RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies. 

https://www.nature.com/articles/s41576-022-00546-w.pdf

Probing the dynamic RNA structurome and its functions

Structured RNAs bind ligands and are attractive targets for small-molecule drugs. A wide variety of analytical methods have been used to characterize RNA-ligand interactions, but our experience is that most have significant limitations in terms of material requirements and applicability to complex RNAs. Surface plasmon resonance (SPR) potentially overcomes these limitations, but we find that the standard experimental framework measures notable nonspecific electrostatic-mediated interactions, frustrating analysis of weak RNA binders. SPR measurements are typically quantified relative to a non-target reference channel. Here, we show that referencing to a channel containing a non-binding control RNA enables subtraction of nonspecific binding contributions, allowing measurements of accurate and specific binding affinities. We validated this approach for small-molecule binders of two riboswitch RNAs with affinities ranging from nanomolar to millimolar, including low-molecular-mass fragment ligands. SPR implemented with reference subtraction reliably discriminates specific from nonspecific binding, uses RNA and ligand material efficiently, and enables rapid exploration of the ligand-binding landscape for RNA targets.

RNA-Ligand Interactions Quantified by Surface Plasmon Resonance with Reference Subtraction.

RNA is challenging to target with bioactive small molecules, particularly those of low molecular weight that bind with sufficient affinity and specificity. In this report, we developed a platform to address this challenge, affording a novel bioactive interaction. An RNA-focused small-molecule fragment collection (n = 2500) was constructed by analyzing features in all publicly reported compounds that bind RNA, the largest collection of RNA-focused fragments to date. The RNA-binding landscape for each fragment was studied by using a library-versus-library selection with an RNA library displaying a discrete structural element, probing over 12.8 million interactions, the greatest number of interactions between fragments and biomolecules probed experimentally. Mining of this dataset across the human transcriptome defined a drug-like fragment that potently and specifically targeted the microRNA-372 hairpin precursor, inhibiting its processing into the mature, functional microRNA and alleviating invasive and proliferative oncogenic phenotypes in gastric cancer cells. Importantly, this fragment has favorable properties, including an affinity for the RNA target of 300 ± 130 nM, a molecular weight of 273 Da, and quantitative estimate of drug-likeness (QED) score of 0.8. (For comparison, the mean QED of oral medicines is 0.6 ± 0.2). Thus, these studies demonstrate that a low-molecular weight, fragment-like compound can specifically and potently modulate RNA targets.

Low-Molecular Weight Small Molecules Can Potently Bind RNA and Affect Oncogenic Pathways in Cells.

The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling (RBRP) methodology, and applied it to uncover transcriptome interactions of a set of FDA-approved small-molecule drugs in vivo. We show that these protein-targeted drugs pervasively interact with the human transcriptome and can exert unintended biological effects on RNA function. In addition, we show that many off-target interactions occur at RNA loci associated with protein binding and structural changes, allowing us to generate hypotheses to infer the biological consequences of RNA off-target binding. The results suggest that rigorous characterization of drugs’ transcriptome interactions may help assess target specificity and potentially avoid toxicity and clinical failures.

/pdf/pervasive-transcriptome-interactions-of-protein-targeted-2hsjet3e.pdf

Pervasive Transcriptome Interactions of Protein-Targeted Drugs

Targeting RNA with small molecules is an emerging field. While several ligands for different RNA targets are reported, structure-based virtual screenings against RNAs are still rare. Here, we elucidated the general capabilities of protein-based docking programmes to reproduce native binding modes of small molecule RNA ligands and to discriminate known binders from decoys by the scoring function. The programmes were found to perform similar compared to the RNA-based docking tool rDOCK and the faced challenges during docking, namely protomer and tautomer selection, target dynamics and explicit solvent, do not largely differ from challenges in conventional protein-ligand docking. A prospective virtual screening with the Bacillus subtilis preQ1-riboswitch aptamer domain performed with FRED, HYBRID and FlexX, followed by microscale thermophoresis assays identified 6 active compounds out of 23 tested virtual screening hits with potencies between 29.5 nM and 11.0 μM. The hits were selected not solely based on their docking score, but for resembling key interactions of the native ligand. Therefore, this study demonstrates the general feasibility to perform structure-based virtual screenings against RNA targets, while at the same time it highlights pitfalls and their potential solutions when executing RNA-ligand docking.

Protein-based Virtual Screening Tools applied for RNA-Ligand Docking identify new Binders of the preQ1-Riboswitch

Significance RNA molecules encode proteins and play numerous regulatory roles in cells. Targeting RNA with small molecules, as is routine with proteins, would create broad opportunities for modulating biology and creating new drugs. However, this opportunity has been difficult to realize because creating novel small molecules that bind RNA, especially using modest resources, is challenging. This study integrates two widely used technologies, SHAPE chemical probing of RNA and fragment-based ligand discovery, to craft an innovative strategy for creating small molecules that bind to and modulate the activity of a structured RNA. The anticipated impact is high because the methods are simple, can be implemented in diverse research and discovery contexts, and lead to realistic druglike molecules.

Auréliane Michaud

Papers

SHAPE-enabled fragment-based ligand discovery for RNA