Fig. 4

AlphaScreen screening strategy (a, b). Normalized percentage inhibition (PIN) of duplicates of each of the 409 primary hits in (a) a confirmatory repeat of the IN–TRN-SR2 AlphaScreen or (b) the GST-His₆ false positive counterscreen. In both cases, the linear regression fit to the data is shown: slopes are not significantly different from 1 and R² values are 0.9675 and 0.9640 for (a) and (b), respectively. c Average percentage inhibition in the GST-His₆ counterscreen plotted against that observed in the IN–TRN-SR2 assay. Hits of interest show > 50% inhibition of the IN–TRN-SR2 interaction and are devoid of quenching at the active concentration (< 20% inhibition of the GST-His₆ signal or 50% stronger inhibition of the IN–TRN-SR2 interaction). Primary hits occupying the different areas of the result space are plotted as dark grey triangles (false positives), light grey squares (quenchers) and black circles (hits of interest). d Titration results and dose–response curve fit obtained for compound MVG059. Duplicates are plotted. e Histogram of the obtained IC₅₀ values for all 98 hits sorted into 5 µM bins. f Hit compound specificity. IC₅₀ values obtained against the IN–TRN-SR2 and LEDGF/p75–JPO2 interactions were plotted against one another. Compounds having an IC₅₀ < 20 µM against the IN–TRN-SR2 interaction and being tenfold less active or having an IC₅₀ > 60 µM against LEDGF/p75–JPO2 are preferred (black circles). Hits that were only between 10 and 3 times more active in the IN–TRN-SR2 assay are less promising (light grey squares). Those that were less potent (IC₅₀ ≥ 20 µM) but did not inhibit the LEDGF/p75–JPO2 interaction (IC₅₀ > 60 µM) were looked at case-by-case. Compounds falling outside of these regions were considered non-specific and not investigated further (dark grey triangles)