Recombinant NS3 Seine Protease From Dengue Virus 2 As A Screen For Small Molecules
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Date
2011-08
Authors
Ab Aziz, Nurohaida
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Dengue infection is re-emerging as a major global disease and is classified as
a Category A priority pathogen. Dengue viruses are estimated to infect 50-100
million people annually and are considered to cause one of the most important
arthropod-borne viral diseases in terms of human morbidity and mortality. Virus
transmission occurs through the bite of the Aedes aegypti mosquito and half the
world’s population is at risk for infection. There is presently no approved vaccine or
antiviral drug that is effective against dengue viruses. The focus of this thesis is to
combine the power of high performance computing with wet lab experiments for the
recombinant NS3 serine protease from dengue virus type 2 as a screen for antiviral
small molecules that can be used either to prevent or treat dengue virus infections.
In collaboration with Dr. Irene Newhouse, Advance Studies for Genomics,
Proteomics and Bioinformatics (ASGPB), University of Hawaii, molecular
modelling and in silico screening of small molecule compound libraries from the
National Cancer Institute (NCI) and ZINC databases that dock into the DENV2
NS2B-NS3 protease binding site was carried out. From the pool of best-fit candidates
(53 potential small molecule inhibitors), the 4 high-scoring water-soluble,
commercially available compounds were selected for in vitro assessment.
The NS2B-NS3 serine protease gene from dengue virus serotype 2 was
cloned and expressed in E. coli as a recombinant hexahistidine tagged protein. The
NS2B-NS3 protease was purified using affinity and gel filtration chromatography. In
vitro assay revealed protease activity toward a fluorogenic peptide substrate
containing two basic amino acid residues. All 4 high-scoring water-soluble
compounds were tested and exhibited in vitro inhibition activity on the recombinant
NS2B-NS3 serine protease. Compound 4 was found to be most active inhibitor with
64% inhibition at 100 M concentration.
Description
Keywords
Recombinant NS3 seine protease , from dengue virus 2