This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.With increasing anti-viral and anti-bacterial resistance the need for novel therapeutic development is ever more pressing. While proteins have classically been targeted to inhibit bacterial and viral cell processes, therapeutics binding to RNA elements remains largely undeveloped. Difficulty in targeting RNA elements stems from their inherent dynamic nature making the equilibrium conformations difficult to characterize and thus Structure Activity Relationship (SAR) ambiguous. The HIV-I Trans-Activation Response element (TAR) regulates transcription in HIV-I making it a prime target for therapeutic development. As a means to explore the conformational space in which HIV- I TAR exists an NMR validated 60 ns explicit solvent Molecular Dynamics (MD) simulation has been generated (using CHARMM) and 44 unique snapshots isolated for use as receptors in fully flexible Computational Docking. A library with > 1500 RNA binding small molecules, which contains all known HIV-I TAR binders, has been created and subjected to virtual screening against the 44 generated snapshots using the docking Software ICM (Molsoft LLC). Chemical shift perturbation experiments have been implemented to determine atom-specific dissociation constant(s) for top binders, and residual dipolar couplings measured in order to ascertain the RNA structure(s) stabilized by the small molecules. To further develop this methodology MD simulations will be run on the HIV-I Dimerization Initiation Site (DIS) and the bacterial A-site RNA elements. Snapshots obtained from these simulations will be subjected to large-scale virtual screening and subsequent experimental validation using NMR.
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