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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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (T(m) ) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner., (© 2011 John Wiley & Sons A/S.)

The T box riboswitch regulates the transcription of many bacterial genes by structurally responding to cognate non-aminoacylated (uncharged) tRNA. The riboswitch contains multiple conserved RNA elements including a key structural element, the antiterminator, which binds the tRNA acceptor end nucleotides. Previous studies identified a lead 1,4-disubstituted 1,2,3-triazole, GHB-7, that disrupted formation of a tRNA-antiterminator RNA model complex. The affinity and molecular interactions of GHB-7 binding to antiterminator model RNA were characterized as part of a comprehensive T box antiterminator RNA-targeted drug discovery project. In-line probing, UV-monitored thermal denaturation and docking studies all consistently indicated that GHB-7 likely binds to the bulge region of the antiterminator, reduces the flexibility of the bulge nucleotides and, overall, stabilizes the RNA secondary structure. These results begin to elucidate possible mechanisms for ligand-induced inhibition of tRNA binding to T box antiterminator RNA and contribute to the knowledge of how small molecules bind relatively simple RNA structural elements such as bulges., (Copyright © 2011. Published by Elsevier Ltd.)

The binding of tRNA to the T box antiterminator RNA element is a critical component of the T box riboswitch mechanism that regulates essential genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened for disruption of the tRNA-T box antiterminator RNA interaction using a fluorescence anisotropy-based assay. Several compounds reduced the anisotropy greater than 50% likely indicating significant competition for binding antiterminator RNA. General structure-activity trends indicated that the substituents at both N-1 and C-4 likely are involved in ligand binding. In addition, the anisotropy of the complex was significantly decreased not only by ligands with the possibility for electrostatic interactions with the RNA, but also by ligands with the potential for π-π stacking or other hydrophobic interactions indicating that these non-electrostatic interactions could possibly be utilized in the future development of compounds that target and disrupt the function of this medicinally important riboswitch., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized, and their binding to the T-box riboswitch antiterminator model RNA has been investigated in detail. Characterization of ligand affinities and binding site localization indicates that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets.