Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibit or in clinical trials as a cancer therapy that has been recently shown to b lock human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cd k inhibitor that is not competitive with ATP. We examined the ability of fl avopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA p olymerase II and the large subunit of the 5, 6-dichloro-1-beta -D-ribofuran osyl-benzimidazole (DRB) sensitivity-inducing factor and found that the IC5 0 determined was directly related to the concentration of the enzyme. We co ncluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry ev en at concentrations of enzyme in the low nanomolar range. These results in dicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb ass ay and demonstrated that the drug remains bound for minutes even in the pre sence of high salt. Flavopiridol remained bound in the presence of a 1000-f old excess of the commonly used inhibitor DRB, suggesting that the immobili zed P-TEFb could be used in a simple screening assay that would allow the d iscovery or characterization of compounds with binding properties similar t o flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded th at P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.