Verification of Tn5 Tnp inhibition using gel shift assays. In this example, an aromatic thiourea is shown to be a modest inhibitor of Tnp-DNA complex assembly with an IC₅₀ of 24 μM. These values are reported in Fig. 3. The gel shift illustrates compound inhibition of Tnp-DNA complexes in a typical assay and is representative of the data collected for the graph, which was used to calculate an IC₅₀ for the inhibitor. The gel shifts observed at low and high inhibitor concentrations are identical to the ones observed for the DMSO only and no-Tnp control reactions, respectively. The data in this graph were obtained from multiple experiments. Each data point represents the results from at least two, typically three, independent experiments. Error is represented as the standard deviation from the fit of the data to the equation used to calculate the IC₅₀.

A large coumarin dimer inhibits IN activity with an IC₅₀ of 15 μM. IC₅₀s were determined by measuring the degree of 3′ strand processing that occurs with increasing concentrations of inhibitor (0, 0.05, 0.5, 5, 25, 50, 75, 100, 150, 250, 375, or 500 μM) added to the IN reactions, as described in the Materials and Methods. The degree of 3′ strand processing is measured by the percentage of the signal obtained for the 3′ strand processing product relative to the total signal per lane. These values are reported in Fig. 5.

Inhibitory compounds block HIV-1 transduction in cells at a point in the viral life cycle consistent with inhibition of integration. The inhibitory compounds were simultaneously assayed for their effects on reverse transcription, transduction, gross cell viability, and expression of a stably integrated luciferase-encoding provirus. A quantitative PCR assay was used to monitor effects of the compounds on reverse transcription in acutely infected cells. The compounds were also measured in parallel for their effects on general cell viability and for their effects on expression of luciferase encoded from proviral DNA in chronically infected cells, as described in Materials and Methods. The results demonstrated that compounds 10, 10-B, and 10-F have no significant inhibitory effect on either reverse transcription (or any earlier step in the viral life cycle), cell viability, or luciferase expression. Reactions were performed in triplicate using 75 μM concentrations of each inhibitor.

Comparison of FP and gel shift synapsis assays. FP is a good measurement of the amount of synaptic complexes being formed. Increasing concentrations of unlabeled DNA (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 8, 10, 20, 50 μM) were used as competitive inhibitors for synaptic complex formation using 160 nM fluorescently labeled DNA and 800 nM Tnp. FP data (bottom) were compared to data obtained using gel shift assays (top). Bands labeled hetero DNA-Tnp represent two dsDNA molecules, one fluorescently labeled and one unlabeled. The lane labeled “no comp.” does not contain any unlabeled competitor DNA. IC₅₀ values were obtained from fitting these data to an exponential decay and determining the concentration of unlabeled DNA that reduced the amount of fluorescently labeled DNA by one half. IC₅₀ values for FP and gel shift assays are 2.6 ± 0.2 μM and 5.1 ± 0.4 μM, respectively, suggesting that FP is a good measurement of the degree of synaptic complex formation. These data were obtained from multiple experiments. Each data point represents the results from at least two, typically three, independent experiments. Error is represented as the standard deviation from the fit of the data to the equation used to calculate the IC₅₀. mP, millipolarization units.

Twenty compounds are identified that inhibit Tn5 Tnp-DNA assembly. Several substructures consisting of coumarin, benzoic acid, and cinnamoyl derivatives were identified within this group. The IC₅₀ values for these compounds range from 3.4 to 46 μM. IC₅₀ values were determined from gel shift assays, similar to those shown in Fig. 2. These data were obtained from multiple experiments. Each value represents the results from at least two, typically three, independent experiments. Error is represented as the standard deviation from the fit of the data to the equation used to calculate the IC₅₀.

Six compounds consisting of a biothionol and coumarin and cinnamoyl derivatives inhibit HIV-1 IN activity. The IC₅₀ values for these compounds range from 9 to 32 μM. These values are calculated from data measuring the percentage of the 3′ strand processing product formed, as described in the legend to Fig. 4. Assay products are observed using polyacrylamide gel electrophoresis, as described in Materials and Methods. Each value represents the results from at least two, typically three, independent experiments. Error is represented as the standard deviation from the fit of the data to the equation used to calculate the IC₅₀.