Suramin inhibits helicase activity of NS3 protein of dengue virus in a fluorescence-based high throughput assay format

• An improved assay is developed to monitor helicase activity of dengue virus NS3.
• This assay is adapted to a high throughput format to screen for potential inhibitors.
• This assay is validated using a chemical library consisting of 1600 compounds.
• From the validation screen, suramin is identified as a potent inhibitor.
• Suramin inhibits helicase activity of NS3 by a non-competitive mode of inhibition.

Dengue fever is a major health concern worldwide. The virus encoded non-structural protein 3 (NS3) is a multifunctional protein endowed with protease, helicase, nucleoside triphosphatase (NTPase) and RNA 5′ triphosphatase (RTPase) activities. Helicase activity of NS3 catalyzes the unwinding of double stranded polynucleotides by utilizing the energy released from ATP hydrolysis. As this activity is essential for replication, NS3 helicase represents an attractive drug target for developing a dengue antiviral drug.Here, we report fluorescence based molecular beacon helicase assay using a duplex RNA substrate that contains a fluorophore on the 5′ end and a quencher on the 3′ end of one of the strands. The assay was optimized with respect to several parameters and adapted to 384-well high-throughput screening format, with an average Z′ factor of 0.65. Assay validation with a small diverse set library of 1600 compounds identified, suramin as a significant inhibitor of the helicase activity of NS3. Helicase activity deficient NS3 K199A was used in a counter-screen to identify compounds interfering with the assay. Suramin inhibited DENV (dengue virus) NS3 helicase activity with a Ki of 0.75 ± 0.03 μM as a non-competitive inhibitor. The molecular beacon helicase assay together with the counter screen and suramin as a tool compound can be used to identify novel inhibitors of DENV helicase.