ABSTRACT
The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3′-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures. RT-qPCR can be harnessed as a small molecule screening platform to read out the effect of small molecules on the structural stability of a variety of RNA targets.
ABSTRACT
The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3'-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures.
ABSTRACT
The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5'-end. Nuclear magnetic resonancebased structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5' untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNAtargeted antivirals.
ABSTRACT
Initial successes in developing small molecule ligands for non-coding RNAs have underscored their potential as therapeutic targets. More recently, these successes have been aided by advances in biophysical and structural techniques for identification and characterization of more complex RNA structures; these higher-level folds present protein-like binding pockets that offer opportunities to design small molecules that could achieve a degree of selectivity often hard to obtain at the primary and secondary structure level. More specifically, identification and small molecule targeting of RNA tertiary and quaternary structures have allowed researchers to probe several human diseases and have resulted in promising clinical candidates. In this review we highlight a selection of diverse and exciting successes and the experimental approaches that led to their discovery. These studies include examples of recent developments in RNA-centric assays and ligands that provide insight into the features responsible for the affinity and biological outcome of RNA-targeted chemical probes. This report highlights the potential and emerging opportunities to selectively target RNA tertiary and quaternary structures as a route to better understand and, ultimately, treat many diseases.