Multivalent inhibitors to block the Neuraminidase of Influenza A Virus

Abstract

The work described in this thesis focuses on the development of inhibitors for the Influenza A virus (IAV) by developing multivalent carbohydrate-based ligands (Figure 1). The neuraminidase enzyme (NA) plays an important role in the virus life cycle and the spread of the virus. This makes blocking NA a very promising direction for inhibition of the flu. We designed new compounds that rely on multivalency. By linking our designed monovalent units to di- and tetravalent scaffolds, inhibitors were obtained with a potent inhibitory effect in several assays. Chapter 2, efforts to obtain a suitable ligand to be used in the synthesis of multivalent NA inhibitors are described, based on the goals mentioned above. We modified the oseltamivir carboxylate (OC) mimic 3-pentyl analogue, by extending one branch of the pentyl part. This way the molecule could be conjugated to a spacer in order to explore multivalency effects. In addition to the difficulties associated with the glycosylation step, the preparation of the target ligand involved a number of other synthetic challenges.Chapter 3 benefited from previous work in our group where a new rigid spacer was designed and synthesized. The rigid spacer was designed by replacing two carbohydrate groups with two phenyl groups. However, the introduction of the phenyl groups posed a solubility problem. To solve this problem, we screened building blocks for a better solubility.Chapter 4, we show the successful synthesis and characterization of dimer with 5 different lengths and rigid spacers. We attempted to synthesize the tetramers, which were designed to bind to the four binding sites of the tetrameric NA. Unfortunately, none of the tetramers could be dissolved in normal solvents (organic & water). We therefore focused on divalent ligands. Their syntheses were possible by a combination of chemical scaffold synthesis, glycosylation and CuAAC ‘click’ conjugation. Besides the development of optimal molecular designs to inhibit NA, effective methods to evaluate the inhibition activities are also crucial for the research. Here in Chapter 5, we focused on the evaluation of the mono- and divalent inhibitors that have been described in chapters 2 and 4. Two kinds of the Influenza virus proteins were used: Spain N9 and Wisconsin N1 which derived from the NA gene of H7N9 and H1N1, respectively. The compounds were first tested for their ability to inhibit NA activity using recombinant soluble tetrameric N1 and N9 proteins and the MUNANA substrate. Next, we analyzed the inhibitory activity of the different compounds on virus particles rather than recombinant NA proteins as the source of NA activity. A 4-day CPE assay was set up to assess the ability of the compounds to inhibit virus infection. MDCK cells in a 96- well format was infected with H1N1 virus in the presence of a dilution range of the different compounds, and the lowest concentration of the compounds that could prevent cytopathogenic effects and killing of the cells resulting from virus replication was determined. In this process we also attempted to analyze whether these compounds showed additional HA inhibition using a BLI assay.