Flaviviruses, particularly dengue virus are a major public health problem in tropical and subtropical areas of the world with up to 100 million people infected annually. Infection with dengue can result in acute and potentially fatal haemorrhagic fever with the incidence of these diseases having increased over the last two decades. Currently, there are no therapeutic agents available for treating flavivirus infections and the identification of our lead compounds is a first step toward their development. This thesis focuses on the dengue virus fusion mechanism as a target for drug development.
Novel lead compounds were identified using virtual screening against the dengue virus E protein and rational design of E and prM derived peptide fusion inhibitors. To gain insight into the molecular mechanism of viral membrane fusion and to identify new avenues for antiviral design a detailed analysis was performed of the sequences and structures of viral fusion proteins from different families.
Small molecules were successfully identified and tested which inhibit the dengue virus fusion mechanism, a process essential for viral infection of cells. The compounds were identified by in silico docking of a compound library against the dengue virus envelope (E) protein and are assumed to inhibit the transition of the E protein from its metastable pre-fusion form to its stable post-fusion form. We tested the compounds in viral proliferation (plaque) and cell cytotoxicity (MTT) assays and identified several compounds with an IC50 of 1-50 μM and no mammalian cell cytotoxicity. These lead compounds are now undergoing further studies for the development of a dengue virus fusion inhibitor. Furthermore, these lead compounds are active against the flavivirus family members Kunjun virus and Yellow fever virus. The high conservation of E protein structural elements amongst the flaviviruses suggests that these compounds may also serve as leads for the development of fusion inhibitors against other members of the flavivirus family, for example West Nile virus and Japanese encephalitis virus.
Peptide dengue virus fusion inhibitors were rationally designed based on previous experimental data and on the analysis of the interactions of the prM and E protein in immature dengue virus particles. The peptides were expressed and purified, their cytotoxicity tested in MTT assays and their impact on dengue virus proliferation tested using plaque assays. One peptide fusion inhibitor was identified with an IC50 of about 100 μM.
The analysis of sequence and structural features of the influenza virus HA and dengue virus E proteins from multiple viral strains suggests that low pH-mediated viral fusion is initiated by histidine protonation. Highly conserved histidine residues are buried in conserved pockets in the pre-fusion forms of the dengue virus E and influenza HA protein. In the post-fusion forms, the presumably protonated histidines form ionic interactions with new residue partners. This suggests that protonation of the histidines in the low pH environment of the endosome provides the free energy to release the histidines from their binding pocket and drives the structural changes in the fusion protein necessary for membrane fusion. This model is supported by preliminary experimental data and molecular dynamics studies simulating the low pH conditions. A clearer understanding of the detailed molecular events driving viral fusion provides valuable targets for the rational design of small molecule inhibitors and provides directions for the construction of attenuated infectious clone mutants that could serve as future vaccine candidates.