The Anti-inflammatory Drug Leflunomide Inhibits NS2B3 Cluster Formation During Dengue Viral Infection as Revealed by Single Molecule Imaging

A prerequisite for Dengue viral infection is the clustering of NS2B3 viral protein in the infected cell. This calls for drugs capable of reversing the biological processes leading to the declustering of NS2B3 viral complex. In this work, we report a new drug (leflunomide) that shows reversal of NS2B3 clustering, post 24 hours of cell transfection with a recombinant probe (Dendra2-NS2B3) containing the viral complex of interest (NS2B3). To study, we constructed a photoactivable recombinant plasmid for visualizing the activity of the target protein-of-interest (Dendra2-NS2B3). This enabled a better understanding of the underlying biological processes involved in Dengue and the role of NS2B3. The study was performed in a cellular system by transfecting the cell (NIH3T3 -mouse fibroblast cell line), followed by drug treatment studies. A range of physiologically relevant concentrations (250 nM − 10 μM ) of the FDA-approved drug (leflunomide) was used. The single molecule super-resolution microscopy ( scanSM LM ) study showed declustering of NS2B3 clusters for concentrations > 250 nM and near complete disappearance of clusters at concentrations > 5 μM . Moreover, the associated critical biophysical parameters suggest a substantial decrease in clustered molecules (from 53.2 ± 1.77% for control to 14.89 ± 4.80% at 250 nM , and further reduction to 10.55 ± 2.91% at 500 nM ). Moreover, the number of clusters reduced from 46 ± 15 to 13 ± 4, and the number of molecules per cluster decreased from 133 ± 29 to 62 ± 3, with a depletion in large clusters (from 24 to 12). The parameters collectively indicate the clustering nature of NS2B3 viral protein during the infection process at a cellular level and the effect of leflunomide in declustering. The results supported by statistical analysis suggest strong declustering promoted by leflunomide, which holds the promise to contain/treat dengue viral infection. Statement of Significance The fact that there is no approved antiviral approach for Dengue makes it life-threatening and calls for ways to tackle viral infection. Hence, understanding Dengue biology at a single molecule level plays a vital role. In the present super-resolution study, we noted the formation of key viral protein (NS2B3) clusters post 24 hours of transfection in a cellular system. We identified a repurposed FDA-approved drug (Leflunomide) that inhibits the clustering process and promotes declustering at higher drug concentrations. This may become the basis of future studies, which may have therapeutic potential against Dengue.