Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P‐TEFb) and Block HIV‐1 Replication

Abstract

The positive transcription elongation factor (P-TEFb) (CDK9/cyclin T1) regulates RNA Polymerase II dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat-transactivation and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription, indicating that P-TEFb could be a potential target for developing anti-HIV therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat-transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, cellular antiviral potency, and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics.

CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses

“…Flavopiridol inhibited the transcription elongation activity and suppressed replication of the influenza virus (46,47). The anti-influenza virus activity of FIT-039 was also examined.…”

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

“…Cell culture, drug treatments, and extract preparation HCT116 cells were grown as previously described ) to ∼70%-80% confluence and subjected to drug treatments with 3-MB-PP1, 2-FP-FP (Ali et al 2009), FP (Sigma), or DRB (EMD). Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS) plus 2 mM EDTA, 50 mM NaF, 1 mM DTT, 1 mM PMSF, and 4 µg/mL leupeptin.…”

“…To test a possible requirement for Cdk9 in Xrn2 phosphorylation in vivo, we first treated HCT116 cells with available Cdk9 inhibitors: flavopiridol (FP); 2-fluorophenyl-flavopiridol (2-FP-FP), an FP analog with increased selectivity for Cdk9; or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) (Marshall et al 1996;Chao and Price 2001;Ali et al 2009). In cells treated with 1 µM FP or 2-FP-FP or 50 µM DRB for 4 h prior to harvest, Xrn2-T439-P signals were diminished relative to DMSO-treated controls ( Fig.…”

Why minimal is not optimal: Driving the mammalian cell cycle—and drug discovery—with a physiologic CDK control network

“…Any selectivity that does occur is usually dictated by CDK-drug interactions outside the ATP-binding site, and computer modeling based on that principle has aided in the development of compounds that are more specific for a selected CDK. 45,46 In one such study, the more specific inhibitor was less cytotoxic than the less specific one on which it was based, even though the IC 50 for the primary target, Cdk9, was the same for both compounds in vitro. 45 Therefore, developing specific CDK inhibitors and identifying…”

“…45,46 In one such study, the more specific inhibitor was less cytotoxic than the less specific one on which it was based, even though the IC 50 for the primary target, Cdk9, was the same for both compounds in vitro. 45 Therefore, developing specific CDK inhibitors and identifying…”