Inhibits TEFb in vivo and CDK2. Although 12i compound inhibits P TEFb GSK-3 in vitro, it has no effect on P or TEFb function in vivo CDK2. The analog 12d, the selective inhibitor flavopiridol in vitro P TEFb is specifically inhibits P TEFb function in vivo and has no effect on CDK2 function. This selectivity T shows in vivo that the antiviral effect of 12d to the inhibition of P-TEFb can be attributed. Selective inhibition of the activity t of P-kinase TEFb flowering bridges HIV-1 Tat transactivation and viral replication, without the cellular Ren transcription and Lebensf Ability of the cells, providing a strong rationale for targeting P TEFb and other cellular re cofactors as a strategy for the development of a potential anti-HIV drugs.
We examined the activity T profile Similar structure with flavopiridol in vitro kinase cellular Ren antiviral and cytotoxicity Tsassays, and identified a number of analogues that potently and selectively inhibit the kinase activity of t of P TEFb in vitro with a high antiviral activity t in cell assays. However, our results show that the total P in vitro kinase inhibitors activity Th TEFb cellular flavopiridol analogues not directly related to her Ren antiviral Kr Correlated forces and culture in vitro kinase Cdk2/cyclin A or P-TEFb activity are not correlated with th hnlichen cytotoxicity th. The deschloroflavopiridol 12a, 12d fluorophenyl analog 2 and 5 methylisoxazole analog 12n all potent inhibitors of HIV virus replication in cell assays, with respective indices of the selectivity T 2, 3 and 14 times h Ago than that of flavopiridol.
The two analog fluorophenyl 12d is a selective inhibitor of P TEFb that flavopiridol in vitro are approximately 40-fold selectivity t in the direction P TEFb compared to other CDKs. We show that compound 12d selectively inhibits P TEFb function in vivo with no effect on the function of CDK2, w While at the same concentration inhibits flavopiridol and CDK2. This result shows that 12d is a selective inhibitor of P TEFb function in vivo, and its antiviral effect most likely due to inhibition of P TEFb. This study provides further evidence that P is TEFb a potential target for drug development in the fight against HIV 1 and that selective inhibitors 12d TEFb as P k Nnte serve as an anti-HIV drugs first The profile of the structure reported anything similar activity t of flavopiridol with the crystal structure recently reported P TEFb complexed with flavopiridol can rationally design selective inhibitors of P TEFb interest generated.
A recombinant baculovirus was prepared using BaculoGold DNA and plasmids pBAC HuCDK9 T1. Sf9 cells were infected with recombinant baculovirus, incubated for 3 days, and harvested by centrifugation. The cell pellets were resuspended in 6 ml of lysis buffer containing protease inhibitor cocktail insects and resuspended min on ice for 45 minutes. The cell lysates were centrifuged at 10,000 g × and the supernatant was removed. Pre-washed Ni NTA beads were added to the whichever type Ligands were added and incubated at 4 h with constant mixing for 3 After incubation, the beads centrifuged at 500 g and to an S Molecules × screening for small subsequent affinity Tschromatographie. Briefly, the beads were washed with 6 ml of 6X His washing buffer containing 50 mM imidazole. The P TEFb was eluted from the beads by