Blog

Screening Strategies for RNA-Targeted Small-Molecule Drugs

The ability to target RNA with small-molecule drugs presents a powerful opportunity for therapeutic intervention. In contrast to traditional drugs, which generally act against proteins, these drugs interact with RNA molecules. In most cases, they function by modulating splicing or blocking translation of a specific gene transcript, although they can also affect RNA localization, post-transcriptional modification, and degradation dynamics.

Risdiplam, a small-molecule drug that alters splicing of the SMN2 gene, was approved by the FDA in 2020 for the treatment of spinal muscular atrophy (SMA), representing a landmark validation of the clinical utility of RNA-targeted small-molecule drugs. While other RNA-targeting modalities exist, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), the adaptation of these technologies to clinical use has been fraught with challenges. Effective delivery is hindered by their large size, negative charge, and hydrophilicity, as well as their susceptibility to nuclease activity. In addition, oligonucleotide-based drugs have been linked to serious side effects, including thrombocytopenia and acute kidney and liver toxicity, and are known immunostimulants. In contrast, small-molecule drugs are orally bioavailable, readily cross the blood-brain barrier, and can be flexibly optimized by leveraging a deep body of existing medicinal chemistry knowledge.

The critical ‘rate-limiting’ step in the development of an RNA-targeting small-molecule drug is the identification of a promising ligand. For protein targets, existing structural data may inform a rational design strategy. However, this approach is generally ill-suited for RNA targets due to their inherent conformational flexibility and dynamic nature, as well as the low abundance at which many RNA species exist in vivo. While many algorithms purport to predict 3D RNA structure, these in silico predictions have been largely underwhelming, and significant biological follow-up is required for validation. Even deep-learning approaches like AlphaFold, which have revolutionized the determination of protein structures, exhibit limited predictive power with respect to RNA.

For these reasons, high-throughput screening is typically the most productive approach for the identification of RNA-targeting small molecules. However, many screening methods are unsuitable for this application. For example, DEL screening requires test compounds to be tagged with DNA barcodes; non-specific oligo-oligo interactions between these barcodes and an RNA target generate confounding false positives. Given that screens against RNA targets typically have hit rates of 0.1% to 0.01%, even a low level of non-specific false positives can overwhelm and obscure any true hits. Similar concerns exist for fluorescence polarization (FP) -based screens, in which false positives commonly arise from interactions between ligands and the reporter fluorophore, rather than the target RNA. While cell-based screens can circumvent some of these issues, they require time-consuming assay development, and the complex cellular environment can make it difficult to determine the pathway through which an effect is mediated without extensive experimental effort.

Affinity selection mass spectrometry (ASMS) is a high-throughput screening method uniquely suited for the identification of RNA-targeting small-molecule drugs. ASMS is a rigorous biophysical approach that can screen diverse libraries of unmodified ligands, avoiding tag-driven false positives and ensuring that hits are immediately available for validation and follow-up. The automated ligand identification system (ALIS) provides a versatile ASMS platform for the solution-phase interrogation of targets without their immobilization (as required for SAMDI- and DEL-based methods). As a result, screening conditions are physiologically relevant, no protein engineering is required, and ligands can interact freely with the target in all orientations. Beyond initial screening, ASMS/ALIS can be used for hit validation, selectivity screens, and additional follow-up experiments, avoiding the need to develop and optimize auxiliary assays. Affinity selection mass spectrometry provides a powerful and flexible strategy for the identification, validation, and development of RNA-targeting small molecules, allowing innovative therapeutics to be efficiently advanced from bench to bedside.

Momentum has helped more than a dozen clients identify RNA-binding small molecules through ASMS using our library of over 300K drug-like compounds. To find out more about how we can advance your drug discovery project, contact us. Or, to learn more about the power of ASMS screening, check out our previous blog post.

 

 

 

Sources

Bernard C, Postic G, Ghannay S & Tahi F. Has AlphaFold 3 reached its success for RNAs? bioRxiv (2024).

Childs-Disney JL, Yang X, Gibaut QMR, Tong Y, Batey RT & Disney MD. Targeting RNA structures with small molecules. Nat Rev Drug Discovery 21, 736-762 (2022).

Deigan Warner K, Hajdin CE & Weeks KM. Principles for targeting RNA with drug-like small molecules. Nat Rev Drug Discovery 17, 547-558 (2018).

Goyenvalle A, Jimenez-Mallebrera C, van Roon W, Sewing S, Krieg AM, Arechavala-Gomeza V & Andersson P. Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides. Nucleic Acid Ther 33, 1-16 (2023).

Hargrove AE. Small molecule-RNA targeting: Starting with the fundamentals. Chem Commun. 56, 14744-56 (2020).

Morgan BS, Forte JE & Hargrove AE. Insights into the development of chemical probes for RNA. Nucleic Acids Research 46, 8025-37 (2018).

Yu A, Choi YH & Tu M. RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges. Pharmacol Rev 72, 862-898 (2020).

Zhu S, Rooney S & Michlewski G. RNA-Targeted Therapies and High-Throughput Screening Methods. Int J Mol Sci 21, 2996 (2020).

Back to Blog