Development And Application Of Dengue Virus Ns2b/Ns3 Protease Inhibition Assay Using Alphascreen® Technology
Loading...
Date
2015-11
Authors
Abduraman, Muhammad Asyraf
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Dengue is an infectious disease caused by the dengue virus and is a global health problem. NS2B/NS3 protease is a non-structural protein which plays a pivotal role in viral replication and maturation of the dengue virus. This complex is a primary target for the development of anti-dengue drugs. In this study, the main objectives were to develop a specific NS2B/NS3 protease inhibition assay using AlphaScreen® technology and subsequently to screen a group of synthesized compounds which were potentially active as determined using in silico studies. Molecular interactions of the potentially active compound within the active site of the NS2B/NS3 protease were also further determined. The development of specific NS2B/NS3 protease inhibition assay involved utilizing a recombinant NS2B/NS3 protease and NS3/NS4A peptide substrate with proprietary StrepTactin® donor beads and nickel chelate acceptor beads. Briefly, a cross-titration experiment was carried out and a typical bell-shaped curve revealed the optimum concentrations of NS2B/NS3 protease and the peptide substrate at 100 nM and 300 nM, respectively. The assay system was subsequently optimized in a 384-well plate format and the optimal assay solution consisted of 10 mM HEPES, 20 mM NaCl, 0.20 % (v/v) BSA, and at pH 9.0. To validate the assay and NS2B/NS3 protease inhibition activities, aprotinin, panduratin A and 15 synthesized compounds were screened using this optimized AlphaScreen® assay. Aprotinin, a known serine protease inhibitor and panduratin A, a natural compound which exhibited inhibitory activity against NS2B/NS3 protease were used as positive controls. Aprotinin was found to actively inhibit NS2B/NS3 protease with 74.00 % of inhibition at the maximum concentration of 5 μM. Meanwhile, panduratin A showed 53.40 % of inhibition at the maximum concentration of 100 μM. Of all 15 compounds, none exhibited good inhibitory activity against NS2B/NS3 protease, except for MH005 which weakly inhibit NS2B/NS3 protease with 50.20 % of inhibition at 1 mM. The average Z’ factor for this assay was 0.5 and the overall signal to background (S/B) ratio of the maximum signal was 196:1. As for the coefficient of variation (CV), the mean values for the maximum signal groups on the day 1, 2, 3 were 3.00 %, 1.90 % and 2.10 %, respectively. The mean values for the minimum signal groups on the same day were 4.20 %, 5.10 % and 7.20 %, respectively, which indicated the reliability of this AlphaScreen® assay for high-throughput screening purpose. A detailed in silico studies of panduratin A and MH005 had identified both hydrogen bonds and hydrophobic interactions with residues reported to be essential for the binding and inhibition of the NS2B/NS3 protease. Further modifications of the compound structure based on MH005 may yield a stronger inhibitor of the NS2B/NS3 protease. As a conclusion, a new high-throughput screening assay for dengue NS2B/NS3 protease inhibition based on AlphaScreen® technology has been successfully developed and utilized in screening of compounds. A possible lead but with weak NS2B/NS3 protease inhibition activity was also identified in this study.
Description
Keywords
Dengue