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Recent advances in our understanding of RNA biology have uncovered crucial roles for RNA in multiple disease states, ranging from viral and bacterial infections to cancer and neurological disorders. As a result, multiple laboratories have become interested in developing drug-like small molecules to target RNA. However, this development comes with multiple unique challenges. For example, RNA is inherently dynamic and has limited chemical diversity. In addition, promiscuous RNA-binding ligands are often identified during screening campaigns. This Tutorial Review overviews important considerations and advancements for generating RNA-targeted small molecules, ranging from fundamental chemistry to promising small molecule examples with demonstrated clinical efficacy. Specifically, we begin by exploring RNA functional classes, structural hierarchy, and dynamics. We then discuss fundamental RNA recognition principles along with methods for small molecule screening and RNA structure determination. Finally, we review unique challenges and emerging solutions from both the RNA and small molecule perspectives for generating RNA-targeted ligands before highlighting a selection of the "Greatest Hits" to date. These molecules target RNA in a variety of diseases, including cancer, neurodegeneration, and viral infection, in cellular and animal model systems. Additionally, we explore the recently FDA-approved small molecule regulator of RNA splicing, risdiplam, for treatment of spinal muscular atrophy. Together, this Tutorial Review showcases the fundamental role of chemical and molecular recognition principles in enhancing our understanding of RNA biology and contributing to the rapidly growing number of RNA-targeted probes and therapeutics. In particular, we hope this widely accessible review will serve as inspiration for aspiring small molecule and/or RNA researchers.

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure–function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule–RNA targeting.

Discoveries of RNA roles in cellular physiology and pathology are raising the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an “RNA-privileged” chemical space. Biennial updates of the database and the establishment of a website platform (rbind.chem.duke.edu) have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA Structure Search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different to that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.

While the opportunities available for targeting RNA with small molecules have been widely appreciated, the challenges associated with achieving specific RNA recognition in biological systems have hindered progress and prevented many researchers from entering the field. To facilitate the discovery of RNA-targeted chemical probes and their subsequent applications, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND). This collection contains an array of information on reported chemical probes that target non-rRNA and have biological activity, and analysis has led to the discovery of RNA-privileged properties. Herein, we developed an online platform to make this information freely available to the community, offering search options, a suite of tools for probe development, and an updated R-BIND data set with detailed experimental information for each probe. We repeated the previous cheminformatics analysis on the updated R-BIND list and found that the distinguishing physicochemical, structural, and spatial properties remained unchanged, despite an almost 50% increase in the database size. Further, we developed several user-friendly tools, including queries based on cheminformatic parameters, experimental details, functional groups, and substructures. In addition, a nearest neighbor algorithm can assess the similarity of user-uploaded molecules to R-BIND ligands. These tools and resources can be used to design small molecule libraries, optimize lead ligands, or select targets, probes, assays, and control experiments. Chemical probes are critical to the study and discovery of novel functions for RNA, and we expect this resource to greatly assist researchers in exploring and developing successful RNA-targeted probes.

SummaryMany enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ∼6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.HighlightsCell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19Drug screen reveals critical role for membrane lipid composition in fusionSpike’s unusual membrane-proximal cysteines and aromatics are essential for fusionCholesterol tunes relative infectivity of SARS-CoV-2 viral particles

Structural studies of the 3'-end of the oncogenic long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) confirmed a unique triple-helix structure. This structure enables accumulation of the transcript, and high levels of MALAT1 are found in several cancers. Here, we synthesize a small molecule library based on an RNA-binding scaffold, diphenylfuran (DPF), screen it against a variety of nucleic acid constructs, and demonstrate for the first time that the MALAT1 triple helix can be selectively targeted with small molecules. Computational analysis revealed a trend between subunit positioning and composition on DPF shape and intramolecular interactions, which in turn generally correlated with selectivity and binding strengths. This work thus provides design strategies toward chemical probe development for the MALAT1 triple helix and suggests that comprehensive analyses of RNA-focused libraries can generate insights into selective RNA recognition.

The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease.