Characterizing HIV-1 Transactivation Response Element Dynamics that GovernLigand Recognition: Direct Applications to Drug Discovery.

RNA Dynamics;Drug Discovery;HIV;NMR;Computational Docking;Biological Chemistry;Science;Chemical Biology

Stelzer, Andrew CorbetWalter, Nils G. ;

University of Michigan

关键词: RNA Dynamics;    Drug Discovery;    HIV;    NMR;    Computational Docking;    Biological Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/76006/stelzer_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

RNAs regulate and affect numerous cellular processes, making them highly sought therapeutic targets. One mechanism to inhibit functional RNAs is to alter their structural-dynamics using small molecule binders. While structure based drug design is often used to discover small molecule inhibitors, difficulties arise because, unlike proteins, RNAs undergo large conformational changes between the free and ligand-bound-states that cannot be determined a priori. The spatial and temporal complexity of these conformational changes precludes accurate characterization that would allow one to visualize the conformational changes. Using NMR and MD we aim to uncover the biophysical principles governing TAR-mediated ligand recognition and discover new TAR-binding small molecules. First, we present a Sample and Select (SAS) method, which combines NMR residual dipolar couplings (RDCs) and MD to provide an accurate all-atom description of RNA dynamics over sub-millisecond timescales. RDCs measured on elongated TAR molecules are used to separate internal and overall motions and impose a helix-anchored reference frame. Using the SAS approach, refined RNA ensembles that re-capitulate experimental RDCs are generated from an MD trajectory. Specific snapshots of the ensemble closely agree with previously determined ligand-bound TAR structures, suggesting that the bound-state conformations are sampled in the absence of ligand. In a second study we investigate the sequence dependence of TAR dynamics and show that a modest mutation greatly perturbs global and local dynamics giving rise to changes in small molecule binding affinity while still forming the same bound-state TAR conformation. Lastly, the SAS ensemble structures are used in RNA structure-based drug discovery. Computational docking simulations are used to discover 11 TAR-binding small molecules, 8 of which have never before been shown to bind TAR and 2 never before been shown to bind RNA. NMR chemical shift perturbations and fluorescence polarization measurements verify that the small molecules bind TAR and inhibit the TAR-Tat interaction with inhibition constants ranging 0.627-300 μM. RDCs measured on TAR bound to the small molecule netilmicin suggest that docking against the SAS structures accurately re-capitulates the bound-state. Remarkably, netilmicin also inhibits TAR-mediated HIV-1 LTR expression and HIV-1 replication in an indicator cell line with an IC50 of 23.1 μM.