Date of Award

2010

Thesis Type

PhD

Document Type

Thesis

Divisions

Faculty of Science

Department

Department of Chemistry

Institution

Universiti Malaya

Abstract

Dengue is a serious infectious disease that is endemic in over 100 countries. There has been an estimate of 50 million infection per year globally, with more than 2.5 billion people are at risk for epidemic transmission. Two principal illnesses associated with dengue are Dengue Fever (DF) and Dengue Haemorrhagic Fever (DHF). To date, there is no licensed vaccine or therapeutic drug available for these illnesses, although there have been reports of some vaccine candidates in clinical trials. Treatments have only been supportive thus far. This thesis describes part of our ongoing effort to search for a lead therapeutic agent for DF/DHF. The early phase of this study involved the attempts to crystallize DEN-2 NS2BNS3 protease complex. Optimization of the protein expression and purification procedures was carried out to yield protein purity suitable for crystallization trials. Protein crystallization techniques involved the hanging drop and sitting drop methods. The crystallization attempts were unsuccessful mainly due to the dynamic nature of the dengue protease which underwent auto-cleavage upon folding into its active conformation and produced degenerative products. The crystallized protease of DEN-2, however, was published in 2006 by D’Arcy et al. Following the report of the crystallized protease, we performed validation of the in silico method that we established in predicting and building of the secondary structure profile of NS3 protease. The approach adopted was able to yield good prediction results and can be very useful, especially for cases where there are low homology sequence relationships between query and template proteins. Subsequent experiments involved the computational docking of six noncompetitive and one competitive inhibitor onto the DEN-2 protease complex. For the non-competitive inhibitors (ligands), blind docking was performed on the rigid ligand structures followed by flexible-ligand docking. Results obtained showed Lys 74 to be the most important residue in the binding site for interaction with the non-competitive inhibitors. Other residues were involved in the interactions with the inhibitors via Hbonds, van der Waals and hydrophobic interactions. Structure activity relationship study yielded the important features of the inhibitors that are responsible for activities to be the rigid structure of flavanone, the presence of the C5 hydroxyl and C7 methoxy groups on ring A, and the phenyl ring (B) in the molecules. Computational docking of the competitive inhibitor, 4-hydroxypanduratin A, onto the protease active site, was done followed by QM/MM study using the ONIOM2 method. Results obtained proposed that the binding of the inhibitor to the active site to be mediated by H-bonding to Ser135. Structural investigation of the protein-inhibitor complex illustrated that the inhibitor was able to block the S1 pocket of the binding site, thus inhibiting the recognition of the pocket for two basic amino acids at the P1 position of a substrate. This blockade seemed to disable the substrate from entering the S1 pocket, rendering the protease inactive. Information obtained from this study will be useful for the design of potential anti-dengue therapeutic agents.

Initial

khm

Additional Information

Thesis (PhD) - Faculty of Science, Universiti Malaya, 2010.

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