Leen Delang

Statins potentiate the in vitro anti‐hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development

Statins are 3‐hydroxyl‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti‐HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon‐alpha (IFN‐α) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short‐term (3 days) antiviral assays. Neither statins, at a concentration of five‐fold their median effective concentration (EC50) value, nor polymerase, protease inhibitors, or IFN‐α, at concentrations 10‐ or 20‐fold their EC50 value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV‐796. Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors. (HEPATOLOGY 2009.)

Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral

OBJECTIVES T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus. METHODS The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir. RESULTS Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general. CONCLUSIONS This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals.

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

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. (HEPATOLOGY 2013)

Prostratin and 12-o-tetradecanoylphorbol 13-acetate are potent and selective inhibitors of chikungunya virus replication

A chemical study of the Vietnamese plant species Trigonostemon howii led to the isolation of a new tigliane-type diterpenoid, trigowiin A (1), along with several known coumarins and phenylpropanoids. The planar structure and the relative configuration of compound 1 were elucidated based on spectroscopic analysis, including 1D- and 2D-NMR experiments, mass spectrometry, and comparison with literature data. Trigowiin A (1) exhibited moderate antiviral activity in a virus-cell-based assay for Chikungunya virus (CHIKV). Since the structure of compound 1 is closely related to those of well-known tigliane diterpenoids such as prostratin (2), phorbol (3), 12-O-tetradecanoylphorbol 13-acetate (TPA) (4), and 4α-TPA (5), the antiviral activity of the latter compounds was also evaluated against CHIKV, as well as in virus-cell-based assays of two additional members of the genus Alphavirus (Sindbis virus, SINV, and Semliki forest virus, SFV). Whereas prostratin inhibited CHIKV replication with a moderate EC(50) of 2.6 μM and a selectivity index (SI) approximating 30, compound 4 proved to be an extremely potent inhibitor, with an EC(50) of ∼3 nM and a SI near 2000. Interestingly, no or very little activity was observed on the replication of SINV and SFV.

Comparative Study of the Genetic Barriers and Pathways towards Resistance of Selective Inhibitors of Hepatitis C Virus Replication

ABSTRACT Hepatitis C virus (HCV) inhibitors include direct-acting antivirals (DAAs) such as NS3 serine protease inhibitors, nucleoside and nonnucleoside polymerase inhibitors, and host-targeting antivirals (HTAs) such as cyclophilin inhibitors that have been developed in recent years. Drug-resistant HCV variants have been reported both in vitro and in the clinical setting for most classes of drugs. We report a comparative study in which the genetic barrier to drug resistance of a representative selection of these inhibitors is evaluated employing a number of resistance selection protocols. The NS3 protease inhibitors VX-950 and BILN 2061, the nucleoside polymerase inhibitor 2′-C-methylcytidine, three nonnucleoside polymerase inhibitors (thiophene carboxylic acid, benzimidazole, and benzothiadiazine), and DEB025 were included. For each drug and passage in the selection process, the phenotype and genotype of the drug-resistant replicon were determined. For a number of molecules (BILN 2061 and nonnucleoside inhibitors), drug-resistant variants were readily selected when wild-type replicon-containing cells were directly cultured in the presence of high concentrations of the inhibitor. Resistance to DEB025 could be selected only following a lengthy stepwise selection procedure. For some DAAs, the signature mutations that emerged under inhibitor pressure differed depending on the selection protocol that was employed. Replication fitness of resistant mutants revealed that the C445F mutation in the RNA-dependent RNA polymerase can restore loss of fitness caused by a number of unfit resistance mutations. These data provide important insights into the various pathways leading to drug resistance and allow a direct comparison of the genetic barriers of various HCV drugs.

Computer-aided identification, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus.

Chikungunya virus (CHIKV) is an Arbovirus that is transmitted to humans primarily by the mosquito species Aedes aegypti. Infection with this pathogen is often associated with fever, rash and arthralgia. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. Albeit considered a tropical pathogen, adaptation of the virus to the mosquito species Aedes albopictus, which is also very common in temperate zones, has resulted in recent outbreaks in Europe and the US. In the present study, we report on the discovery of a novel series of compounds that inhibit CHIKV replication in the low μM range. In particular, we initially performed a virtual screening simulation of ∼5 million compounds on the CHIKV nsP2, the viral protease, after which we investigated and explored the Structure-Activity Relationships of the hit identified in silico. Overall, a series of 26 compounds, including the original hit, was evaluated in a virus-cell-based CPE reduction assay. The study of such selective inhibitors will contribute to a better understanding of the CHIKV replication cycle and may represents a first step towards the development of a clinical candidate drug for the treatment of this disease.

Towards antivirals against chikungunya virus

Abstract Chikungunya virus (CHIKV) has re-emerged in recent decades, causing major outbreaks of chikungunya fever in many parts of Africa and Asia, and since the end of 2013 also in Central and South America. Infections are usually associated with a low mortality rate, but can proceed into a painful chronic stage, during which patients may suffer from polyarthralgia and joint stiffness for weeks and even several years. There are no vaccines or antiviral drugs available for the prevention or treatment of CHIKV infections. Current therapy therefore consists solely of the administration of analgesics, antipyretics and anti-inflammatory agents to relieve symptoms. We here review molecules that have been reported to inhibit CHIKV replication, either as direct-acting antivirals, host-targeting drugs or those that act via a yet unknown mechanism. This article forms part of a symposium in Antiviral Research on “Chikungunya discovers the New World.”

The role of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate during viral replication.

Abstract Phosphoinositides (PI) are phospholipids that mediate signaling cascades in the cell by binding to effector proteins. Reversible phosphorylation of the inositol ring at positions 3, 4 and 5 results in the synthesis of seven different phosphoinositides. Each phosphoinositide has a unique subcellular distribution with a predominant localization in subsets of membranes. These lipids play a major role in recruiting and regulating the function of proteins at membrane interfaces [1]. Several bacteria and viruses modulate and exploit the host PI metabolism to ensure efficient replication and survival. Here, we focus on the roles of cellular phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4-kinases (PI4Ks) during the replication cycle of various viruses. It has been well documented that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ, EC 2.7.1.67) is indispensable for viral RNA replication of several picornaviruses. Two recruitment strategies were reported: (i) binding and modulation of GBF1/Arf1 to enhance recruitment of PI4KIIIβ and (ii) interaction with ACBD3 for recruitment of PI4KIIIβ. PI4KIII has also been demonstrated to be crucial for hepatitis C virus (HCV) replication. PI4KIII appears to be directly recruited and activated by HCV NS5A protein to the replication complexes. In contrast to picornaviruses, it is still debated whether the α or the β isoform is the most important. PI4KIII can be explored as a target for inhibition of viral replication. The challenge will be to develop highly selective inhibitors for PI4KIIIα and/or β and to avoid off-target toxicity.

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.

Identification of [1,2,3]Triazolo[4,5-d]pyrimidin-7(6H)-ones as Novel Inhibitors of Chikungunya Virus Replication

Chikungunya virus (CHIKV) is a re-emerging Alphavirus that is transmitted to humans by Aedes mosquitoes. Currently, there are still no drugs or vaccines available for the treatment or prevention of this disease. Although traditionally restricted to Africa and Asia, the adaptation of the virus to Aedes albopictus, a mosquito species with an almost worldwide distribution, has contributed to the geographical spread of this virus in the past decade. Here, we report on a new family of compounds named [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones that inhibit CHIKV replication in the low micromolar range with no toxicity to the host (Vero) cells. The most potent compound in this series has an EC50 value below 1 μM with no cytotoxicity detected up to 668 μM, therefore affording a selectivity index greater than 600. Interestingly, the compounds have little or no antiviral activity on the replication of other members of the Togaviridae family. The exploration and study of this class of selective inhibitors of CHIKV replication will contribute to deeper insights into the CHIKV life cycle and may be a first step toward the development of a clinical drug candidate.