Neutron experiments end 40-year debate on enzymes for drug development

In just two neutron experiments, scientists have discovered remarkable details about the function of an enzyme that may help in the development of drugs to combat aggressive cancers.

The scientists, working at the Department of Energy's Oak Ridge National Laboratory, used neutrons from the Spallation Neutron Source and the High Flux Isotope Reactor to precisely determine the chemical composition of serine hydroxymethyltransferase (SHMT), a metabolic enzyme necessary for cell division, at the atomic level.

Cancer hijacks chemical reactions in the metabolic pathway involving SHMT and other key enzymes, turning the entire process into a runaway train that rapidly reproduces cancer cells. Developing an inhibitor to block the function of the enzyme that occurs early in the pathway could thwart cancer's attempts to overtake it. The findings were published in Chemical Science.

“I think neutrons are going to be in high demand in future structure-based drug development,” said ORNL's Victoria Drago, lead author and a biochemist who works with Andrey Kovalevsky, a renowned R&D scientist at ORNL who uses neutron diffraction to illuminate protein structures.

“This paper is a good example of how quickly neutrons can produce information that has been the subject of debate for a very long time. Studies on SHMT function and its catalytic mechanism date back to the early 1980s.”

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

“The neutron data clearly show that the glutamate, which is an acid, is carrying the proton,” said co-author Robert Phillips, a professor of chemistry at the University of Georgia. “You might expect that it has already given up its proton. But because it's able to carry that proton around, it can transfer it back and forth. So it acts as both an acid and a base.”

This enzyme works in a process called one-carbon metabolism in the mitochondria of a cell, the 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 produces building blocks for the synthesis of nucleic acids such as DNA and RNA and other biological molecules that are crucial 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 map its protein structure before its interaction with tetrahydrofolate. In the current experiment, the researchers imaged the enzyme in the next step and were thus able to gain certainty about how the enzyme's reaction mechanism actually works.

Painting the picture with neutrons

Neutrons detect light elements like hydrogen, and X-rays detect 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 gave the team the insights they needed to clearly characterize the enzyme's chemical reaction.

“Neutrons allow us to see hydrogen atoms, and hydrogen drives chemistry,” Drago said. “Enzymes are 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 see the positions where there is hydrogen and, just as important, the positions where there is no hydrogen. You get the whole picture.”

As shown in the animation, the mitochondria of cancer cells produce too much of the SHMT enzyme, a tetramer consisting of four identical peptide chains or protomers (shown in gray). SHMT works by using pyridoxal 5′-phosphate 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 different 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, thus stopping the one-carbon pathway in cancer cells.

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

Cells contain thousands of enzymes that act as catalysts, speeding up biochemical reactions necessary for bodily functions—from breathing to hormone production 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 acts earlier in this pathway and offers the opportunity to stop cancer sooner.

However, the difficulties in treating cancer are partly related to the stealthy attacks cancer cells make on metabolic processes. Unlike drug resistance in infectious diseases, when one pathway is not working well, cancer recalibrates other metabolic processes, producing too many cancer cells.

“Now that we know the atomic details of SHMT, we can advance the development of an inhibitor that targets this specific protein as part of a combination therapy,” said Kovalevsky.

“Compared to treating infectious diseases, this is much more difficult because with chemotherapy for cancer, you usually attack your own proteins, which is why patients have side effects. In infectious diseases, the proteins you attack belong to the viruses or bacteria. But with cancer, you have to kill your own cells. The idea is to kill the cancer faster and put less strain on the patient.”

Accelerate the pace of discovery

The team used neutrons at the MaNDi instrument at the SNS and the IMAGINE instrument at the HFIR for their research. ORNL's recent Proton Power Upgrade project added more powerful beams to all instruments at the SNS. More powerful proton beams mean more neutrons. More neutrons mean shorter data collection times on smaller samples, resulting in faster responses that help scientists develop smarter drugs to treat disease.

“Discovery research is absolutely necessary,” said William Nelson, director of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, who was not an author on any of the ORNL-led studies. “We're getting closer and closer to the point where, using artificial intelligence, we'll be able to sequence a gene in a cancer, predict what the protein structure would look like, and make a drug that fits into it; it'll 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 the actual protein structure, the chemical structure and the way things interact, the better we can train AI models to predict things we don't immediately know.”