Anti-HIV drugs have primarily focused on inhibition of essential viral enzymes such as reverse transcriptase or viral protease. However, these drugs fail in long-term treatment of HIV-1 due to viral drug resistance. Alternative targets of attack of the HIV-1 would be targeting host cell transcription factors. Transcription of HIV-1 viral genes is uniquely dependent on both viral proteins and host cell factors. The hypothesis of this proposal is that iron chelators may inhibit HIV-1 replication by affecting host cell transcription factors. To address the hypothesis, the iron chelators, desferrioxamine, 2-hydroxy-1-naphthylaldehyde isonicotinoylhydrazone (311) and ICL670 will be utilized to chelate intracellular iron.
The first aim of the proposal will investigate the effect of the iron chelators on HIV-1 gene expression and replication in several cell lines, including HeLa, HeLa-MAGI, CEM T, 293T and HIV-1 infected lymphocytic cells. HIV-1 transcription and viral replication will be measured with the use of reporters or infection with pseudotyped HIV-1 viruses. The extent of the iron chelation will be evaluated by measuring labile Iron. Also, Lactate DeHydrogenase (LDH)-Cytotoxicity Assay will be utilized to test if chelators render any damage to the cells. The second part of the proposal will focus on understanding the mechanism for HIV-1 inhibition. Our lab has previously shown that cyclin dependent kinase 2 (CDK2)/cyclin T1 is important for HIV-1 transcription. Therefore, the second aim will study if iron chelators inhibit expression and cellular activities of the cyclin dependent kinase 9 (CDK9) and CDK2. Tat phosphorylation by CDK2/cyclin E will be measured to determine if iron chelators inhibit the activity of CDK2. The ability of HIV-1 to replicate is also shown to depend on nuclear factor (NF)-kappa ? activation, mediating inducible HIV-1 gene transcription. NF-kappa ? activation, regulating proviral transcription, can be influenced by iron. Thus, the effect of the iron chelators on NF-kappa ? level, activation and target genes will also be studied. These findings will provide information in the search for alternative therapeutic approaches that are hopefully more effective and efficient to treat HIV-1. Iron chelators could add to improve the treatment of HIV disease. ? ? ?
| Debebe, Zufan; Nekhai, Sergei; Ashenafi, Meseret et al. (2012) Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers. J Chromatogr B Analyt Technol Biomed Life Sci 906:25-32 |
| Debebe, Zufan; Ammosova, Tatyana; Breuer, Denitra et al. (2011) Iron chelators of the di-2-pyridylketone thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series inhibit HIV-1 transcription: identification of novel cellular targets--iron, cyclin-dependent kinase (CDK) 2, and CDK9. Mol Pharmacol 79:185-96 |
