Ontwikkeling van krachtige en breed-spectrum remmers van rhinovirussen voor de behandeling/prophylaxis van rhinovirus infecties bij patiënten met asthma en COPD.Development of potent and broad-spectrum inhibitors of rhinoviruses forthe treatment/prophylaxis of rhinovirus infections of patients with asthma and COPD.

In the fifties, rhinoviruses (HRV) were identified as the cause of the common cold, a self-limiting upper respiratory tract illness. The role of these respiratory viruses in the onset of virus-induced exacerbations of lower respiratory tract diseases like asthma and chronic obstructive pulmonary disease (COPD) became evident in the last decades. Vaccinationto prevent rhinovirus infections seems not to be feasible, since to date more than 150 different genotypes are known and the number is still rising. Development of small molecule inhibitors of rhinovirus replicationis ongoing since the 80s; however no drugs are yet available for the treatment of these infections. In the 80s and 90s, a number of rhinovirus inhibitors were evaluated in clinical studies for the treatment of common colds in otherwise healthy persons but none of them was approved. Nowadays, the new indication for the treatment of asthma and COPD exacerbations renewed the interest in the development of rhinovirus inhibitors. Since the path towards successful therapy of rhinovirus-induced COPD exacerbations not only involves (a) potent and broad-spectrum inhibitor(s), the different challenges on this road are discussed in ChapterI.The rhinovirus replication cycle consists out several stages that, theoretically, all can be the interfered with during drug treatment. The rhinovirus capsid is one of the best characterized antiviral targets in the viral lifecycle. A group of chemically diverse compounds, known as capsid binders, inhibit uncoating (and in some cases receptor attachment) of the virus through stabilization of the capsid. The capsid binders, including pleconaril, pirodavir and vapendavir, all interactwith a hydrophobic pocket underneath a canyon that surrounds the 5 fold axis in the capsid. In a large-scale, cell-based antiviral screening effort, we identified a benzonitrile analogue (LPCRW_0005, Chapter II) as a selective inhibitor of human rhinovirus 14 (HRV14) replication. Further characterisation of the compound confirmed that it acts as a capsid binder. However, the compound has a chemical structure that is markedly smaller than that of other known capsid binders. In Chapter II.annex, we explored the antiviral activity of a panel of novel pirodavir analogues with modifications of the central hydrocarbon chain. The activity of these analogues against six prototype strains of the enterovirus genus was evaluated. Additionally, we characterised the antiviral activity and mechanism of action of ca603, one of the most active congeners in the series.Due to the lack of proofreading of the viral polymerase, RNA-virus strains are not genetically uniform but consist of a quasispecies swarm. When drug pressure is applied tothis quasispecies, the drug-insensitive variants or the ones less susceptible to the drug will still be able to replicate. The rise of drug-resistant variants during therapy is a well-known problem during the treatment of chronic viral infections such as those caused by human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Also for acute viral infections such as those caused by influenza viruses, drug-resistance has been observed. During the clinical development of capsid binders, drug-resistant rhinovirus variants were rapidly detected. In vitro, resistance selection is however a useful tool to identify the target of the compound. In Chapter III, we describe two resistance selectionmethods used for both capsid binders and non-capsid binders. In the first method, the virus is passaged in suboptimal compound concentrations which are gradually increased between the passages. Rhinovirus variants resistant to capsid binders are relatively easy to obtain by this method.However, a higher number of passages is needed to obtain virus resistant to a non-capsid binder, such as a protease inhibitor. Therefore a second method based on clonal selection was used and this method has proven to be a fast way to determine the primary compound-resistant mutations in a rhinovirus, compared to serial passage.In a large-scale, cell-based antiviral screening effort, we identified R030987 as a broad-spectrum inhibitor of a representative panel of the two species HRV-A and HRV-B (Chapter IV). R030987 acted, like the capsid binders, at an early stage of the replication cycle. Surprisingly, R030987 did notstabilise the capsid and pleconaril-resistant HRV14 variants did not show cross resistance to R030987, suggesting a different mode of action. R030987-resistant HRV14 carried mutations in the inner capsid protein VP4, which is involved in both uncoating and assembly of the virus. Furtherexperiments are ongoing to understand the involvement of this small hydrophobic protein the mechanism of action of the rhinovirus inhibitor R0303987.Viral proteases are crucial in the replication of the virus and are therefore promising drug targets. In Chapter V, we discuss the antiviral activity of SG85, a peptidic α,β-unsaturated ethyl ester that was developed during a structure-based design of Michael acceptor inhibitors of the enterovirus 68 3C protease.Designed to be a broad-spectrum enterovirus inhibitor, SG85 inhibitsin vitro both rhinovirus A genotypes from the minor and major receptor group, and rhinovirus B genotypes.A low-level SG85-resistant HRV14 variant was obtained by clonal selection and genotyping of this variant revealed a double amino acid mutation in the 3C protease. One of these residues was also reported to be mutated in HRV14 resistant to rupintrivir, another 3C protease inhibitor. In a crystal structure of the enterovirus 68 3C protease complexed with SG85, the corresponding residue to the second mutated amino acid was observed to interact with the compound. The absence of a robust rhinovirus mouse model to evaluate antiviral agents led to the use of a surrogate virus, Coxsackie virus B4 (CVB4), ina pancreatitis mouse model. Since that SG85 is only active in a µMrange against this related enterovirus CVB4 compared to the activity against the rhinovirus strains (nM range),we therefore did not expected a potent effect in this infectious mouse model. Treatment with SG85 did not reduce viral replication in the treated mice, however a modest effect on the clinical course of the disease was observed.In conclusion, we describe four small molecule inhibitors of rhinovirus replication. With these antiviral molecules, different stages of the viral lifecycle were targeted. Capsid binders LPCRW_0005 and ca603 are early-stage inhibitors that stabilise the capsid and thereby prevent rhinovirus replication. R030987 was also identified as an early-stage inhibitor, however thecompound has a mechanism of action that differs from that of the capsidbinder pleconaril. The precise involvement of the inner capsid protein VP4 in the mechanism of action of R030987 has yet to be elucidated. However, seen the crucial role of VP4 during uncoating and RNA release, we identified an inhibitor class with a new and promising target in the replication cycle. Another well-known viral target is the 3C protease. The 3C protease inhibitors, such as the herein characterised SG85, inhibit the vital processing of the viral polyprotein. Rhinoviruses do not only cause mild and self-limiting upper respiratory tract infections, but are also responsible for a great percentage of virus-induced asthma and COPD exacerbations. To lower the burden of these respiratory infections, the development of small molecules as described in this thesis is urgently needed.nrpages: 141status: publishe