The American Cancer Society estimates there will be about 20,050 new cases of acute myeloid leukemia (AML) in the United States in 2022. Stanford School of Medicine professor Kathleen Sakamoto, MD, PhD, has been working on the development of therapeutics against AML and other blood disorders. Her team, however, was hampered in their search by a subtle gap between two technologies, X-ray crystallography and cryogenic electron microscopy.

Their findings are published in the journal ACS Central Science in a paper titled, “Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia.”

“Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases,” the researchers wrote. “However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein that has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell.”

Researchers at Stanford University’s Schools of Medicine and Engineering and the Department of Energy’s SLAC National Accelerator Laboratory have found a way to bridge that gap by using a kind of molecular cage to stabilize certain medium-sized proteins so they can be imaged for the first time with cryo-EM, which can reveal almost atomic-level details.

The solution came to the researchers when they would sandwich batches of KIX proteins between a central, ball-shaped molecule and an outer molecular cage. Because this “double shell” was much larger than individual KIX molecules, it would be easier to spot and orient in cryo-EM images, and that would make it easier to get high-resolution images of the KIX molecules themselves. In addition to seeing KIX’s structure, the researchers were able to add other molecules to the mix to see if they might bind to and potentially inhibit KIX’s function.

The team’s results also suggest this method could prove useful for other proteins of in-between sizes that are hard to study with either cryo-EM or X-ray crystallography, including, perhaps, some viral proteins. “We are moving forward to expand the applicability of the approach,” explained Soichi Wakatsuki, PhD, SLAC and Stanford professor.