Virtual Screening for RNA-Interacting Small Molecules - Biophysical Approaches to Translational Control of Gene Expression - page 238

Fig. 12.1 −1 frameshifting ef fi ciencies induced by biotin aptamer RNA pseudoknot in the presence
of compounds (250 mM) determined by SDS-PAGE ( a ) and dual luciferase assay ( b ). −1 FS %
values are shown at the bottom of the autoradiogram of SDS-PAGE. Each −1 FS % value from
dual luciferase assay is the average of triplicate experiments
−1 FS is essential for the synthesis of enzymatic proteins. The stability of the RNA
pseudoknot that induces −1 FS in the SARS-CoV (SARS-pseudoknot) also has a
dramatic effect on −1 FS efficiency. Therefore, the RNA secondary structure in
the −1 FS site has emerged as an attractive target for drug development (Baranov
et al 2005 ; Plant et al 2005 ; Su et al. 2005 ) .
Park first conducted virtual screening against the RNA pseudoknot in the −1 FS
site to discover ligands that change −1 FS efficiency (Park et al. 2008 ) . In this pilot
study, they used the −1 FS system containing biotin aptamer RNA pseudoknot as
the RNA secondary structure element. Biotin RNA aptamer was the only ligand-
bound RNA pseudoknot structure (PDB id: 1F27) determined by X-crystallography
(Nix et al. 2000 ). Park et al. used the conventional 2D and 3D pharmacophore search
program Unity (Martin 1992 ) for primary database filtering and the FlexX docking
program for final docking screening. RNA flexibility was not considered and ligand
flexibility was given during FlexX run. Out of about 80,000 compounds in the
chemical DB, they obtained 37 hits which increased −1 FS. Compound h4 showed
the highest activity in the in vitro transcription and translation coupled assay
(Fig. 12.1 ). The FlexX-docked pose of h4 is shown in Fig. 12.2 . The docking mode
of h4 is similar to that of biotin; however, h4 forms a stronger interaction with the
receptor RNA (Fig. 12.2a ). Compound h4 forms hydrogen bonds with O4¢ and the
2-carbonyl oxygen of uracil ring of U7 which is one of the critical residues for inter-
action with biotin in the X-ray structure, and an additional hydrogen bond with
ribose O2¢ atom of A16 (Fig. 12.2b ). These interactions may alter the stability of the
RNA pseudoknot and increase the stalled time of the ribosome on the slippery site,
thus increasing the rates of −1 FS.