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. Author manuscript; available in PMC: 2013 Jun 14.
Published in final edited form as: J Med Chem. 2012 May 24;55(11):4968–4977. doi: 10.1021/jm201442t

Figure 2. Overview of the strategy for identifying compounds that disrupt HIV-1 nucleocapsid/RNA interaction.

Figure 2

(A) Outline of the experimental procedure. (B) Enumeration of Hits during library screening. Utilizing a fluorescence polarization assay to detect small molecule displacement of 3'-6-carboxyfluorescein-labeled SL-2 DNA (FSL-2) from binding to the nucleocapsid (NC’) protein, 14,400 compounds were screened and 101 compounds (Hits) were identified as displacing FSL-2. The 101 Hits were retested for NC’-FSL-2 displacement and 65 did not show activity or demonstrated < 60 % FSL-2 displacement activity and were eliminated from further study. Of the remaining 36 compounds (Selected Hits), 18 failed to show a significant shift in thermal stability when the compound-Gag complexes were evaluated by differential scanning fluorometry assay, 8 displayed promiscuous β-galactosidase inhibition, and 5 were excluded due to low affinity binding to NC’. Of the 5 remaining Selected Hits, 3 Hits showed cellular toxicity at higher compound concentrations, whereas, 2 compounds demonstrated both low cellular toxicity and inhibition of HIV-1 replication in CD4+ T cells.