Inhibition of Human Immunodeficiency Virus-1 by Iron Chelators: Effect of Iron Chelators on HIV-1 transcription and Estimation of In-vitro Bioavailability Employing Caco-2 Monolayer

In recent years, research advances have resulted in ample knowledge on HIV molecular biology, the pathogenesis of the disease, and the impact of the disease progression on immune function. However, these findings have translated into anti-HIV drugs for only a limited number of viral targets, i.e. reverse transcriptase and protease. The currently adapted long term anti-HIV drug therapy with combinations of drugs active against these targets have succumbed to the development of resistance, metabolic disturbances, and toxicities. Therefore, it is crucial that additional therapeutic targets/strategies need to be identified and aggressively studied.;Transcription of HIV-1 viral genes is uniquely dependent on both viral proteins and host cell factors. Studies have shown that increased iron stores correlate with faster HIV-1 progression in HIV-1 positive patients. Also, excess of iron induces HIV-1 replication and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. It is known that HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cycle-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional co-activators to the HIV-1 promotor. Recently, DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) have been shown to inhibit expression of proteins that regulate cell-cycle progression including cycle-dependent kinase 2 (CDK2) in cells.;Here, we evaluated the effect of iron chelators on HIV-1 transcription and the possible mechanisms of inhibition. The expression and activities of CDK9 and CDK2 after iron chelation was determined using 293T cells. We utilized the iron chelators DFO, 311, Deferasirox (ICL670), and the novel di-2-pyridylketone thiosemicarbazone (DpT)-based iron chelator Dp44mT, and 2-benzoylpyridine thiosemicarbazone (BpT)-based tridentate iron chelators, Bp4eT and Bp4aT to chelate intracellular iron. Our results indicate that the chelators inhibit Tat-induced, pseudotyped and also active HIV-1 transcription and replication in PBMC, CEM-T, THP-1, 293T and HeLa-MAGI cells. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1 and disrupted the interaction of CDK9 and cyclin T, suggesting a possible mechanism for inhibition of CDK9.;One of the key concerns in further developing the iron chelators for the clinical use has been their absorption in the body. Therefore, we studied the permeability of the chelators in Caco-2 cells. We developed a simple and sensitive HPLC method to measure Bp4eT and Bp4aT and utilized it to estimate the compounds' transport across Caco-2 cell monolayers. HPLC method was validated and optimized to measure the interconvertible Z and E isomers of iron chelators Bp4eT and Bp4aT in HBSS buffer during the transport studies across CaCo2 monolayers. The HPLC method was successfully applied to study the transport of both compounds in Caco-2 monolayer using transwell plates. Our bidirectional transport study showed that both chelators permeate fairly well. With CaCo2 permeability being an excellent in-vitro correlator for oral drug absorption, our data indicates a promise for future oral dosage form development.;In conclusion, our findings show that iron chelators inhibit HIV-1 transcription possibly by de-regulating CDK2 and CDK9, and Bp4eT and Bp4aT could possibly be good candidates for oral therapy.