Scientists from the Case Western Reserve University School of Medicine report that using a special type of electron microscope with samples cooled to extremely cold temperatures provides critical information for drug developers seeking to reduce nausea and vomiting side effects of cancer treatments. Published in Nature Communications, the study (“Molecular mechanism of setron-mediated inhibition of full-length 5-HT3A receptor“) offers a glimpse into how widely-used anti-nausea drugs attach to their target protein in the gastrointestinal tract. High-resolution images obtained by this method provide details about how the drugs attach into a binding pocket on the protein and offer clues into how their design might be improved.

“Serotonin receptor (5-HT3AR) is the most common therapeutic target to manage the nausea and vomiting during cancer therapies and in the treatment of irritable bowel syndrome. Setrons, a class of competitive antagonists, cause functional inhibition of 5-HT3AR in the gastrointestinal tract and brainstem, acting as effective anti-emetic agents. Despite their prevalent use, the molecular mechanisms underlying setron binding and inhibition of 5-HT3AR are not fully understood,” the investigators wrote.

“Here, we present the structure of granisetron-bound full-length 5-HT3AR solved by single-particle cryo-electron microscopy to 2.92 Å resolution. The reconstruction reveals the orientation of granisetron in the orthosteric site with unambiguous density for interacting sidechains. Molecular dynamics simulations and electrophysiology confirm the granisetron binding orientation and the residues central for ligand recognition. Comparison of granisetron-bound 5-HT3AR with the apo and serotonin-bound structures, reveals key insights into the mechanism underlying 5-HT3AR inhibition.”

Setrons are generally well-tolerated, but some cancer patients do not respond to them, explained study lead Sudha Chakrapani, PhD, associate professor of physiology and biophysics at Case Western Reserve University School of Medicine.

“Cancer patients who have vomiting later in their treatment plans—delayed emesis—don’t tend to respond to setrons,” Chakrapani said. “There is a constant need for better drugs.” Drug improvement has been stalled by a lack of models showing exactly how drugs like setrons attach to their target protein in the body—the serotonin (3) receptor. Without a precise model, drug developers have been unable to understand exactly which elements of setron-receptor interactions are most important, and how to enhance them.

The new study provides the highest-resolution images to date of a setron settling inside the binding pocket of a serotonin (3) receptor. Researchers tracked the receptor-drug interactions, to less than a billionth of a meter using a cryo-electron microscope. Cryo-electron microscopy (cryo-EM) has only recently become available for small protein targets and was the focus of the 2017 Nobel prize in chemistry.

Cryo-EM images revealed setrons use the same attachment site as the receptor’s natural binding partner in the body, serotonin, but take a slightly different pose that changes the receptor shape slightly. The differences helped the researchers build a more precise model of how setrons work on a molecular level.

Sandip Basak, PhD, co-first author on the paper, noted that, “In the past, we didn’t have the confidence to model the drug in its binding pocket. Now we can precisely do that. We can also watch the drug move in the pocket using molecular dynamics simulations.”

Chakrapani collaborated with colleagues at Mt. Sinai to identify the most stable interactions between setrons and serotonin receptors. The team watched as setrons twisted and turned in the pocket, revealing key portions of the drug and the receptor that are required for a tight connection. They then mutated the key portions, which eliminated setrons’ affinity for the serotonin receptors. Together, the experiments helped reveal which portions of setrons and serotonin receptors are most important and might be most promising to enhance therapeutically.

“Identifying the binding pocket and the interactions that are most important, and the orientation of the drug in the binding pocket, lays the foundation for designing drugs that are going to be more efficient,” said Yvonne Gicheru, PhD, who is a co-first author on the paper.