4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine.

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

RNA sequence and ligand binding alter conformational profile of SARS-CoV-2 stem loop II motif.

Antiviral drug discovery continues to be an essential complement to vaccine development for overcoming the global pandemic caused by SARS-CoV-2. The genomic RNA of SARS-CoV-2 contains structural elements important for viral replication and/or pathogenesis making them potential therapeutic targets. Here we report on the stem-loop II motif, a highly conserved noncoding RNA element. Based on our homology model we determined that the G to U transversion in the SARS-CoV-2 stem-loop II motif (S2MG35U) forms a C-U base-pair isosteric to the C-G base-pair in the early 2000's SARS-CoV (S2M). In addition, chemo-enzymatic probing and molecular dynamics simulations indicate the S2MG35U conformational profile is altered compared to S2M in the apical loop region. We explored S2MG35U as a potential drug target by docking a library of FDA approved drugs. Enzymatic probing of the best docking ligands (aminoglycosides and polymyxins) indicated that polymyxin binding alters the conformational profile and/or secondary structure of the RNA. The SARS-CoV-2 stem-loop II motif conformational differences due to nucleotide transversion and ligand binding are highly significant and provide insight for future drug discovery efforts since the conformation of noncoding RNA elements affects their function.

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

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 Kd 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.