A detailed understanding of human proteins is often key to designing drugs that target them. For decades, drug developers have typically relied on X-ray crystallography for this purpose, a technique that captures the three-dimensional structure of protein crystals by exposing them to X-rays and analyzing the resulting diffraction patterns. This reveals protein structures in unprecedented detail, sometimes with an "atomic resolution" of at least 1.2 Å, high enough to discern individual atoms.
X-ray crystallography is crucial for structure-based drug design, in which drugs are designed based on the chemical structure of the protein of interest. For example, detailed studies of the protease used by the human immunodeficiency virus (HIV) have led to the development of enzyme-inhibiting drugs for the treatment of HIV. But the technique has a major drawback. It requires proteins to be in a crystalline state, which means going through certain physical processes to force them into an ordered lattice, a cumbersome process that doesn't work for all proteins.
That's where cryo-electron microscopy (cryo-EM), a new technique that doesn't require crystallization, comes into play. Instead, protein samples in solution are simply snap-frozen and bombarded with electrons; images are produced from patterns of electrons projected onto detectors. Still, the technique has long been derided as "hematology" because of the relatively low-resolution images it captures. Until the late 1990s, these images had a resolution of about 20 angstroms and could only show the general shape of the protein.
Fortunately, persistent scientists have managed to improve the resolution of cryo-EM over time. The development of better electronic detection hardware and efficient algorithms for constructing 3D structures from multiple protein images finally sparked a "resolution revolution" in cryo-EM around 2015.1In 2020, researchers obtained the impressive 1.25 Å structure of apoferritin, demonstrating that cryo-EM can observe proteins in atomic detail, at least in some cases.2In awarding the 2017 Nobel Prize in Chemistry to scientists Jacques Dubochet, Joachim Frank and Richard Henderson for their work developing cryo-electron microscopy, the Royal Swedish Academy of Sciences said the technique "brought biochemistry into a new era".3
Among drug developers, cryo-EM has quickly become a popular tool for analyzing the structure of target proteins, especially those of large proteins (such as the membrane-bound proteins that make up most drug targets. Modern drugs), which are difficult to analyze. Solved by X-ray crystallography. Cryo-EM has not only expanded the range of proteins that can be targeted by structural drug design, but has also become a key imaging tool in drug development.createChatted with various entities at the forefront of this cryo-EM movement—microscope manufacturers, cryo-EM service companies, drug manufacturers, and data storage experts—to learn more about how cryo-EM is reshaping drug discovery .
Application of cryo-EM in Drug Structure Design
Nanoimaging Services is a San Diego, CA-based contract research organization that has been performing cryo-electron microscopy exclusively for pharmaceutical and biotechnology companies since its founding in 2001. Initially, Nanoimaging Services used the technique to image drug delivery vehicles such as nanoparticles to ensure that each batch contained intact particles of the same size, said Dr. Giovanna Scapin, the company's chief scientific officer.4
Higher resolutions are now possible, and one of the company's priorities is determining the structure of proteins that drugmakers are targeting. The goal is often to examine how lead candidates bind to proteins during or after initial drug design to better understand how the drug and target interact.5
Scapin has found that there is a high demand for cryo-EM structures of membrane proteins and protein complexes, which are often too large, too flexible, or too few in number to crystallize. Another advantage of cryo-EM, he notes, is that it doesn't require proteins to crystallize for specific confirmation. "We can observe the protein in its true natural state," he emphasizes.
The average resolution of cryo-EM structures is in the range of 2 to 2.5 Å. Scapin acknowledges that this is lower than the typical resolution of X-ray crystallography, but adds that computer modeling can be used to refine atomic models of proteins. However, the biggest challenge of cryo-EM lies in the preparation of protein samples. Often it is necessary to attach targets to other molecules. For example, a very flexible protein might need to be stabilized, or a protein too small to be clearly visualized alone might need to be part of a larger protein. In principle, cryo-EM "can be used for anything," says Scapin, "[but] we have to make proteins work for cryo-EM."
Protein Complex Analysis
Dr. Lei Jin, COO of Wuxi Biortus Biosciences, a contract research organization focused on protein structure analysis, agrees that sample preparation remains a challenge. However, the main advantage of cryo-EM is its speed, he points out. For many popular drug targets, especially large proteins and protein complexes, "crystallography can take years to obtain a structure," he explained.
Many of these proteins have been taken up by cryo-EM, according to scientists at Wuxi Biortus. These include, for example, cyclin-dependent kinase 7 (CDK7), an enzyme involved in cell cycle regulation and a popular target in oncology. The scientists mapped, at 2.5 Å resolution, the large complex that CDK7 forms with two other proteins, as well as a small molecule drug designed to inhibit CDK7.6These studies "can tell us a lot about the nature of drug-target interactions," Jin said. "We can even use this information to further optimize small molecules."
Other cryo-EM themes include proteolytically directed chimeras (PROTACs), large complexes that exploit the body's own proteolytic machinery to destroy disease-causing proteins that are difficult to address with small-molecule drugs. Imaging such complexes using cryo-EM can help optimize PROTAC molecules, for example, by making them more specifically target proteins of interest. Cryo-EM research can also provide unique insights into how known drugs work against new targets, such as the drug remdesivir, which has been used against the novel coronavirus SARS-CoV-2.7
As Jin sees it, the only major limitation of cryo-EM is that the protein under study must be larger than 80 kDa (or contain more than 700 amino acids) to reliably determine its structure, although Jin adds that the Biortus scientists managed to lower the limit to 70 kDa. X-ray crystallography "probably does a better job" if the protein is smaller than that, he notes.
Cryo-EM Meets Fragment-Based Drug Design
Cambridge, UK-based Astex Pharmaceuticals began experimenting with cryo-electron microscopy in 2016. The Medical Research Council Laboratory of Molecular Biology in the UK collaborated with FEI, a microscope manufacturer based in Hillsborough, Oregon. Astex and the University of Cambridge Center for Nanoscience have been working to help pharmaceutical companies explore the use of cryo-EM for early drug discovery research.8
"At first, it was 'very challenging' to learn the technology," said Pamela Williams, Ph.D., senior director of molecular science at Astex. This is especially true because she and her colleagues are used to high-throughput, automated workflow, while X-ray has been developed for crystallography for many years. "[The cryo-electron microscope] is an incredible technical device," he added.
In 2020, Williams and his colleagues conducted a proof-of-concept study with encouraging results. This study demonstrates the utility of cryo-electron microscopy for the design of specific drugs based on Astex fragments.9This approach looks for small fragments and studies how they bind to proteins individually; this fragment can be iteratively expanded to build a molecule that binds the protein efficiently. This study demonstrates that cryo-EM can be used to study molecular interactions between small fragments and proteins, in this case the popular oncology target pyruvate kinase 2, at a resolution comparable to that achieved by crystallography .
Astex isn't pursuing that goal, but the company is using cryo-electron microscopy to apply fragment-based drug design to membrane protein targets, such as those involved in central nervous system disorders. According to Williams, cryo-EM could be combined with X-ray crystallography, for example, in the design of drugs that interfere with the binding event between two proteins. Cryo-EM can be used to image protein complexes, while subsequent X-ray crystallography can be used to examine each individual protein in detail. "These two technologies," he insisted, "have the potential to work together."
Cryo-EM Data Storage Troubleshooting
Dr. Björn Kolbeck, CEO of Quobyte, a data warehouse specialist in Santa Clara, Calif., has seen in recent years that many areas of the life sciences have begun to generate massive amounts of data, especially cryo-EM data. . Because cryo-EM is used to collect high-resolution images of many proteins to enable good 3D reconstructions of proteins, the amount of data, Kolbeck points out, is "far greater than other imaging techniques in the life sciences."
In this case, it is crucial to have a robust and efficient data storage system that can accommodate the volume of data generated, allowing continuous operation of the microscope and fast image processing. But storage is often an afterthought for scientists working with cryo-EM, Kolbeck points out. Many people store their data on separate servers, creating a single point of failure if the system fails. Smaller pharmaceutical and biotech companies typically store data in the cloud first. According to Kolbeck, this approach can quickly become prohibitively expensive and doesn't work well when data must be transferred from field devices such as microscopes.
That's where Quobyte's software comes into play.10The system essentially aggregates data from multiple servers into one large storage system. This allows more servers to be added as needed, and the storage is still available if one server fails or needs service.
Since the company started selling its cryo-EM software two years ago, it has drawn interest from academic groups and drug developers. Kolbeck predicts that as cryo-EM technology continues to advance, the need for data storage will grow. "From a storage standpoint," he said, "[the situation] is only going to get worse."
The Future of Cryo-EM
Dr. Melanie Adams-Cioaba, senior director and general manager of pharmaceuticals at Thermo Fisher Scientific, said cryo-EM technology has indeed come a long way. Headquartered in Waltham, Massachusetts, the company offers a variety of laboratory instruments and supplies, including cryo-electron microscopes. The company has been selling cryo-EM microscopes since its 2016 acquisition of FEI, an industry leader in electron microscope development. These instruments include the Titan Krios microscope, which in 2020 captured the legendary atomic-resolution images of apoferritin.
Adams-Cioaba noted that advances in cryo-EM technology have established the technique as the method of choice for difficult-to-crystallize targets, and cryo-EM applications are now rapidly expanding. One of the most popular applications of cryo-EM is the imaging of large, flexible biopharmaceuticals such as therapeutic antibodies. Cryo-EM could prove useful "anywhere we really want to understand protein structure, how proteins interact with other proteins, and how modifications to those proteins affect protein function," Adams-Cioaba added.11
For example, drug makers can use cryo-electron microscopy to help detect the multitude of antibodies they produce against a particular protein. This structural analysis facilitates the rapid classification of antibodies based on their structure and the selection of those with the greatest therapeutic promise. Historically, structural biology was rarely used in the early stages of antibody development. This is difficult in part because proteins do not crystallize easily. "But because cryo-EM is faster, the technique allows structural studies to guide antibody development earlier in the process."
Adams-Cioaba predicts that cryo-EM will continue to find new applications in drug development. "The combination of innovations in the field really continues to break down my cognitive barriers," he said. "[Cryo-EM's capabilities] are only limited by our persistence and our imagination."
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