Nieuwe enterovirus remmers; virale en cellulaire doeleiwitten

The genus Enterovirus of the family Picornaviridae includes many important human pathogens, such as poliovirus (PV), coxsackievirus, enterovirus 71 and rhinovirus. These viruses can cause a wide variety of diseases, ranging from mild to potentially fatal. Efficient vaccines against PV have almost succeeded to globally eradicate PV, but development of vaccines against all enteroviruses is not feasible due to the enormous amount of serotypes (almost 300 identified to date). At the moment, no antiviral drugs are available and treatment is limited to symptomatic and supportive care. For these reasons, antiviral drugs are urgently needed. In addition, antiviral drugs may be beneficial to contain any posteradication outbreaks of PV which may occur after stopping of the global vaccination program. Therefore, the aim of this thesis was to identify and characterize targets in the replication cycle of enteroviruses for antiviral therapy and compounds with antiviral activity against enteroviruses. These compounds may either exert their antiviral activity by targeting viral proteins or host factors that are essential for virus replication. Furthermore, by studying the mechanism of action of enterovirus inhibitors, we aim to increase our knowledge of the viral replication process and the requirements. This information may in turn benefit the search for new antiviral drugs, for example by identifying new drug targets. In CHAPTER 2, we have studied the effects of two putative inhibitors of the cellular protein GBF1 on enterovirus replication. The guanine nucleotide exchange factor GBF1 is an essential host factor for replication of enteroviruses. At the early secretory pathway, GBF1 activates the GTPase Arf1 by stimulating GTP exchange. Activated Arf1 then regulates membrane trafficking through the recruitment of effector proteins. Inhibition of GBF1 by the fungal metabolite Brefeldin A (BFA) strongly impairs enterovirus replication. The compound Golgicide A (GCA) was proposed to be an inhibitor of GBF1, with a similar mechanism of action as BFA, but with higher specificity. AG1478 was also reported to inhibit GBF1 activity, but in a different, probably indirect manner. Consequently, we investigated whether these compounds would share the antiviral effect of BFA. We found that GCA inhibited enterovirus replication, but this was not the case for AG1478, supporting the idea that this latter compound does not inhibit GBF1 in the same manner as the other compounds. A further major finding supporting this idea is that the effects of AG1478 on the function and integrity of the secretory pathway could be rescued by overexpression of Arf1, which was not the case for BFA and GCA. Both GCA, which unlike BFA does not target other large Arf activators, and AG1478, which disrupts secretory pathway integrity and function by a different mechanism than BFA and GCA, provide important new tools to further dissect the role of GBF1 in enterovirus RNA replication. CHAPTER 3 describes the identification and characterization of a newantiviral compound, TTP-8307. The spectrum of antiviral activity of TTP-8307 encompassed most, but not all, enteroviruses. To study the mechanism of action of the compound, TTP-8307-resistant coxsackievirus B3 (CVB3) mutants were selected by passaging 10 times in the presence of TTP-8307. These mutants harbored mutations in the viral protein 3A, a membrane protein implicated in the viral modification of secretory pathway membranes. Previously, a variety of mutations in 3A had been shown to confer resistance to enviroxime, an antiviral compound identified in the late 1970s of which the mechanism of action remained unknown. We showed that the TTP-8307-resistant mutants were cross-resistant to enviroxime, suggesting that these antiviral compounds have a similar or related mechanism of action. Subsequently, in CHAPTER 4 we showed that GW5074 also is an inhibitor of enterovirus replication, and that this kinase inhibitor, as well as enviroxime, act by inhibiting the activity of PI4KIIIß, a lipid kinase that had just been identified as an essential host factor for enterovirus replication3. We also showed that enviroxime- and GW5074-resistant viruses (i.e., the viruses with the single point mutations identified in CHAPTER 3) could replicate in the absence of high levels of PI4P, the product of PI4KIIIß. These results demonstrate that viruses are so versatile that they can even bypass the need for host factors for virus replication. In CHAPTER 5, NIH Clinical Collection compound library, a collection of small molecules that have a history of use in human clinical trials, was screened for inhibitors of CVB3. This screen revealed that itraconazole (ITZ), a compound which is in use as an antifungal drug, has antiviral activity. Known targets of ITZ, such as hCYP51, CYP3A4 or the hedgehog pathway were investigated if they mediated the antiviral activity of ITZ, but this appeared not to be the case. Interestingly, the antiviral activity of ITZ could be counteracted by the same mutations in 3A that provided protection against PI4KIIIß inhibitors, suggesting a similar mechanism of action. However, ITZ had no effect on PI4KIIIß activity. Also, unlike PI4KIIIß inhibitors, ITZ inhibited virus replication of cardioviruses, providing further evidence for a different target. CHAPTER 6 reports on the antiviral activity of fluoxetine, an anti-depressant (brand name Prozac) that was also identified in a screen of the NIH Clinical Collection. The antiviral spectrum of activity of fluoxetine encompassed some enteroviruses, but not all. The observation that mutations in the viral protein 2C of CVB3 offered protection to the compound, suggested that fluoxetine targets 2C. 2C has been implicated in many different aspects of virus replication, but its exact function is unknown. 2C is an ATPase, but fluoxetine had only a modest effect on 2C’s ATPase activity. This finding suggests that fluoxetine targets another activity of 2C. In CHAPTER 7, we studied the antiviral activity of the small molecule GPC-N114. GPC-N114 inhibited virus replication of both enteroviruses and cardioviruses at the RNA replication stage. No resistant CVB3 variants were obtained despite many passages in the presence of GPC-N114. In the cardiovirus EMCV, mutations in the viral RNA-dependent RNA polymerase 3Dpol conferred resistance to the compound. Accordingly, GPC-N114 inhibited in vitro elongation activity of CVB3 and EMCV 3Dpol. Analysis of the three-dimensional structure of 3Dpol in complex with GPC-N114 revealed that the compound, a non-nucleoside analog, bound at the site where the templating nucleotide is located during the RNA replication process. This result suggests that GPC-N114 may interfere with template binding. This hypothesis was confirmed in functional studies. Collectively, these findings show that GPC-N114 inhibits picornavirus replication by affecting the binding of the RNA template to 3Dpol, making this the first picornavirus inhibitor with this mechanism of action. Finally, in CHAPTER 8 we describe the development of a cell-based assay for determining the capability of compounds to inhibit picornaviral protease activity. Inhibition of protease activity results in the induction of expression of a firefly luciferase reporter. A major advantage of this assay is that it is cell-based, in contrast to most protease assays available, allowing to select for non-toxic and cell-permeable compounds. We showed that the assay could accurately predict the potential of compounds to inhibit virus replication, with IC50 values obtained for several compounds in the same range as those determined with virus infections. In addition, the assay was versatile, applicable to CVB3 3Cpro and 2Apro and could easily be extended to 3Cpro of different genera. In combination with the excellent properties for use in a high-throughput setting, this assay could be a valuable tool for the discovery and validation of protease inhibitors.Chapter 1 General introduction and outline of this thesis Chapter 2 Mutations in the non-structural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307 Chapter 3 Differential effects of the putative GBF1 inhibitors golgicide A and AG1478 on enterovirus replication Chapter 4 Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication Chapter 5 The antifungal itraconazole inhibits picornavirus RNA replication by targeting OSBP and ORP4 Chapter 6 Selective serotonin reuptake inhibitor fluoxetine inhibits replication of Human Enterovirus B and D by targeting viral protein 2C Chapter 7 A non-nucleoside inhibitor of the RNA-dependent RNA polymerase of enteroviruses and cardioviruses that affects binding of the RNA template Chapter 8 Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases Chapter 9 Summary & General discussion Chapter 10 Nederlandse samenvatting Appendices Acknowledgments - Dankwoord; Curriculum vitae; Bibiographynrpages: 220status: publishe