Advancing RNA drug discovery with Cryo-EM

RNA is a novel therapeutic target for small-molecule discovery, yet advancement has been constrained by limited structural data. Cryo-EM can address this limitation, however it favors larger molecular weight targets. By employing an RNA scaffold, Evotec resolved the structure of an 85-nt branched RNA identified in Nipah virus, obtaining a 2.9 Å global map with approximately 4 Å local resolution on the target RNA.

The perception of RNA as an ‘undruggable’ target is rapidly changing. Advancements in computational instruments, combined with diversification in compound mode of action, have started to produce promising results with the arrival of the first FDA-approved RNA-targeting compound (risdiplam) on the market alongside a growing list of compounds in clinical trials.¹

Despite these developments, drugging RNA remains extremely difficult due to the lack of hydrophobic pockets, conformational heterogeneity, and low sequence complexity, as RNA is composed of just four different nucleotides.

In addition, the Protein Data Bank (PDB) lacks experimentally determined RNA structures, with just ~0.8% of deposited structures comprising RNA-only structures.

This low number of structures substantially restricts the training sets of artificial intelligence (AI) instruments, limiting their ability to correctly predict target RNA three-dimensional structures. For this reason, experimentally derived structural data plays a key role in facilitating structure-based drug discovery (SBDD), which helps to both reduce risk and accelerate RNA pharmaceutical discovery programs.

However, RNA structural biology continues to pose significant challenges. NMR model determination requires homogeneous samples of isotopically labeled RNA, ideally with low molecular weight (<15 kDa).

X-ray crystallography requires the production of well-ordered crystals that diffract to high resolution and, unlike most protein targets, continues to face the phase problem, which must be addressed with heavy atom soaks.

Cryo-electron microscopy (Cryo-EM), however, faces different challenges, instead restricted to a minimum target molecular weight (although RNA versatility still makes it a capricious target). To address the low-molecular-weight challenge, electron microscopists can fuse the target to a rigid RNA scaffold to increase the molecular weight of the targets, making them sufficiently large for cryo-EM.

Inspired by recent developments in RNA scaffold development, Evotec used a literature Tetrahymena ribozyme scaffold to determine the structure of a new RNA sequence.² A judicious construct design enabled the fusion of a new 85-nucleotide viral branched RNA sequence, identified from the highly pathogenic Nipah virus genome, to the Tetrahymena ribozyme scaffold.

After completing in-house in vitro transcription, cryo-EM grids were prepared and optimized using Evotec’s in-house FEI Vitrobot. A total of 8000 exposures were then collected on eBIC’s industrial access Titan Krios. CryoSPARC was subsequently used to process data, generating a map with 2 Å average resolution and 4 Å local resolution for the target viral RNA. 

Generating the first 3D structure of the target provided valuable insights into possible druggable pockets and accelerated Evotec’s chemistry program, which is focused on discovering innovative antivirals that bind to and inhibit the specific RNA sequence.

Figure 1. Cryo-EM structure of branched Nipah virus RNA (pink) fused to an RNA scaffold (blue). RNA shown in cartoon form with electron density envelope in transparent gray. Image Credit: Evotec

This successful and time-efficient strategy, which achieved full structure determination in 16 weeks, will serve as a template to structurally enable future RNA targets.

References and further reading

1. Chen, S., et al. (2024). RNA-Binding Small Molecules in Drug Discovery and Delivery: An Overview from Fundamentals. Journal of Medicinal Chemistry. DOI:10.1021/acs.jmedchem.4c01330. https://pubs.acs.org/jmcmar/article-abstract/67/18/16002/171269/RNA-Binding-Small-Molecules-in-Drug-Discovery-and?redirectedFrom=fulltext.
2. Conner J, L. and Kieft, J.S. (2023). A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM. Nucleic Acids Research, 51(20), pp.e100-e100. DOI:10.1093/nar/gkad784. https://academic.oup.com/nar/article/51/20/e100/7288835. 

Acknowledgments

Produced from materials originally authored by Ciaran McFarlane, PhD, Tamas Yelland, PhD, Mateusz Mieczkowski, PhD, and Mélissanne de Wispelaere, PhD, from Evotec.

This work was supported by Coefficient Giving (previously Open Philanthropy) and Good Ventures Foundation, under award number GV673602866.

About Evotec

Evotec’s mission is to discover and develop highly effective therapeutics and make them globally available to the patients who need them. To this end, Evotec has established itself as a global platform company, leveraging its data-driven multimodality platform for both proprietary as well as partnered research, and applying a unique combination of innovative technologies for the discovery and development of first-in-class and best-in-class pharmaceutical products. Its network of partners includes all Top 20 Pharma and hundreds of biotechnology companies, academic institutions, and other healthcare stakeholders. Evotec has strategic activities in a broad range of currently underserved therapeutic areas, including, e.g., neurology, oncology, as well as metabolic and infectious diseases. Within these areas of expertise, Evotec aims to create the world-leading co-owned pipeline for innovative therapeutics and make them accessible to patients worldwide. To-date, the Company has established a portfolio of more than 200 proprietary and co-owned R&D projects from early discovery to clinical development.

Evotec operates globally with more than 4,500 highly qualified people at 17 sites in six countries across Europe and the USA. The Company’s sites in Hamburg (HQ), Cologne, Goettingen, Halle/Westphalia and Munich (Germany), Lyon and Toulouse (France), Abingdon and Alderley Park (UK), Modena and Verona (Italy), Orth (Austria), as well as in Branford, Princeton, Redmond, Seattle and Framingham (USA) offer highly synergistic technologies and services and operate as complementary clusters of excellence.

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