Antiviral activities of 15 dengue NS2B-NS3 protease inhibitors using a human cell-based viral quantification assay

• There are an estimated 50–100 million dengue infections annually worldwide.
• The viral NS2B-NS3 protease is deemed a plausible drug target.
• 15 inhibitors published between 2003 and 2013 were screened using our viral titer assay.
• The most potent compound, an anthraquinone, reduced viral titer 10-fold at 1 μM.
• This could serve as a plausible scaffold for further medicinal chemistry efforts.

The dengue virus is a mosquito-borne pathogen responsible for an estimated 50–100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor’s ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 μM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 μM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay.