Rana Abdelnabi

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.”

Inhibition of Chikungunya Virus-Induced Cell Death by Salicylate-Derived Bryostatin Analogues Provides Additional Evidence for a PKC-Independent Pathway

Chikungunya virus (CHIKV) has been spreading rapidly, with over one million confirmed or suspected cases in the Americas since late 2013. Infection with CHIKV causes devastating arthritic and arthralgic symptoms. Currently, there is no therapy to treat this disease, and the only medications focus on relief of symptoms. Recently, protein kinase C (PKC) modulators have been reported to inhibit CHIKV-induced cell death in cell assays. The salicylate-derived bryostatin analogues described here are structurally simplified PKC modulators that are more synthetically accessible than the natural product bryostatin 1, a PKC modulator and clinical lead for the treatment of cancer, Alzheimers disease, and HIV eradication. Evaluation of the anti-CHIKV activity of these salicylate-derived bryostatin analogues in cell culture indicates that they are among the most potent cell-protective agents reported to date. Given that they are more accessible and significantly more active than the parent natural product, they represent new therapeutic leads for controlling CHIKV infection. Significantly, these analogues also provide evidence for the involvement of a PKC-independent pathway. This adds a fundamentally distinct aspect to the importance or involvement of PKC modulation in inhibition of chikungunya virus replication, a topic of recent and growing interest.

Chikungunya virus infections: time to act, time to treat

Chikungunya virus (CHIKV) is a re-emerging alphavirus that caused massive outbreaks of Chikungunya fever in several countries and regions in Africa, Asia and more recently in Central and South America. An acute CHIKV infection is usually associated with fever and arthritis and it is rarely fatal. However, 15-60% of patients suffer from chronic polyarthralgia for weeks, months or even for several years after the acute infection. There are currently no vaccines or antivirals available for the prevention or treatment of CHIKV infections. The development of potent and safe antivirals for prophylaxis (e.g., during travel to CHIKV-endemic regions) and treatment of CHIKV infections is urgently needed. We here review some of the recently reported CHIKV inhibitors, both directly-acting and host-targeting compounds.

Simplified Bryostatin Analogues Protect Cells from Chikungunya Virus-Induced Cell Death

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.

Characterization of a panel of monoclonal antibodies toward mouse PAI-1 that exert a significant profibrinolytic effect in vivo.

INTRODUCTION PAI-1 is the main physiological inhibitor of t-PA and u-PA. Elevated PAI-1 levels have been implicated in the pathogenesis of several thrombotic and non-thrombotic diseases. The effect of PAI-1 inhibition can be studied in mouse models, when appropriate immunological tools are available. The majority of the available monoclonal antibodies against PAI-1 have been raised against human PAI-1. Even though some of these antibodies cross-react with non-glycosylated PAI-1 from different species, these antibodies often do not cross-react sufficiently with glycosylated mouse PAI-1. Moreover, the antibodies that cross-react with glycosylated mouse PAI-1 often have decreased inhibitory properties in the presence of vitronectin. Our objective was the generation of a panel of monoclonal antibodies reacting with vitronectin-bound glycosylated mouse PAI-1. RESULTS Five monoclonal antibodies revealed binding to glycosylated mouse PAI-1 and exerted a strong (i.e., 58-80% inhibition of PAI-1 activity) inhibitory effect toward mouse PAI-1. Similar inhibitory effects were seen in the presence of a 33-fold molar excess of vitronectin. The PAI-1 inhibitory potential of the antibodies in vivo was demonstrated in a thromboembolism model, in which the evaluated antibodies significantly increased the percentage of mice with normal physical activity in comparison to mice treated with negative control antibody. CONCLUSIONS To the best of our knowledge this is the first panel of monoclonal antibodies that can inhibit mouse PAI-1 in the presence of vitronectin and that show a profibrinolytic effect in vivo. Therefore these antibodies provide excellent immunological tools to further investigate the role of PAI-1 in mouse models.

Antiviral strategies against Chikungunya virus

In the last few decades the Chikungunya virus (CHIKV) has evolved from a geographically isolated pathogen to a virus that is widespread in many parts of Africa, Asia and recently also in Central- and South-America. Although CHIKV infections are rarely fatal, the disease can evolve into a chronic stage, which is characterized by persisting polyarthralgia and joint stiffness. This chronic CHIKV infection can severely incapacitate patients for weeks up to several years after the initial infection. Despite the burden of CHIKV infections, no vaccine or antivirals are available yet. The current therapy is therefore only symptomatic and consists of the administration of analgesics, antipyretics, and anti-inflammatory agents. Recently several molecules with various viral or host targets have been identified as CHIKV inhibitors. In this chapter, we summarize the current status of the development of antiviral strategies against CHIKV infections.

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

ABSTRACT 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.

Discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication

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.

Favipiravir as a potential countermeasure against neglected and emerging RNA viruses

&NA; Favipiravir, also known as T‐705, is an antiviral drug that has been approved in 2014 in Japan to treat pandemic influenza virus infections. The drug is converted intracellularly into its active, phosphoribosylated form, which is recognized as a substrate by the viral RNA‐dependent RNA polymerase. Interestingly, besides its anti‐influenza virus activity, this molecule is also able to inhibit the replication of flavi‐, alpha‐, filo‐, bunya‐, arena‐, noro‐, and of other RNA viruses, which include neglected and (re)emerging viruses for which no antiviral therapy is currently available. We will discuss the potential of favipiravir as a broad‐spectrum countermeasure against infections caused by such neglected RNA viruses. Favipiravir has already been used off‐label to treat patients infected with the Ebola virus and the Lassa virus. Because of the particular set‐up of the clinical trials during these outbreaks, clear conclusions on the efficacy of favipiravir could not be made. For several viruses, it was demonstrated that the barrier of resistance development against favipiravir is high. Favipiravir has been shown to be well tolerated in healthy volunteers and in influenza virus‐infected patients; however, caution is needed because of the teratogenic risks of this molecule. Because of its antiviral activity against different RNA viruses and its high barrier for resistance, the potential of favipiravir as a broad‐spectrum antiviral seems promising, but safety and potency issues should be overcome before this drug or similar molecules could be used to treat large patient groups.

Glutathione is a highly efficient thermostabilizer of poliovirus Sabin strains

Glutathione (GSH) is the most abundant thiol peptide in animal cells and has a critical role in antioxidation. GSH was reported to be essential for stabilization of some enteroviruses, including poliovirus (PV), during viral morphogenesis. Here, we explored the potential use of GSH as a thermostabilizer of oral poliomyelitis vaccine (OPV) formulations. GSH significantly protected the three types of PV from heat-inactivation in a concentration-dependent manner. At a GSH concentration of 20mM, nearly complete protection was observed against heating temperatures up to 53°C for 2min.GSH also markedly protected PV1 from heat-inactivation and this up to 6 h at temperatures of 44°C and 46°C and 3 h at 48°C. The fact that GSH is naturally present at high concentration in the human body makes it an efficient candidate stabilizer for OPV formulations.