Analysis by EMSA of the ELs interference on the Tat-TAR complex

ELs were tested as inhibitors of the Tat-TAR complex formation. ELs analysis was not possible to be performed by FQA due to a quenching effect observing after the addition of ELs to fluoresceinated Tat. The analysis of the ELs interference on the Tat-TAR complex was therefore performed by EMSA.

The Electrophoresis Mobility Shift Assay (EMSA) allows the evaluation of the in vitro activity of compounds to interfere with the Tat-TAR complex formation. This assay is based on the different electrophoretic mobility of the free and complexed TAR, and in particular an experimental protocol that uses the staining with the fluorescent molecule SybrGreen II was performed and validated in our previous research [50].

With this method the band of the Tat-TAR complex is not detectable, because, when TAR is bound to Tat, the dye cannot intercalate RNA; however, the complexation reaction can be monitored observing the disappearance of the band corresponding to free TAR. The addition of increasing concentrations of compound determines, if the compound impairs the complex formation, the re-appearance of the free TAR band.

Tat 10aa was incubated with increasing concentrations of each compound at room temperature for 15 minutes, then folded TAR was added for other 15’ minutes. The gel shifts obtained are reported below (Figure 1.45, 1.46, 1.47, 1.48, 1.49 and 1.50).

Identification of anti-HIV agents Results and Discussion

Figure 1.45 EMSA to evaluate the inhibition of the Tat-TAR complex formation by 1186 (on the left) and 1182 (on the right) compounds. Controls: 60ng TAR folded in TNMg 1X in the presence and in the absence of

Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Figure 1.46 EMSA to evaluate the inhibition of the Tat-TAR complex formation by 1186 (on the left) and 1185 (on the right) compounds. Controls: 60ng TAR folded in TNMg 1X in the presence and in the absence of

Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Figure 1.47 EMSA to evaluate the inhibition of the Tat-TAR complex formation by UA (on the left) and UB (on the right) compounds. Controls: 60ng TAR folded in TNMg 1X in the presence and in the absence of

Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Identification of anti-HIV agents Results and Discussion

Figure 1.48 EMSA to evaluate the inhibition of the Tat-TAR complex formation by GA. Controls: 60ng

TAR folded in TNMg 1X in the presence and in the absence of Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Figure 1.49 EMSA to evaluate the inhibition of the Tat-TAR complex formation by 1184 (on the left) and 1181 (on the right) compounds. Controls: 60ng TAR folded in TNMg 1X in the presence and in the absence of

Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Figure 1.50 EMSA to evaluate the inhibition of the Tat-TAR complex formation by EA (on the left) and 1187 (on the right) compounds. Controls: 60ng TAR folded in TNMg 1X in the presence and in the absence of

Tat 10aa (10µM). the Tat-TAR complex formation was analyzed in the presence of increasing concentrations (1, 5, 10, 50, 100 µM) of compound. Electrophoresis on a 10% polyacrylamide gel in TBE 1X. After electrophoresis nucleic acids on the gel were stained with SybrGreenII; nucleic acids fluorescence in the gel were detected on a Geliance 600 Imaging System (PerkinElmer).

Identification of anti-HIV agents Results and Discussion

In all the experiments, the Tat-TAR complex formation is verified: in the presence of Tat, the free TAR band disappears and is replaced by a weakly detectable smeared band.

1182 compound (Figure 1.45, on the right) and 1185 compound (Figure 1.46, on the right) are inactive for the inhibition of the Tat-TAR complex formation.

In Figure 1.47 are reported the results of UA (on the left) and of UB (on the right): they are inactive.

GA (Figure 1.48) and EA (Figure 1.50, on the left) do not improve the Tat-TAR complex formation.

In Figure 1.49 are reported the analysis of 1184 (on the left) and of 1181 (on the right) that result to have no activity as Tat-TAR complexation inhibitors.

In Figure 1.45 (on the left) and in Figure 1.46 (on the left) are reported the gel shifts in which 1186 compound was analyzed. The addition of 100 µM of 1186 compound determines the completely re-appearance of the free TAR band. In the presence of high concentration of 1186 the Tat-TAR complex does not occur. Only compound 1186 can act as Tat-TAR complex formation inhibitor.

Identification of anti-HIV agents Conclusions