteins. We demonstrated that exogenously added geranylgeranyl pyrophosphate (GGpp) reversed the anti-HCV activity of statins, whereas cholesterol was unable to resort this effect. Furthermore, statins altered the localization of Rab5, a geranylgeranylated host protein which is a necessary host factor for HCV replication. This effect was reversed by exogenous GGpp but not by exogenous cholesterol. Since the intracellular localization of Rab5 is essential for its correct function, we hypothesize that statin treatment hampers the function of Rab5 in the cell and that this altered Rab5 function probably disturbs efficient HCV replication. Of course, further study will be needed to provide more insights in the role of prenylated Rab5 in the replication of HCV. Other host-targeting approaches could be considered in anti-HCV therapy. A good host target may be phosphatidylinositol 4-kinase IIIalfa (PI4KIIIalfa), which was reported across several studies to be essential for HCV replication. Interestingly, enviroxime and enviroxime-like inhibitors were shown to inhibit RNA replication of picornaviruses by inhibiting a similar lipid kinase, PI4KIIIß. We therefore characterized in chapter 7 the in vitro anti-HCV activity of enviroxime and a newly identified enviroxime-like inhibitor, compound A. To this end, we generated enviroxime-resistant and compound A-resistant replicons. Enviroxime and compound A proved cross-resistant, suggesting a similar mechanism responsible for resistance. Furthermore, we demonstrated that the resistant phenotype is associated with the viral genome by transfecting the RNA from resistant replicon cultures into naïve hepatoma cells. Genotyping of the resistant replicon populations revealed that the resistant replicons shared two mutations in NS4B (V38M, D167E) and one in NS5A (D389N). Swapping of the NS5A gene of the compound Ares replicon into a wild-type genome did not result in a transfer of the resistant phenotype. Therefore, mutations in NS5A are probably not responsible for resistance. The key mutation(s) responsible for the resistant phenotype thus remain to be identified. Altogether, we demonstrated that a new class of HCV HTA, the enviroxime-like compounds, can inhibit the in vitro replication of HCV. The main target of this class of inhibitors remains to be identified, but our data combined with previously published results provide strong evidence that PI4KIIIalfa is involved.status: publishe