Interrogating the Small Molecule Modulation of Viral RNA Secondary Structures

Abstract

RNA viruses have been the cause of viral outbreaks, epidemics and pandemics worldwide. However, many viruses do not have specific antiviral treatments. As such, there is an imperative need for the development of viral therapeutics. The recent advances in our understanding of RNA have shown that RNA plays a critical role in various disease states and has properties that make it an attractive potential therapeutic target. One property of interest is the structure-function relationship of RNA, where the structure and dynamics of viral RNA has been found to significantly impact biological function. Additionally, small molecules are tunable therapeutics that have commonly been used to target different biomolecules. Therefore, there is an opportunity to exploit the structure-function relationship in viral RNA by using small molecules to target viral RNA structure and modulate its biological function. However, the impact small molecules have on functional, viral RNA secondary structures has been underexplored. Different RNA viruses such as Enterovirus 71 (EV71), Hepatitis C Virus (HCV), Human immunodeficiency virus (HIV), and Flaviviruses contain different RNA secondary structures that have been found to have biological function. In this work, we used the functional RNA secondary structures found in these RNA viruses and two different methods, Förster energy transfer resonance (FRET) and fluorescence indicator displacement (FID), to investigate small molecule binding to viral RNA secondary structures and the impact small molecules have on their dynamics. FRET is a method that has been found to detect conformational change in biomolecules and a modular FRET-based system has been previously developed for an RNA switch in HCV. We hypothesized that the FRET system could be adapted to similar viral RNA targets and used to detect conformational change. Therefore, we tried to apply the FRET system to a viral RNA switch in EV71 that has the same secondary structure and a similar function to the RNA switch in HCV. We found that while we could not use the modular FRET system to detect conformational change, a conventional similar FRET-based system could be used to detect conformational change in the viral RNA targets. In addition to evaluating if a FRET-based system could be used to detect conformational change in a novel RNA target, we used high-throughput screening to identify small molecules that bind to or induced conformational change in viral RNA targets containing RNA secondary structures. We used a FID screen to identify small molecules that bound to viral RNA targets containing the bulge and three-way junction structural motif and found hit small molecules for each RNA secondary structure. Additionally, a FRET screen was developed and used to determine small molecules that prompted a linear or bent conformational change in two different RNA switches. After we identified the small molecules that bound to or induced conformational change in the viral RNA targets, we used computational analysis to analyze the cheminformatic properties that were important for the interaction. The computational analysis revealed that cheminformatic properties such as surface area and the number of hydrogen bond donors were important for binding to viral RNA targets containing the bulge and three-way junction structural motif and for prompting conformational changes in viral RNA switches. The findings from this work furthered our understanding of viral RNA secondary structures and has the potential to aid in the development of novel antiviral treatments.