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Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2010 (English)In: Journal of Biomedical Science, ISSN 1021-7770, E-ISSN 1423-0127, Vol. 17, 68- p.Article in journal (Refereed) Published
Abstract [en]


 The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is not intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of disigning inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.


 Alanine substitutions were introduced by site directed mutagenesis at residues L115, D129, G133, T134, T150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters K-m, K-cat and K-cat/K-m were determined.


 Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activiey. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole.


 Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

Place, publisher, year, edition, pages
2010. Vol. 17, 68- p.
National Category
Pharmaceutical Sciences Medical and Health Sciences Biological Sciences
URN: urn:nbn:se:uu:diva-134695DOI: 10.1186/1423-0127-17-68ISI: 000282337300001OAI: oai:DiVA.org:uu-134695DiVA: diva2:373531
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2012-06-11Bibliographically approved
In thesis
1. Studies of Retroviral Reverse Transcriptase and Flaviviral Protease Enzymes as Antiviral Drug Targets: Applications in Antiviral Drug Discovery & Therapy
Open this publication in new window or tab >>Studies of Retroviral Reverse Transcriptase and Flaviviral Protease Enzymes as Antiviral Drug Targets: Applications in Antiviral Drug Discovery & Therapy
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Viruses are a major threat to humans due to their unique adaptability, evolvability and  capability to control their hosts as parasites and genetic elements. HIV/AIDS is the third largest cause of death by infectious diseases in the world, and drug resistance due to the viral mutations is still the leading cause of treatment failure. The flaviviruses, such as Dengue virus (DEN) and Japanese encephalitis virus (JEV), represent other major cause of morbidity and mortality, and the areas where these viruses are endemic are spreading rapidly. No curative therapy for any flavivirus could be made available as yet.

The first part of this thesis focuses on the HIV-1 drug resistance caused by mutations in a major HIV drug target, the HIV-1 reverse transcriptase (RT) as a response to the largest class of clinically used anti-retrovirals, the NRTIs. A robust proteochemometric model was created to analyse the complex mutation patterns in RT drug resistance. The model identified more than ten frequently-occurring mutations, each conferring at least two-fold decrease in susceptibility for one or several NRTIs. Using our prediction server (hivdrc.org), the model can be applied to propose optimum combination therapy for patients harbouring mutated HIV variants.

The second part of the thesis encompasses studies on a promising drug target, the NS2B(H)-NS3pro, in two flaviviruses, namely the dengue virus (DEN) and Japanese encephalitis virus (JEV). Functional determinants of DEN NS2B(H)-NS3pro were identified by site-directed mutagenesis. Further, peptide inhibitors were designed using proteochemometrics (PCM) and statistical molecular design (SMD), synthesized and assayed on DEN proteases, which resulted in some novel peptides with low micromolar or sub-micromolar inhibitor activity. The very poorly characterised JEV NS2B(H)-NS3pro  was cloned, purified and the kinetic parameters of this attractive drug target were determined for a series of model substrates and inhibitor. The results identified the role in target-ligand interaction of different residues on specific positions in the target (NS2B(H)-NS3pro) and ligands (substrates/inhibitors).

Overall, the findings in this thesis contribute to rational antiviral drug discovery and therapy.

Place, publisher, year, edition, pages
Acta Universitatis Upsaliensis: Uppsala universitet, 2012. 68 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 163
Virus, enzymes, HIV/AIDS, retroviral reverse transcriptase, flaviviral protease, NRTIs, proteochemometrics, drug resistance, DEN, JEV, NS2B(H)-NS3pro, antiviral, drug targets, drug discovery, drug therapy.
National Category
Pharmaceutical Sciences
Research subject
urn:nbn:se:uu:diva-173504 (URN)978-91-554-8388-3 (ISBN)
Public defence
2012-06-14, C4:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Available from: 2012-05-23 Created: 2012-04-25 Last updated: 2012-08-01Bibliographically approved

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