Dengue is an emerging mosquito-borne viral infection with an estimated 2.5 billion people being at risk. The virus is found in tropical and subtropical areas around the globe and is transmitted by the main vector Aedes aegypti. According to WHO there are an estimated 50-100 million infections every year worldwide with an estimated 500,000 cases being hospitalized annually. Currently there is no treatment available, thus there is an urgent need for discovering new drugs.
The dengue NS3 serine protease is a promising target for new drugs since it is involved in viral polyprotein processing together with NS2B and thus important for viral replication. Crystal structures of NS2B-NS3pro bound to a peptide inhibitor recently revealed a pocket located at the opposite side of the protein from the active site. Residues from both NS2B and NS3 are lining the pocket, which is larger than the active site. Conservation in West Nile virus structures suggests functional importance. Based on these findings this study aims to characterize the NS2B-NS3 protease with its large allosteric pocket in more depth. Mutagenesis studies of different residues lining the pocket should help to understand the functional role of the pocket as a whole, as well as the impact on function for single residues in viral replication. The findings could further be used in drug development to specifically target residues that are crucial for viral replication.
Mutagenesis studies of selected residues to alanine resulted in impaired or abolished protease activity for most of the mutants. The five mutants V078, W089, T118, G124 and N152 were completely inactive. Mutants M084 and I165 were barely active compared to WT. Only mutant Q167 showed slightly higher activity than WT. Furthermore protease activity could be restored for two selected mutants in additional conservative mutagenesis studies. The hydroxyl group in the threonine of position 118 seems to be the main factor affecting protease activity since introduction of a serine lead to restorage of activity by 60%. For mutant M084 the introduction of a phenylalanine restored activity in a similar range than mutant T118, suggesting that hydrophobicity to be a main factor influencing activity. In general in vitro studies on viral replication were able to confirm results obtained from protease activity assays. In particular, protease activity could surprisingly not be restored for mutants N152D and I165L, even though the introduced amino acids differ only slightly from the WT residue. This suggests that those two residues are especially important for protease function.
Overall, the results obtained from this study helped to identify residues within the allosteric pocket that are crucial for protease activity and viral replication. The pocket is therefore an attractive target and could potentially be targeted for the design of antiviral compounds.
|School:||National University of Singapore (Singapore)|
|Department:||Infectious Diseases, Vaccinology And Drug Discovery|
|School Location:||Republic of Singapore|
|Source:||DAI-B 77/06(E), Dissertation Abstracts International|
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