Quantification of live cells by microscopic evaluation of trypan

Quantification of live cells by microscopic evaluation of trypan blue stained samples revealed a significant decrease in live cell numbers for the HIV infected MT4 LTR EGFP IIIB cells, whereas the number of uninfected www.selleckchem.com/products/Imatinib(STI571).html control cells remained constant. In order to test whether the observed cyto toxic effect on virus producing cells was due to enhanced HIV PR activity we added 200 nM of the PI darunavir to infected and uninfected cells in the presence and absence of VRX 480773. DRV treatment impaired Gag processing and comple tely reversed the cytotoxic effect of VRX 480773 in MT4 LTR EGFP IIIB cells, supporting the interpretation that the observed NNRTI induced cell killing was mediated by HIV PR.

By quantification of intracellular GFP fluorescence of drug treated MT4 CMV EGFP and MT4 TR EGFP IIIB cells, respectively, we compared the relative Inhibitors,Modulators,Libraries effect of dif ferent NNRTIs on viability of infected versus uninfected cells. Differential effects, corre lating with the biochemical data obtained on 293T cells, were revealed. The most potent compounds, IDX 12899, GW 678248 and VRX 480773, displayed CC50 values in the submicromolar range on MT4 LTR EGFP IIIB cells. Cytotoxicity on uninfected MT4 CMV EGFP control cells was undetectable for IDX 12899 and GW 678248 in the tested range. VRX 480773, displayed detectable unspecific toxicity, albeit with a 10 fold higher CC50 than on virus producing cells. EFV was less cytotoxic on the infected cells, but this effect was again specific as indicated by the observation that MT4 CMV EGFP cells were not affected.

The remaining compounds showed no specific effect in the tested concentration range TMC 120 displayed toxicity on the virus produ cing cells, but also showed comparable toxicity on unin fected control cells, while the remaining compounds had no detectable effect on total EGFP expression on either cell line. Inhibitors,Modulators,Libraries In all cases the specific NNRTI induced cyto toxicity on virus producing cells was completely reverted by addition of DRV. These results support the hypothesis Inhibitors,Modulators,Libraries that NNRTIs can exert a dose dependent, inhibitor specific activation of intracellular HIV PR by stabilizing Gag Pol dimers. In order to obtain further evidence for this model, we ana lyzed the effect of the various NNRTIs on RT dimeriza tion in a mammalian two hybrid system.

We found that, while lower absolute concentrations were required in this context, the relative effects of the various Inhibitors,Modulators,Libraries com pounds on RT dimer formation paralleled their Inhibitors,Modulators,Libraries effects on intracellular Gag processing IDX 12899, GW 678248 and VRX 480773 promoted RT dimerization in the low nM range, whereas a fivefold higher concentration was required for EFV, and EC50 values for the remaining compounds were higher than 100 nM. thereby This correlation lends further support to the proposed mechanism of action.

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