RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function

Abstract

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived K_d values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.

Keywords: Allosteric inhibition; Conformational restriction; Drug discovery; RNA; T-box riboswitch.

Figure 1.

Design for conformationally restricted compounds

Figure 2.

a) T-box riboswitch antiterminator model RNA, AM. b) Correlation between experimental ΔG = RTln(K_d) and predicted binding energy (Glide-derived E_model) at pH 6.5. Best fit linear regression indicated by solid line (R² = 0.8, 95% confidence intervals shown as dashed lines).

Figure 3.

a) and b) Ligand effect on formation of tRNA-AM complex as monitored by changes in anisotropy of fluorescently-labeled AM. Average of triplicate data for 3a-d and 1a-d (100 μM). c) and d) Ligand effect on T-box riboswitch transcription readthrough in the presence (filled bars) or absence (open bars) of tRNA induction. Average of triplicate data (100 μM). p values of unpaired t-test are indicated by * (0.01<p<0.05), ** (0.001<p<0.01) and *** (0.0001<p<0.001).

Figure 4.

a) Relative fluorescence ratios for 1a-d and 3a-d binding antiterminator model RNAs. b) Lowest energy Glide-docked structure for 3a-d binding to AM.

Scheme 1