Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex.
BACKGROUND
The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.


RESULTS
We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD50/IC50) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication.


CONCLUSIONS
Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures.
Biglione
Sebastian
S
Interdisciplinary Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA, USA. biglione@cbrinstitute.org
Byers
Sarah A
SA
Price
Jason P
JP
Nguyen
Van Trung
VT
Bensaude
Olivier
O
Price
David H
DH
Maury
Wendy
W
D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D010880:Piperidines / Q000494:pharmacology*; D042925:Positive Transcriptional Elongation Factor B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014779:Virus Replication
D004791:Enzyme Inhibitors; D005419:Flavonoids; D010880:Piperidines; D011687:Purines; D014764:Viral Proteins; D000077546:Roscovitine; C077990:alvocidib; D004004:Dichlororibofuranosylbenzimidazole; D042925:Positive Transcriptional Elongation Factor B; D042863:Cyclin-Dependent Kinase 9