There are currently 40-100 million individuals in the world infected with human immunodeficiency virus (HIV), and nearly 16,000 new infections occur worldwide each day based on World Health Organization estimates. The introduction of highly active anti-retroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality. Unfortunately, up to 25% of patients discontinue their initial HAART regimen because of treatment failure (inability to suppress HIV viral replication to below the current limit of detection, 50 copies/mL), toxic effects or non-compliance within the first few months of therapy. Therefore, today it is widely believed that the success in HIV-1 treatment will require targeting of other HIV and/or host cellular proteins. The pathogenesis of HIV -induced disease is complex and multifactoral. Several key HIV and cellular proteins have been assigned to be necessary for the course of infection including the transactivator Tat. Viral clones deficient in Tat will not replicate in vitro or in vivo to high liters. Also, cellular activation (a process where cells go from G0 to the Gi/S stage of the cell cycle) of latently infected cells by various stimuli leads to viral expression followed by progeny formation. Minimal activated transcription from the HIV-1 long terminal repeat (LTR) occurs in GO quiescent cells. We previously have shown that HIV-1 in latently infected cells can down modulate the natural cyclin dependent kinase (cdk) inhibitors, and in turn is able to control the primary cdk targets such as cdk2/cyclin E complex. Therefore, inhibition of the Gi/S kinases, could be a possible target for inhibition of HIV-1 replication. More importantly, cdk2 and cyclin E are not essential for the growth of normal (as seen in cdk2"""""""""""""""" or cyclin E'A mice), non-cancerous cells, and therefore, use of inhibitors against the cdk2/cyclin E complex may pose a viable option to inhibit HIV-1 in infected cells. Our current preliminary data indicates that cdk2/cyclin E could phosphorylate the RNA Pol IICTD, can be found in the HIV- 1 elongation complexes, recombinant cdk2/cyclin E stimulates Tat-dependent HIV-1 transcription in a reconstituted transcription assay and immunodepletion of cdk2/cyclin E from nuclear extracts blocks Tat-dependent transcription. Also, RNAi experiments show that cdk2 downregulation affects HIV-1, but not HTLV-1, CMV, or IgH promoters. Here, we propose to use an ATP analog and a new derivative which can inhibit cdk activity. The target of these drugs in HIV-1 infected cells is the cdk2/cyclin E complex at an IC50 of 0.009-0.15 uM.
Our specific aims i nclude:
Aim 1 : Effect of CYC202 and its derivative on HIV-1 elongation factor occupancy and splicing. To define the mechanism of inhibition, transcription elongation factor occupancy in presence of drugs will be tested in vivo using ChIP assays. The effect of CYC202 and its derivative on HIV-1 singly or multiply spliced messages will be evaluated in vitro and in vivo using HIV-rtTA (KWK and KYK), PBMCs and Tat variants (wild type, C22G, and L43A).
Aim 2 : Effect of CYC202 and its derivative compound #4 on HIV-1 variants, and possible cell toxicity. Effect of drug treatment on HIV-1 variants will be tested in wild type and recombinant viruses as well as LTR and Tat transfections. Determine any significant alterations in cellular gene expression following CYC202 or derivative #4 treatments using Microarrays. Therefore, the current proposal is aimed at determining whether drugs such as ATP analogs are a viable option in treating HIV-1 infected cells both in vitro and in vivo. ? ? ?
| Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C et al. (2015) Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells. J Neurovirol 21:257-75 |
| Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C et al. (2015) Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells. Virology 485:1-15 |
| Easley, Rebecca; Van Duyne, Rachel; Coley, Will et al. (2010) Chromatin dynamics associated with HIV-1 Tat-activated transcription. Biochim Biophys Acta 1799:275-85 |
| Klase, Zachary; Winograd, Rafael; Davis, Jeremiah et al. (2009) HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression. Retrovirology 6:18 |
| Chen, Hao; Van Duyne, Rachel; Zhang, Naigong et al. (2009) A novel binding pocket of cyclin-dependent kinase 2. Proteins 74:122-32 |
| Klase, Zachary; Kale, Prachee; Winograd, Rafael et al. (2007) HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 8:63 |
| Berro, Reem; de la Fuente, Cynthia; Klase, Zachary et al. (2007) Identifying the membrane proteome of HIV-1 latently infected cells. J Biol Chem 282:8207-18 |
| Kehn, K; Berro, R; Alhaj, A et al. (2007) Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Oncogene 26:5060-9 |
