Stanford Researchers Make Cancer Breakthrough – Clever Synthesis of Rare Cancer-Fighting Compound


The researchers succeeded in synthesizing the anti-cancer compound EBC-46.

A new and improved way to produce an acclaimed anti-cancer compound.

Stanford University researchers have found a rapid and sustainable approach to synthesizing a promising cancer-fighting compound in the lab. Because there is only one plant species that produces the substance naturally, and that species only grows in a small region of rainforest in northeastern Australia, the availability of the compound has been limited.

The compound EBC-46, also known as tigilanol tiglate, works by promoting a localized immune response against tumors. The response breaks the tumor’s blood vessels, ultimately killing the cancer cells. Following its very high success rate in treating a particular type of cancer in dogs, clinical trials testing EBC-46 in humans have recently begun.

However, due to its complicated structure, EBC-46 seemed synthetically inaccessible, meaning that no conceivable approach to making it practically in the lab seemed to exist. However, Stanford scientists have demonstrated how to chemically convert an abundant plant-based raw material into EBC-46 for the first time using a clever process.

Another advantage of this approach is the ability to create “analogues” of EBC-46, which are chemically similar to the parent compound but may be much more effective and able to treat a surprising number of other serious diseases. These diseases, which include AIDS, multiple sclerosis and Alzheimer’s disease, all share biological pathways that are influenced by the target of EBC-46, a key enzyme known as protein kinase C, or PKC.

“We are very pleased to announce the first scalable synthesis of EBC-46,” said Paul Wender, Francis W. Bergstrom Professor in the School of Humanities and Science, Professor of Chemistry and, by courtesy, Chemistry and systems biology at Stanford. , and corresponding author of a study describing the results in the journal natural chemistry. “Being able to make EBC-46 in the lab really opens up huge research and clinical opportunities.”

The study’s co-authors are Zachary Gentry, David Fanelli, Owen McAteer and Edward Njoo, all of whom are PhD students in Wender’s lab, as well as former member Quang Luu-Nguyen.

Wender expressed the immense satisfaction the research team felt with the breakthrough in EBC-46 synthesis. “If you were to have visited the lab the first few weeks after their success,” Wender said, “you would have seen my brilliant colleagues grinning from ear to ear. They were able to do something that many people had considered impossible.

From a distant region

Tigilanol tiglate initially emerged through an automated drug candidate screening process by QBiotics, an Australian company. In nature, the compound appears in the seeds of the pink fruit of the blushwood tree, picrosperma fountain. Marsupials such as muskrat-kangaroos that eat blushwood fruits avoid seeds rich in tigilanol tiglate, which when ingested trigger vomiting and diarrhea.

Injection of much lower doses of EBC-46 directly into some solid tumors alters cell signaling by PKC. Specifically, EBC-46 is proposed to activate certain forms of PKC, which in turn influence the activity of various proteins in cancer cells, eliciting an immune response by the host body. The resulting inflammation makes the tumor’s vasculature, or blood vessels, leaky, and this hemorrhage causes the tumor growth to die. In the case of external skin malignancies, the tumors crust over and fall off, and ways to deliver EBC-46 to internal tumors are being explored.

In 2020, the European Medicines Agency and the United States Food and Drug Administration approved a drug based on EBC-46, sold under the brand name Stelfonta, to treat mast cell cancer, skin tumors more common in dogs. One study showed a 75% cure rate after a single injection and an 88% rate after a second dose. Clinical trials have since begun for skin, head and neck, and soft tissue cancers in humans.

Based on this emerging research and the clinical needs associated with the geographic limitations of the source seeds, scientists have considered establishing special plantings for blushwood trees. But this poses a host of problems. For starters, trees require pollination, which means the right kind of pollinating animals must be on hand, and trees must be planted at appropriate densities and distances to facilitate pollination. In addition, seasonal and climatic variations affect trees, as well as pathogens. Setting aside plots for blushwood trees further poses land use issues.

“For sustainable and reliable production of EBC-46 in the quantities we need,” Wender said, “we really need to go the synthetic route.”

Make EBC-46 from scratch

A good starting point for making EBC-46, Wender and his colleagues realized, is the plant-derived compound phorbol. Over 7,000 plant species produce phorbol derivatives worldwide, and phorbol-rich seeds are commercially inexpensive. The researchers selected Croton tigliumcommonly known as purgative croton, an herb used in traditional Chinese medicine.

The first step in preparing EBC-46, Wender says, is a daily experience. “You buy a bag of these seeds, and it’s kind of like making coffee in the morning,” Wender said. “You grind the seeds and run a hot solvent through them to extract the active ingredient,” in this case a phorbol-rich oil.

After processing the oil to produce phorbol, the researchers then had to find a way to overcome the previously insurmountable challenge of coating part of the molecule, called the B ring, with carefully placed oxygen atoms. This is necessary to allow EBC-46 to interact with PKC and alter the activity of the enzyme in cells.

To guide their chemical and biological studies, the researchers relied on instrumentation from the Stanford Neuroscience Microscopy Service, the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource, and the Stanford Sherlock Cluster for computer modelling.

Using this guidance, the team succeeded in adding additional oxygen atoms to the B ring of phorbol, first via a so-called ene (pronounced “een”) reaction conducted under flowing conditions, where the reactants mix as they flow together through the tubes. The team then introduced other cyclic B groups in a gradual and controlled manner to achieve the desired spatial arrangements of the atoms. In total, only four to six steps were needed to obtain EBC-46 analogs and a dozen steps to reach EBC-46 itself.

Wender hopes that the much wider availability of EBC-46 and its PKC-influencing cousin compounds afforded by this breakthrough approach will accelerate the search for potentially breakthrough new treatments.

“As we learn more and more about how cells work, we learn more about how we can control this functionality,” Wender said. “This control of functionality is particularly important for treating cells that spin out of control in diseases ranging from cancer to Alzheimer’s disease.”

Reference: “Practical summary of tigilanol tiglate and its analogues therapeutic avenues” by Paul A. Wender, Zachary O. Gentry, David J. Fanelli, Quang H. Luu-Nguyen, Owen D. McAteer and Edward Njoo, October 3, 2022, natural chemistry.
DOI: 10.1038/s41557-022-01048-2

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