Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo *

By Sheng Chao, D. Price. Journal of Biological Chemistry, 2001

679 citations recorded by the source corpus.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block 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 Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC50determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate 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 assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.

Subfields: Molecular Biology, Oncology

View Paper: https://www.semanticscholar.org/paper/eb10044041aba531b9b5ede8d9453c1af50d2e8f

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Citations in the corpus

Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. (2010, methodology): “To assess the role of P-TEFb-dependent pause release in global transcriptional control, we repeated the ChIP-Seq experiment for total Pol II in mES cells treated with flavopiridol (FP), an inhibitor of Cdk9 kinase activity (Chao et al., 2000; Chao and Price, 2001).”
Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription (2013, methodology): “We initially tested two different elongation inhibitors: the cyclin-dependent kinase (cdk) inhibitor flavopiridol, which at low concentrations preferentially inhibits the Cdk9 activity of P-TEFb (Chao and Price, 2001); and IBET151, a selective inhibitor of BET (bromodomain and extra terminal domain)”
c-Myc regulates transcriptional pause release (2010, methodology): “To assess the role of P-TEFb-dependent pause release in global transcriptional control, we repeated the ChIP-Seq experiment for total Pol II in mES cells treated with ﬂavopiridol (FP), an inhibitor of Cdk9 kinase activity (Chao et al., 2000; Chao and Price, 2001).”
Defining mechanisms that regulate RNA polymerase II transcription in vivo (2009, neutral): “at most (if not all) genes; inhibition of P-TEFb dramatically decreases global transcriptio”
Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells (2007, methodology): “The specificity of H5 and 4H8 for Ser 2P and Ser 5P, respectively, was validated using flavopiridol, a potent inhibitor of CDK9, an enzyme that phosphorylates Ser 2 residues, but a weak inhibitor of CDK7, which targets Ser 5 (ref”