Cryo-electron microscopy (cryo-EM) makes it possible to image important proteins that were once inaccessible at high resolution using other methods. In recent years, an increasing number of pharmaceutical researchers have been turning to this method to gain insights into the structure and function of a wide range of small molecules in complex with their drug targets, paving the way toward the development of safer and more effective drugs.
One promising area of research encompasses G protein-coupled receptors (GPCRs) found in the cell membranes of humans and other organisms. These proteins function like gatekeepers, opening or closing the door to control the traffic of molecules that can enter the cell. This function of detecting molecules outside the cell and activating cellular responses makes them ideal drug targets.
Until recently, researchers thought GPCRs worked like simple switches, turning pathways “on” or “off.” However, using techniques like cryo-EM, scientists have discovered they actually react to different stimuli and demonstrate a range of responses. By uncovering the mechanisms behind how GPCRs work, pharmaceutical companies can advance their understanding of the protein binding receptor interface in GPCR complexes. This, in turn, will enable them to develop more effective drugs with fewer side effects for chronic diseases including diabetes, asthma, Parkinson’s Disease, schizophrenia, depression and various types of cancers.
Currently, GPCRs are targets of approximately 35 percent of drugs approved by the U.S. Food and Drug Administration, accounting for $180 billion in global drug sales. Yet, so far, drugs have only been developed against less than 13 percent of the approximately 800 potentially useful therapeutic GPCR modalities expressed in humans.
Until recently, X-ray crystallography was the standard method for small molecule structure-based drug design. While this technique has revolutionized our understanding of many proteins, most GPCRs cannot be crystallized, thus preventing their structure determination using this method. In other cases, the movement of certain parts of some GPCRs meant they could only be imaged at low resolution, making it impossible to see which part of the protein was being attacked or bonding to molecules coming from outside the cell.
Cryo-EM solves these two problems by vitrifying GPCRs in their near-native state. Once the GPCR’s moving parts are stabilized, they can be imaged at high resolution. Today, cryo-EM is the only method that can yield the detailed structure of many GPCRs and the bonding of drug candidates to these proteins on a routine basis. Many pharmaceutical researchers have generated breakthrough results by turning to this technique.
According to a review of the impact of GPCR structures on drug development written by researchers at Sosei Heptares, cryo-EM is driving structure-based drug design by making it easier and faster to resolve high-resolution GPCR structures. “Since 2017, more structures of GPCRs in the active state coupled to [other] proteins have been determined by cryo-EM than by X-ray crystallography,” the researchers wrote. “This is because cryo-EM can now determine structures of smaller proteins than previously, due to developments in direct electron detectors and computer programs for structure determination.”
Patrick Sexton, of Monash University, has published 10 GPCR structures in the last eight months. He is now in the process of developing a “chemical toolbox,” which will facilitate additional understanding of GPCR signaling pathways.
The following examples provide a glimpse into recent revelations in GPCR research:
- Researchers at Monash University used cryo-EM to determine two integral structures in the GLP-1 receptor in complex with agonists, proving that cryo-EM can explain the different effects of compounds on multiple GPCR signaling pathways. GLP-1 promotes the release of insulin and is helpful in the search to create a pill, rather than an injection, to treat type II diabetes. Researchers achieved a resolution of 2.5A and 2.1A for the PF-06882961 (Pfizer) and OWL-833 (Chugai/Eli Lilly) compounds, respectively, setting a new cryo-EM record. They provided proof that GPCR structures, previously thought impossible to solve, can be routinely visualized at high resolution with cryo-EM. They showed, with mechanistic insight, how PF-06882961 mimics the pharmacology of GLP-1 while OWL-833 does not. Cryo-EM provides structural insights that increase the likelihood of creating more effective, specific drug candidates.
- Researchers at Roche used cryo-EM to uncover the structure of the rhodopsin-Gi-Fab16 complex. The density map revealed the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of the Gβ as scaffolding for recruiting Gα subunits and G protein-receptor kinases. By comparing the complexes, the researchers found a small set of common anchoring points that are G protein-subtype specific. Taken together, their analysis provides a new structural basis for the molecular events of the GPCR signaling pathway.
Cryo-EM structure of the rhodopsin-Gi-Fab16 complex
- Researchers at Roche also used cryo-EM to recognize a mode of antibody that stabilizes G-protein subtypes with minimal protein engineering. Using cryo-EM, the researchers were able to describe how the antibody fragment mAb16 stabilizes GPCR/G-protein complexes and is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.
- Researchers at Eli Lilly used cryo-EM to discover a GPCR ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a “molecular glue.” Cryo-EM determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The researchers’ findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.
These are just a few examples of the many pharma companies, big and small, involved in development of novel drugs based on GPCRs due to their therapeutic effectiveness and fewer side effects. Stay tuned for our upcoming blogs that will showcase how cryo-EM is used in the pharmaceutical industry for structure-based drug design of small molecules and biologics!
Register for our virtual event on September 29 where Patrick Sexton, of Monash University, will elaborate on his GPCR research, including his recent publication on the GLP-1 receptor.
Visit our Cryo-EM in the Pharmaceutical Industry webpage to learn more about how other pharmaceutical companies are using cryo-EM to accelerate their drug discovery.
To find out how you can incorporate cryo-EM into your pharmaceutical research, please see our Bio-pharmaceutical Research with Electron Microscopy page.
Aleksander Stefanovic is a Senior Market Development Manager at Thermo Fisher Scientific.
Speak with an expert: https://www.thermofisher.com/blog/microscopy/speak-with-an-expert/