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; this indicates that P-TEFb could be a potential target for developing anti-HIV-1 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 the 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, and tested their 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.

Figure 1

Structures of CDK inhibitors DRB 1, flavopiridol 2, and seliciclib (CYC202) 3.

Figure 2

Structures of flavopiridol mimics: olefin analogue 4, thio- and oxaflavopiridol 5 and 6, 2-benzylidene-benzofuranone 7, and P276-00 8 (exact structure not disclosed).

Figure 3

Flavopiridol analogue 12d specifically and potently inhibits P-TEFb-regulated genes. P-TEFb activity was examined by using qPCR to measure relative levels of c-Fos, Hsp70, and Mcl-1 mRNA, whereas CDK2 activity was assessed by measuring relative levels of Cdc2 and cyclin A mRNA. HeLa cells were treated overnight with 10 nM flavopiridol, 12d, and 12i, or transfected with CDK9 or CDK2 siRNAs. RNAs were harvested and reverse-transcribed, and c-Fos, Hsp70, Mcl-1, Cdc2, and cyclin A transcripts were measured by qPCR; expression levels are calculated by normalizing to GAPDH. A. CDK9 siRNA specifically inhibits c-fos, hsp70, and mcl-1 genes with no effect on expression of CDK2, Cdc2 and cyclin A. B. Flavopiridol and analogue 12d (10 nM) specifically inhibit the P-TEFb-regulated genes c-fos, hsp70, and mcl-1, but have no effect on Cdc2 and cyclin A expression. Compound 12i (10 nM) does not inhibit CDK9 or CDK2 in vivo. C. Flavopiridol at high concentration (200 nM) significantly reduces expression of Cdc2 and cyclin A, but analogues 12d and 12i have no effect.

Scheme 1

Synthesis of chiral flavopiridol C-ring analogues from chiral acetophenone 9. (a) NaH, DMF, RCO₂Me, 0 °C to RT, overnight; (b) dry HCl (gas), CHCl₃, RT, 1 h, 60–90%; (c) BBr₃, 1,2-dichloroethane, 90 °C, 14 h; (d) 2N HCl, MeOH, 60–70%.

Scheme 2

Synthesis of flavopiridol D-ring olefin analogues from olefin acetophenone 13. (a) NaH, DMF, RCO₂Me, 0 °C to RT, overnight; (b) dry HCl (gas), CHCl₃, RT, 1 h, 60–90%; (c) Pyridine-HCl, quinoline, 180 °C, 2 h; (d) 2N HCl, MeOH, 60–70%.