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Structure-based small molecule drug discovery using cryo-EM
Small molecule drug discovery relies on understanding how compounds interact with their target proteins at the molecular level. Structural insight into ligand binding and mechanisms of action is critical for selecting, optimizing, and differentiating candidate molecules across the drug discovery workflow.
Cryo-electron microscopy (cryo-EM) supports multiple stages of the small molecule drug discovery workflow, from early structural characterization to lead optimization and candidate selection.
Overview of the small molecule drug discovery workflow, highlighting key stages from target identification and structure-based drug design to clinical development and commercialization.
At key stages within this workflow, cryo-EM enables high-resolution visualization of small molecule–protein complexes under near-native conditions, including targets that are difficult to study using traditional approaches such as X-ray crystallography. By directly revealing ligand binding modes, interaction networks, and conformational changes, cryo-EM provides actionable insights that support hit validation, structure-based drug design, and lead optimization.
Beyond structure-based design, advances in cryo-EM are expanding its role across the small molecule drug discovery workflow, including fragment-based screening and emerging modalities such as targeted protein degradation. This enables access to challenging targets such as membrane proteins and multi-protein complexes, supporting faster and more informed decision-making.
Ligand binding analysis and mechanism of action
Understanding how small molecules interact with their target proteins is central to small molecule drug discovery. Structural characterization of ligand–protein complexes provides detailed insights into binding modes, interaction geometry, and the molecular basis of specificity and affinity.
Cryo-EM enables high-resolution visualization of small molecule–protein complexes under near-native conditions, including systems that are difficult to study using traditional structural biology methods. This allows analysis of both ligand-bound and unbound states, supporting the identification of conformational changes and interaction patterns.
These structural insights are critical for understanding mechanism of action (MoA) and provide a foundation for downstream applications such as structure-based drug design and lead optimization.
Visualization of a protein–ligand complex showing a peptide–small molecule conjugate bound within the target binding pocket, illustrating ligand binding interactions and structural context. Image adapted from Muratspahić et al., 2023.
Structure-based drug design (SBDD) in small molecule drug discovery
Structure-based drug design (SBDD) uses high-resolution structural information to guide the design and optimization of small molecule therapeutics. Within small molecule drug discovery, integrating structural data with medicinal and computational chemistry approaches enables researchers to refine compound properties such as binding interactions, specificity, and overall molecular behavior.
Cryo-EM has expanded the applicability of SBDD by enabling structural characterization of challenging targets that are difficult to study using traditional methods, including membrane proteins such as GPCRs, transporters, ion channels, and multi-protein complexes. Because cryo-EM does not require crystallization, it provides access to previously intractable systems and enables structure-based design across a broader range of targets.
This expanded access to structurally complex targets enhances the impact of structure-based drug design across the drug discovery workflow. Advances in automation and data acquisition are improving throughput, enabling more efficient structural characterization within drug discovery workflows. This supports the generation of structural data across multiple compounds and conditions, contributing to iterative design and optimization processes.
For example, a high-throughput single-particle cryo-EM workflow developed in collaboration with Carrick Therapeutics, Imperial College London, and the Institute of Cancer Research enabled structural analysis of the CDK-activating kinase in complex with multiple inhibitors. Within approximately one hour of data acquisition, drug–protein interactions were resolved at ~3.5–4.5 Å resolution, with extended acquisition supporting higher-resolution structures.
Cryo-EM workflow for evaluating small molecule compounds, enabling rapid structural characterization of drug–target complexes from screening to high-resolution structure determination.
These structural insights support detailed understanding of target–inhibitor interactions, including water-mediated networks within the active site, supporting rational drug design and the development of next-generation therapeutics.
Cryo-EM for fragment-based drug discovery (FBDD) in small molecule drug discovery
In addition to structure-based drug design, fragment-based drug discovery (FBDD) is widely used to identify and optimize small molecule ligands.
Fragment-based drug discovery (FBDD) identifies and optimizes small molecule ligands by screening libraries of low-molecular-weight compounds. These fragments typically exhibit weak binding affinities and require sensitive methods to detect and characterize their interactions with target proteins.
Structural characterization of fragment–protein complexes is a critical step in FBDD. It enables the identification of binding modes and guides the development of higher-affinity lead compounds. While X-ray crystallography has traditionally been used for this purpose, it can be limited by the need for well-diffracting crystals and challenges associated with certain target classes.
Cryo-EM provides a powerful approach for structural analysis of fragment–protein complexes, particularly for targets that are difficult to crystallize, such as membrane proteins and multi-protein assemblies. By enabling direct visualization of ligand densities and interaction patterns, cryo-EM supports the identification and validation of fragment binding in structurally complex systems.
For example, a single-particle cryo-EM workflow established at Astex Pharmaceuticals demonstrated the feasibility of fragment screening for multimeric protein targets. At resolutions below 2.5 Å, ligand densities were clearly resolved, enabling accurate modeling of fragment binding and supporting subsequent lead optimization.
Cryo-EM structures showing fragment ligand densities bound to protein targets, supporting identification of binding interactions and guiding fragment-based drug discovery. Image adapted from Saur et al., 2020.
Targeted protein degradation in small molecule drug discovery
Beyond traditional ligand binding approaches, targeted protein degradation (TPD) is an emerging strategy in small molecule drug discovery for eliminating disease-relevant proteins. Many targets, such as transcription factors, RNA-binding proteins, and multi-protein complexes, lack well-defined binding sites and are therefore difficult to address using conventional small molecule inhibitors.
Two major classes of degrader molecules are widely used: proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs). PROTACs are heterobifunctional molecules that bring a target protein into proximity with an E3 ubiquitin ligase, enabling ubiquitination and proteasomal degradation. In contrast, molecular glue degraders stabilize interactions between a target protein and an E3 ligase without requiring a linker.
Cryo-EM enables structural characterization of protein–protein interactions by resolving ternary complexes formed between the target protein, degrader molecule, and E3 ligase. This provides direct insight into binding interfaces, conformational changes, and degradation mechanisms without the need for crystallization.
Cryo-EM also provides detailed insight into molecular glue degraders by revealing how these molecules stabilize specific protein–protein interactions. Structural studies show how small molecules promote interactions between target proteins and E3 ligases, enabling degradation and supporting understanding of mechanism of action.
Cryo-EM structure showing ternary complex formation between a target protein, degrader molecule, and an E3 ubiquitin ligase, enabling targeted protein degradation. Image adapted from Radhakrishnan et al., 2022.
For example, structural analysis of DCAF15–RBM39 complexes using cryo-EM has revealed how molecular glue degraders induce target degradation, demonstrating the value of cryo-EM in guiding rational design of targeted protein degradation therapies.
In addition to human therapeutic systems, cryo-EM has also been used to study bacterial targeted protein degradation strategies, including emerging approaches such as BacPROTACs, highlighting its broader applicability.
Cryo-EM structure of a molecular glue degrader bound to an E3 ligase complex, revealing how small molecules stabilize protein–protein interactions to enable targeted degradation. Structure recreated from PDB 6SJ7.
Electron microscopy for small molecule drug formulation and characterization
Downstream in the small molecule drug discovery workflow, formulation plays a critical role in ensuring the safe and effective delivery of active pharmaceutical ingredients (APIs). Understanding formulation structure and composition is essential for optimizing stability, performance, and manufacturability.
Electron microscopy (EM) techniques, including scanning electron microscopy (SEM) and focused ion beam scanning electron microscopy (FIB-SEM), provide high-resolution imaging of particle morphology, surface structure, and internal organization. These approaches enable detailed characterization of key formulation attributes such as particle size distribution, porosity, and the spatial distribution of APIs.
By providing structural and compositional insights, electron microscopy supports formulation development, quality control, and optimization of drug delivery systems. These capabilities complement upstream structural biology approaches, including cryo-EM, by extending structural analysis from target identification and drug design through to final formulation and characterization.
SEM and FIB-SEM imaging of pharmaceutical microparticles, revealing particle morphology, surface architecture, and internal structure for drug formulation characterization. Image adapted from Janich et al., 2019.
Cryo-EM instruments
Electron microscopy instruments support small molecule drug discovery across the full workflow, from high-resolution structure determination of drug–target complexes to downstream formulation characterization. By combining cryo-EM with complementary room-temperature electron microscopy techniques, researchers can generate structural insights across multiple stages of drug development.
Cryo-EM systems are designed to support different stages of structural analysis, from screening to high-resolution structure determination. These systems support structure-based drug design by enabling high-resolution analysis of protein–ligand complexes, including challenging targets.
Glacios 3 and Krios 5 Cryo-TEMs supporting cryo-EM workflows from screening to high-resolution structure determination.
Complementary techniques, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), and focused ion beam scanning electron microscopy (FIB-SEM), provide detailed characterization of drug formulations and related materials. Together, these technologies enable a continuous workflow from early discovery through structural analysis and downstream formulation development.
Phenom Desktop SEM and Apreo ChemiSEM System for rapid imaging and compositional analysis of formulations and materials.
Resources
Explore selected resources on cryo-electron microscopy applications in small molecule drug discovery, including structure-based drug design, fragment screening, and targeted protein degradation.
References
- Cushing, V. I., Koh, A., Feng, J., et al. High-resolution cryo-electron microscopy of the human CDK-activating kinase for structure-based drug design. bioRxiv (2023). https://doi.org/10.1101/2023.04.07.536029
- Saur, M., Hartshorn, M. J., Dong, J., Reeks, J., et al. Fragment-based drug discovery using cryo-EM. Drug Discovery Today 25(3) (2020): 485–490. https://doi.org/10.1016/j.drudis.2019.12.006
- Radhakrishnan, S., Hoff, O., Muellner, M. K. Current challenges in small molecule proximity-inducing compound development for targeted protein degradation. Molecules 27 (2022): 8119. https://doi.org/10.3390/molecules27238119
- Morreale, F., Kleine, S., Leodolter, J., et al. BacPROTACs mediate targeted protein degradation in bacteria. Cell 185(13) (2022). https://doi.org/10.1016/j.cell.2022.05.009
- Bussiere, D. E., Xie, L., Srinivas, H., et al. Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex. Nature Chemical Biology 16 (2020): 15–23. https://doi.org/10.1038/s41589-019-0411-6
- Janich, C., Friedmann, A., Souza e Silva, J. M., et al. Risperidone-loaded PLGA–lipid particles with improved release kinetics: Manufacturing and detailed characterization by electron microscopy and nano-CT. Pharmaceutics 11(12) (2019): 665. https://doi.org/10.3390/pharmaceutics11120665
- Muratspahić, E., Deibler, K., Han, J., et al. Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor. Nature Communications 14(1) (2023): 8064. https://doi.org/10.1038/s41467-023-43718-w
Frequently asked questions
Cryo-EM enables high-resolution visualization of protein–ligand complexes, providing structural insights that support drug design, fragment screening, and targeted protein degradation.
Yes. Cryo-EM can visualize small molecule binding within protein complexes, enabling analysis of binding modes and mechanism of action.
FBDD identifies small, low-molecular-weight compounds (fragments) that bind weakly to target proteins, and it optimizes them into higher-affinity drug candidates.
Cryo-EM enables visualization of fragment binding at high resolution, supporting identification of binding modes and optimization of lead compounds.
PROTACs are molecules that recruit a target protein to an E3 ligase for degradation, while molecular glues stabilize protein–protein interactions to induce degradation.
Cryo-EM resolves ternary complexes and protein–protein interactions, providing insight into degradation mechanisms and guiding degrader design.
Cryo-EM is particularly useful for challenging targets such as membrane proteins, multi-protein complexes, and flexible systems.
Electron microscopy techniques such as SEM and FIB-SEM characterize particle morphology, structure, and composition to support formulation development and quality control.
The appropriate instrument(s) depends on the stage of the workflow and required resolution, from cryo-EM for structural biology to SEM and FIB-SEM for formulation and materials characterization.
For Research Use Only. Not for use in diagnostic procedures.