Effect of different NNRTIs on intracellular Gag processing In order to characterize NNRTI induced PR activation, conditions were optimized for detection of increased, rather than decreased Gag processing. Assuming that the degree of stimulation of Gag Pol dimer formation is inversely correlated with the intracellular concentration of Gag Pol, b Gal activity and Gag processing of cells Enzalutamide clinical trial were measured in cells expressing different amounts of HIV derived proteins in the presence or absence of 5 uM EFV as a prototype NNRTI. No effect of EFV was seen at high Gag and Gag Pol concentrations, whereas transfection of lower amounts of pCHIV. MAa resulted Inhibitors,Modulators,Libraries in detectable Inhibitors,Modulators,Libraries increase of b Gal activity in lysates of EFV treated cells.
Under optimized conditions enhancement of intracellu lar Gag processing and a significant increase in b Gal activity were induced by the addition of 5 uM EFV. Cells transfected with a pCHIV. MAa variant in which PR was inactivated due Inhibitors,Modulators,Libraries to a D25A mutation in the PR active site displayed no increase in Gag processing or b Gal activity when grown in the presence of 5 uM EFV. As a control mimicking enhanced PR activity we used an HIV 1 derivative expressing Inhibitors,Modulators,Libraries an artificially linked PR dimer. Duplicating the PR monomer coding region in the proviral context and connecting the two PR mono mers by a flexible 8 amino acid linker leads to premature activation of HIV PR resulting in greatly enhanced intra cellular Gag processing and prevention of virus forma tion.
Low PI doses, which interfere with infectivity of wild Inhibitors,Modulators,Libraries type HIV, partially rescue HIV replication by restoring an appropriate level of Gag processing, while high concentrations of PI completely block the activity of the artificially activated PR and lead to the production of non infectious virus. Transfection of a construct encoding the 2PR coding sequence in the context of pCHIV. MAa led to nearly complete intracellular Gag processing, while very low levels of CA were released into the supernatant. No effect of EFV on b Gal activity was observed in this case, presumably because Gag and Gag Pol were already completely processed in the absence of EFV. Taken together, these results indicate that the EFV mediated increase in b Gal activity was PR dependent. In order to identify the most potent available compound we next employed the established assay for a detailed com parison of a series of NNRTIs.
We included NNRTIs pre viously compared qualitatively with respect to activation of Gag processing, namely EFV, ETV, NVP and TMC 120, as well as second generation kinase inhibitor Pacritinib NNRTIs not cur rently in clinical use IDX 12899, GW 678248 VRX 480773 and UK 453061. 293T cells co transfected with pCHIV. MAa and pCMVwere grown in the presence of the respective NNRTI at concen trations ranging from 0. 03 to 10 uM. At 44 h post trans fection, cell lysates were analyzed for b Gal activity.