Small Molecule | Structure-Based Drug Design | Cryo-EM in Drug Discovery

Small molecule drug discovery and the importance of cryo-EM

The growth of structural biology and pharmaceutical innovation is accelerating the adoption of cryo-electron microscopy, or cryo-EM, for structure-based drug design. Beginning in the 1980s and accelerating in the years since, the use of protein structure information in small molecule drug discovery has significantly reduced the time and cost of bringing molecules to the clinic. The most well-known successes of structure-based drug design (SBDD) efforts are the first HIV/AIDS protease inhibitors, which were quickly followed by numerous cancer treatments targeting a range of molecular targets. Despite the growth of biologics modalities, small molecule therapeutics continue to be high-value drug assets and compose the majority of new drug approvals by the US Food and Drug Administration (FDA), Pharmaceuticals and Medical Devices Agency (PMDA), the Korean Ministry of Food and Drug Safety (MFDS), and other regulatory bodies.

Today, SBDD and fragment-based drug discovery (FBDD) approaches have been greatly expanded to new targets and new small molecule modalities thanks to the “resolution revolution” in cryogenic electron microscopy (cryo-EM).

Structure-based drug design

In a structure-based drug design (SBDD) approach, determining the three-dimensional structure of a ligand bound to its target protein makes it possible to define and optimize suitable small molecule ligands. Medicinal and computation chemists, together with structural biologists, leverage structures derived from either X-ray crystallography or cryo-EM to improve the drug-target interaction by modifying the shape, size, and charge of drug molecules while also identifying areas where ADME profiles can be improved.

Small molecule drug discovery timeline from therapy concept to final drug.

Cryo-EM workflows have dramatically expanded the protein space accessible to structure-based methods, becoming the gold standard for membrane protein structure determination (e.g., GPCRs, transporters, and channels) and multi-protein complexes. Because single-particle cryo-EM can reveal high-resolution structures of non-crystallizable target proteins, such as membrane proteins and multi-protein complexes, more and more small molecule drugs have been and will be generated due to the advances in workflow automation for high-throughput SBDD using cryo-EM.

Thermo Fisher Scientific worked with Carrick Therapeutics, the Imperial College London, and the Institute of Cancer Research to develop and publish a high-throughput single particle cryo-EM workflow using the cancer target CDK-activating kinase. With only one hour of data acquisition, it was possible to elucidate drug-protein interactions at resolutions of about 3.5 to 4.5 Å resolution. Extended data acquisition subsequently supported structure determination at atomic resolution up to 1.8 Å.

Workflow used for evaluating CDK-targeting compounds. Rapid sample optimization with the Thermo Scientific Glacios Cryo-TEM is followed by high-resolution data collection on a Thermo Scientific Glacios or Krios Cryo-TEM.

These structures provided detailed insights into target-inhibitor interactions and form the basis for the rational design of next-generation cancer therapeutics. This approach demonstrates how automated cryo-EM workflows can accelerate structure-based drug design in research environments that demand high throughput and reproducibility.

Fragment-based drug discovery

Fragment-based drug discovery (FBDD) identifies small chemical fragments that bind weakly to target proteins and uses them as starting points for designing potent lead compounds. Traditionally, X-ray crystallography has been central to this approach, but its limitations with membrane proteins and large complexes have made cryo-EM an increasingly powerful complementary method. Biophysical methods such as SPR or ITC can pre-screen fragments before structure determination using cryo-EM.

Building on these advances, Astex Pharmaceuticals established a FBDD single-particle cryo-EM workflow and created a proof of concept by screening fragments for challenging protein targets. At an atomic resolution below 2.5 Å, all ligand densities can be clearly identified and used as building blocks for lead compound generation and optimization.

Figure from Saur, et. al, under a creative commons license.

Targeted protein degradation

Only 20% of target proteins are considered druggable with small molecules. Targeted protein degradation (TPD) provides an alternative therapeutic approach to access previously undruggable targets. Heterobifunctional protein degraders (PROTACs) and molecular glue degraders (MGDs) use small molecules to bring target proteins into proximity with E3 ubiquitin ligase, leading to selective degradation.

Targeted protein degradation through proximity-inducing compounds. A bivalent molecular degrader forms a ternary complex with an E3 ligase (pink) and a target protein (blue) which leads to attachment of ubiquitin chains (yellow) and subsequent proteasomal degradation. Figure from Radhakrishnan et. al., used under Creative Commons license.

Novartis used single particle cryo-EM to elucidate the structure of the anticancer degrader indisulam bound to its target-ligase complex, revealing its mechanism of action and demonstrating the value of cryo-EM in rational design of novel degraders.

Structure of the human DDB1-DDA1-DCAF15 E3 ubiquitin ligase bound to RBM39 and indisulam. Structure recreated from PDB 6SJ7.

Cryo-EM instruments

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