New findings on serine hydroxymethyltransferase help in the development of cancer drugs

In just two neutron experiments, scientists discovered remarkable details about the function of an enzyme that may help develop drugs against aggressive cancers.

The scientists, working at the Department of Energy's Oak Ridge National Laboratory, used neutrons at the spallation neutron source and high-flux isotope reactor to identify the precise atomic-scale chemistry in serine hydroxymethyltransferase (SHMT), a metabolic enzyme necessary for cell division.

Cancer hijacks chemical reactions in the metabolic pathway involving SHMT and other critical enzymes, turning the entire process into a runaway train that rapidly multiplies cancer cells. Developing an inhibitor to block the enzyme's function, which occurs early in the pathway, could thwart the cancer's attempts to outrun it. The Royal Society of Chemistry published the team's results in CHemic science.

“I think neutrons will be in high demand in future structure-based drug development,” said ORNL’s Victoria Drago, the lead author and a biochemist who works with Andrey Kovalevsky, a distinguished research and development scientist at ORNL who uses neutron diffraction to illuminate protein structures . “This article is a good example of how quickly neutrons can produce information that has long been the subject of debate. Studies on SHMT function and its catalytic mechanism date back to the early 1980s.”

The precise catalytic mechanism and the role of various amino acid residues in the active site of the enzyme have been debated for decades. In the current study, researchers observed that only one amino acid residue, a glutamate, regulates the chemical reactions of this enzyme.

The neutron data clearly show that glutamate, an acid, carries the proton. One might expect that it has already released its proton. But because it can carry the proton around with it, it can transport it back and forth. So it acts as an acid and a base.”

Robert Phillips, co-author, Professor of Chemistry, University of Georgia

In a pathway known as one-carbon metabolism, this enzyme works in a cell's mitochondria, or energy producers. It converts the amino acid serine into another amino acid called glycine by transferring a carbon atom to tetrahydrofolate, a reduced form of folic acid. This reaction creates building blocks for the synthesis of nucleic acids such as DNA and RNA, as well as other biological molecules that are important for cell division. Glutamate controls this process.

In a previous experiment, the team combined two techniques, neutron and X-ray crystallography at physiologically relevant room temperature, to understand SHMT and image its protein structure before its interaction with tetrahydrofolate. In the current experiment, the researchers managed to capture the enzyme in the next step and thus gain certainty about how the enzyme's reaction mechanism actually works.

Paint the picture with neutrons

Neutrons see light elements like hydrogen and X-rays see heavier elements like carbon, nitrogen and oxygen. Neutron diffraction at SNS and HFIR, internal X-ray diffraction at ORNL, and synchrotron X-ray diffraction at Argonne National Laboratory's Advanced Photon Source provided insights the team needed to definitively characterize the enzyme's chemical reaction.

“Neutrons allow us to see hydrogen atoms, and hydrogen powers chemistry,” Drago said. “Enzymes consist of about 50% hydrogen atoms. In terms of electrostatics, hydrogen also carries a positive charge that determines the environment of the enzyme. Once you have a crystal that diffracts neutrons, you have everything you need. You can see the positions where hydrogens are and, equally important, the positions where hydrogens are missing.

As shown in the animation, the mitochondria of cancer cells overproduce the SHMT enzyme, a tetramer composed of four identical peptide chains, or protomers (shown in gray). SHMT works by using pyridoxal-5′-phosphate, which is covalently bound to SHMT, and tetrahydrofolate, shown in gold and purple, respectively. Tetrahydrofolate acts as a substrate that binds to the active sites of all four protomers. The green flashing hydrogen atoms revealed the precise catalytic mechanism and the role of various amino acid residues in the enzyme's active sites. Once the enzyme releases tetrahydrofolate, an inhibitor shown in blue could be developed to block further chemical reactions at these sites, stopping the one-carbon pathway in cancer cells.

“The positions of the hydrogen atoms determine the protonation states of certain chemical groups within the active sites of the enzyme,” Kovalevsky said. “They therefore provide information about the electrical charge distribution or electrostatics. This knowledge is crucial for the development of small molecule inhibitors that would bind to SHMT, replace tetrahydrofolate and stop enzyme function.”

Cells contain thousands of enzymes that act as catalysts, speeding up biochemical reactions necessary for body functions. from breathing to the production of hormones to nerve function. Enzymes also provide a place to store chemicals that target specific processes. Other enzymes in the one-carbon pathway are already known targets for cancer drugs, such as methotrexate and fluorouracil. However, SHMT comes earlier this way and offers the possibility of stopping cancer sooner.

However, the difficulties in treating cancer are partly related to its stealthy attacks on metabolic processes. Unlike drug resistance in infectious diseases, when one pathway is not functioning well, cancer causes other metabolic processes to be recalibrated to cause overproduction of cancer cells.

“Now that we know the atomic details of SHMT, we can influence the development of an inhibitor that targets this specific protein as part of a combination therapy,” Kovalevsky said. “If you compare it to the treatment of infectious diseases, it is much more difficult because in cancer chemotherapy you usually target your own proteins, which is why patients experience side effects. In infectious diseases, the proteins targeted are viruses or bacteria.” . But with cancer, you have to kill your own cells to kill the cancer sooner and have less impact on the patient.

Accelerate the pace of discovery

The team used neutrons on the MaNDi instrument at SNS and the IMAGINE instrument at HFIR for their research. ORNL's recent Proton Power Upgrade Project added stronger beams for all instruments at SNS. Stronger proton beams mean more neutrons. More neutrons mean faster data collection times on smaller samples, accelerating answers that help scientists develop smarter drugs to treat diseases.

“Discovery research is absolutely essential,” said William Nelson, director of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins. “We are getting closer and closer to the point where, using AI, we will be able to sequence a gene in a person's cancer, predict what the protein structure would be, and create a drug to deliver it; “This will work great.”, and we'll do it in an hour and a half. But we're not there yet. So the more we know about actual protein structure, chemical structure, and the way things interact, the better able we will be to train AI models to predict things we don't know right away .”

Nelson was not an author on any of the ORNL-led studies. As director of the Sidney Kimmel Comprehensive Cancer Center and professor at the Johns Hopkins School of Medicine, he teaches urology, medicine, pathology, and radiation oncology and molecular radiation sciences.

SNS and HFIR are user facilities of the DOE Office of Science at ORNL.