The HIV Tat protein, a potent activator of HIV gene expression, is essential for integrated viral genome expression and represents an attractive antiviral target. Tat binds the 5' terminal region of HIV mRNA's stem-bulge-loop structure, the Trans-activation Responsive (TAR) element, to activate transcription. We have recently discovered that didehydro-Cortistatin A (dCA), an analogue of a natural compound isolated from an Indonesian marine sponge, potently inhibits Tat-mediated trans-activation of the integrated HIV-1 promoter by binding specifically to the TAR-binding domain of Tat. Working at subnanomolar concentrations, dCA reduces Tat mediated transcriptional elongation from the viral promoter to inhibit HIV-1 replication in acutely and -more importantly- in chronically infectd cells. Despite the immense success of anti-retroviral therapy (ART) to reduce HIV replication to very low levels, it fails to eradicate the virus. HIV persists in latently and productively infecte CD4+T cells in infected subjects under ART. Significant efforts at HIV eradication are focused on the reactivation and killing of latently infected cells, however these strategies have had only limited success. We hypothesize that by means of a Tat inhibitor, we can further shutdown viral replication, therefore preventing viral reactivation from latently infected resting cells, and production from active persistent reservoirs. We have found that in vitro dCA abrogates low-level virus replication from latently infected primary cells isolated from patients undergoing ART and prevents reactivation upon homeostatic stimulation with cytokines such as IL-15 and antigenic stimulation by CD3/CD28. These results define dCA as a novel anti-HIV drug that could decrease residual viremia during ART. This project aims to structurally and biophysically characterize the interaction between dCA and the HIV-1 Tat protein and to obtain the crystal structure of Tat*dCA and TatP-TEF*bdCA complex. The specific knowledge of the molecular binding and strength of the dCA-Tat interaction, will allow for structure- activity relationship around the Cortistatin A pharmacophore necessary for efficient clinical development. In addition, our preliminary biophysical data suggested that Tat is stabilized by dCA. Tat is a highly flexible protein. This flexibility renders difficult the determination of the Tat structure, explaining the ack of full-length Tat X-ray crystallography study. Our results strongly encouraged the hypothesis that Tat in complex with dCA might be amenable to crystallization. Accordingly we propose two specific aims:
Aim 1. Characterize the site and binding affinity of dCA*Tat interaction.
Aim 2. Determine the structure of Tat*dCA complex Combining approaches of classic biophysics and biochemistry with HDX, NMR and X-ray crystallography we will obtain a clear picture of the molecular interactions between Tat and dCA and possibly the first crystal structure of the entire Tat protein.
The HIV-1 viral protein Tat is a potent activator of HIV transcription and a potential antiviral target. We found that didehydro-Cortistatin A (dCA) is a potent inhibitor of Tat. This application seeks to characterize the biophysical interaction between Tat and dCA.
