Targeting Dynamic Structures of RNA Using Experiment and Computation.

RNA Dynamics;High Throughput Screening;Virtual Screening;R1rho Relaxation Dispersion;Chemistry;Science;Chemistry

Lee, JanghyunMapp, Anna K. ;

University of Michigan

关键词: RNA Dynamics;    High Throughput Screening;    Virtual Screening;    R1rho Relaxation Dispersion;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/108771/janghlee_1.pdf?sequence=1&isAllowed=y

瑞士|英语

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

【 摘 要 】

RNA is growing in its importance as a drug target and new approaches are needed in order to efficiently screen small molecules that bind and modulate the function of RNA. Targeting RNA with small molecules opens a door to the therapeutics of diseases that have not been approachable by targeting proteins and can also increase the effectiveness of existing treatments. In the present thesis, we used experimental high throughput screening utilizing a fluorescence-based displacement assay and ensemble-targeted virtual screening to screen a library of ~100,000 small molecules in search of compounds that inhibit the interaction between the transactivation response element of HIV-1 (TAR) and the Tat protein, a viral RNA-protein complex that is responsible for the transcription elongation. We showed that dimethyl sulfoxide (DMSO), a commonly used solvent to prepare small molecules for biochemical assays, has a small but significant effect on RNA structure and RNA-ligand binding that involves local melting of base-pairs near non-canonical motifs. The experimental and computational screens yielded complimentary small molecule hits, which includes 11 compounds with <+2 formal charge at neutral pH, average molecular weight of 480 g/mol, and IC50s ranging between 30 μM and 178 μM. One of the compounds showed significant transcriptional inhibition in cell-based assays utilizing TZM-bl reporter cell lines. Finally, we characterized a transient state structure of HIV-1 TAR using NMR relaxation dispersion experiment and mutagenesis that simultaneously modifies both a bulge and apical loop motif that are separated by four base-pairs. This transient structure provides the basis for long-range communication between remotely positioned motifs and represents a new TAR target for developing ant-HIV therapeutics. The combination of experimental and virtual screening provides a general strategy for rapidly and effectively screening broad regions of chemical space in search of compounds that bind RNA and modulate their activity.