Small Molecule Targeting of Disease Relevant RNA Secondary and Tertiary Structures

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The discovery of the many active roles of RNA in diseased pathways propelled the current ‘RNA Renaissance’. As a result, RNA is no longer regarded as a passive intermediate between DNA and proteins, but as a key player in human disease and an attractive therapeutic target. Small molecules are uniquely poised for RNA-targeting due to their potential for favorable bioavailability, cell permeability, and spatio-temporal control, overcoming the current limitations of gene editing and sequence-based technologies. Nevertheless, questions regarding favorable properties that render small molecules specific for RNA and, ideally, selective for one RNA target over others are far from being answered. Thus, to elucidate possible trends that can expedite future targeting efforts we leveraged both synthetic and commercially available approaches in targeting disease relevant structures in mammalian and viral RNAs. We designed and synthesized a focused library and evaluated it using a holistic approach against the RNA triple helix motif present at the 3’-end of the mammalian oncogenic long non-coding RNA MALAT1. The development of triple helix-focused assays enabled us to assess small molecules’ affinity and their effect on the triplex structural stability and enzymatic digestion. This study resulted in the first example of synthetically tuned small molecules that can bi-directionally modulate the susceptibility of the triplex to enzymatic degradation. In a subsequent study we leveraged the well-established relationship between the structural stability of the MALAT1 triple helix and its refractivity to degradation as a case-study to develop a high-throughput platform that directly reports on the effects of mutations or small molecules on the stability of the triple helix. Optimization and application of this platform to RNA G-quadruplexes and pseudoknots corroborates the potential of this assay to bridge the current gap between affinity-based assays and cell-based activity. With the emergence of SARS-CoV-2, we focused our efforts in identifying potential RNA targets within the viral genome that would be amenable to small molecule targeting with the goal of developing new antiviral agents. Specifically, we first focused on conserved RNA structures at the 5’-end of the viral genome and utilized an in-house focused library based on the known RNA-binding scaffold amiloride. Through evaluation of small molecules leads using a plethora of in vitro and in cellulo assays we identified three small molecule leads that inhibited viral replication by targeting structures at the 5’-end of the genome. This study represented the first example of RNA-targeted SARS-CoV-2 antivirals. We then focused on an RNA structure present at the interface of two overlapping open reading frames and responsible for programmed ribosomal frameshifting, and essential process in coronavirus lifecycle. Screening of an RNA-biased commercially available library curated by our laboratory resulted in the identification of a small molecule selective for the CoV-2 pseudoknot over other disease-relevant triple helices. The work presented herein provides essential insights into RNA:small molecules molecular recognition events and provides tools and workflows that will significantly expedite future targeting efforts against the RNA structures presented in this work and others yet to be identified or explored.






Zafferani, Martina (2022). Small Molecule Targeting of Disease Relevant RNA Secondary and Tertiary Structures. Dissertation, Duke University. Retrieved from


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