Primary antiviral activity of ALLINIs was observed during virion maturation, where they inhibit IN-RNA interactions [14], we examined the ability of STP0404 to inhibit recombinant IN binding to synthetic TAR RNA using Alpha screen based assay [14]. Briefly, different concentrations of STP0404 were incubated with 100 nM His6 tagged IN in buffer containing 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1mg/mL BSA, 25 mM Tris, pH 7.4 at 4°C for 2 hrs. This mixture was then added to the nickel acceptor beads while biotinylated-TAR RNA was added to the streptavidin donor beads. After 2-hrs incubation at 4°C, the RNA mixture was added to the IN-drug mixture and the reading was taken after 1 hr incubation at 4°C by PerkinElmer Life Sciences Enspire multimode plate reader. The IC50 values were calculated by OriginLab software.

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