Insights, Assays, and Strategies for Small Molecule-Based Modulation of the MALAT1 RNA Triple Helix

Abstract

Recent discoveries of myriad of endogenous RNA transcripts that do not code for proteins has led to a paradigm shift in our understanding of human biology. As the subsequent “RNA revolution” continues to elucidate the disease-related functions and structures of mammalian non-coding RNA molecules, interest in developing small molecule probes and drug leads for these targets is on the rise. For example, the long non-coding RNA MALAT1 (Metastasis-Associated-Lung-Adenocarcinoma-Transcript-1) is overexpressed in and is critical for metastasis in a variety of cancers. A recently characterized triple helix structure at the 3'-end of MALAT1 was shown to be pivotal in stabilizing the transcript, and deletions or a single base substitution - proposed to destabilize this structure - led to a significant decrease in MALAT1 accumulation. While this unique structure represents a novel opportunity for therapeutic intervention, there is a gap in knowledge regarding effective strategies for targeting RNA in general and, in particular, triple helices.Towards elucidating these principles, this work takes a three-pronged approach: i) synthesis and evaluation of scaffold-based small molecule libraries to deduce fundamental ligand-triple helix recognition principles, ii) development of robust and high-throughput screening assays to discover functional probes, and iii) structural characterization of the MALAT1 triple helix conformational landscape for small molecule-based regulation. In the first area, we have discovered first small molecule ligands for the MALAT1 triple helix and gained insights into the role of three-dimensional small molecule shape and intramolecular interactions in binding events. Additionally, we established a correlation between ligand-induced thermal stabilization and prevention of exonucleolytic degradation of this target and elucidated binding properties of ligands with triplex-stabilizing functions. In the second area, we developed a high-throughput screening (HTS) technique based on differential scanning fluorimetry and curated an RNA-targeted library to identify novel small molecules with a destabilizing effect on the triple helix. Such thermal melting profiles were predictive of accelerated degradation profiles of the triple helix, thereby providing a robust discovery tool for identifying functional small molecule modulators of this target. In the third area, we conducted chemical probing experiments to characterize secondary structures that may exist in equilibrium with the triple helix and hence represent attractive targets for small molecule stabilization. Together, this work has and will continue to build a set of small molecule tools, rapid assays, and novel design strategies to enable chemical biology-based interrogation of the MALAT1 triple helix role in cancer biology. This knowledge and tools can be utilized to interrogate the protective mechanism of the MALAT1 triple helix, to identify and develop therapeutic leads, as well as to apply these strategies for the study of other therapeutically relevant RNA.