Stanford University researchers have discovered a rapid and sustainable way to synthetically produce a promising cancer-fighting compound right in the lab. The compound’s availability has been limited because its only currently known natural source is a single plant species that grows solely in a small rainforest region of Northeastern Australia.
The compound, designated EBC-46 and
technically called tigilanol tiglate, works by promoting a localized immune
response against tumors. The response breaks apart the tumor’s blood vessels
and ultimately kills its cancerous cells. EBC-46 recently entered into human
clinical trials following its extremely high success rate in treating a kind of
cancer in dogs.
Given its complex structure, however,
EBC-46 had appeared synthetically inaccessible, meaning no plausible path
seemed to exist for producing it practically in a laboratory. However, thanks
to a clever process, the Stanford researchers demonstrated for the first time
how to chemically transform an abundant, plant-based starting material into
EBC-46.
As a bonus, this process can produce
EBC-46 “analogs” – compounds that are chemically similar, but which could prove
even more effective and potentially treat a surprisingly wide range of other
serious maladies. These diseases, which include AIDS, multiple sclerosis, and
Alzheimer’s disease, all share biological pathways impacted by EBC-46’s target,
a key enzyme called protein kinase C, or PKC.
“We are very excited to report the first scalable
synthesis of EBC-46,” said Paul Wender, the Francis W.
Bergstrom Professor in the School of
Humanities and Sciences,
professor of chemistry and, by courtesy, of chemical and systems biology at
Stanford, and corresponding author of a study describing the
results in the journal Nature
Chemistry. “Being able to make EBC-46 in the lab
really opens up tremendous research and clinical opportunities.”
Co-authors of the study are Zachary
Gentry, David Fanelli, Owen McAteer, and Edward Njoo, all of whom are PhD
students in Wender’s lab, along with former member Quang Luu-Nguyen.
Wender conveyed the immense satisfaction the research team felt over the EBC-46 synthesis breakthrough. “If you were to have visited the lab the first few weeks after they succeeded,” said Wender, “you would’ve seen my stellar colleagues smiling from ear to ear. They were able to do something many people had considered impossible.”
From a remote region
Tigilanol tiglate initially turned up 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, Fontainea picrosperma. Marsupials such as musky rat-kangaroos that eat
blushwood fruit avoid the tigilanol tiglate-rich seeds, which when ingested
trigger vomiting and diarrhea.
Injecting far smaller doses of EBC-46
directly into some solid tumors modifies the cellular signaling by PKC.
Specifically, EBC-46 is proposed to activate certain forms of PKC, which in
turn influence the activity of various proteins in the cancerous cells,
attracting an immune response by the host’s body. The resulting inflammation
makes the tumor’s vasculature, or blood vessels, leaky, and this
hemorrhaging causes the tumorous growth to die. In the case of external,
cutaneous malignancies, the tumors scab up and fall off, and ways of delivering
EBC-46 to internal tumors are being investigated.
In 2020, both the European Medicines
Agency and the Food and Drug Administration in the United States approved an
EBC-46–based medication, sold under the brand name Stelfonta, to treat mast
cell cancer, the most common skin tumors in dogs. A study showed a 75% cure
rate after a single injection and an 88% rate following a second dose. Clinical
trials have since commenced for skin, head and neck, and soft tissue cancers in
humans.
Based on these emerging research and
clinical needs coupled with the source seeds’ geographical limitations,
scientists have considered setting up special plantations for blushwood trees.
But doing so presents a host of issues. For starters, the trees require
pollination, meaning the right sort of pollinating animals must be on hand, plus
trees must be planted in appropriate densities and distances to aid
pollination. Furthermore, seasonal and climate variations affect the trees,
along with pathogens. Setting aside plots for blushwood trees further poses
land use problems.
“For sustainable, reliable production of EBC-46 in the quantities we need,” Wender said, “we really need to go the synthetic route.”
Making EBC-46 from scratch
A good starting point for making EBC-46, Wender and
colleagues realized, is the plant-derived compound phorbol. More than 7,000
plant species produce phorbol derivatives worldwide and phorbol-rich seeds are
commercially inexpensive. The researchers selected Croton tiglium,
commonly known as purging croton, an herb used in traditional Chinese medicine.
The first step in preparing EBC-46,
Wender explains, jibes with an everyday experience. “You buy a sack of these
seeds, and it’s not unlike making coffee in the morning,” said Wender. “You
grind up the seeds and run some hot solvent through them to extract the active ingredient,”
in this case a phorbol-rich oil.
After processing the oil to yield
phorbol, the researchers then had to figure out how to overcome the previously
insurmountable challenge of bedecking a part of the molecule, called the B
ring, with carefully placed oxygen atoms. This is required to enable EBC-46 to
interact with PKC and modify the enzyme’s activity in cells.
To guide their chemical and biological
studies, the researchers relied on instrumentation at the Stanford Neuroscience
Microscopy Service, the Stanford Cancer Institute Proteomics/Mass Spectrometry
Shared Resource, and the Stanford Sherlock cluster for computer modeling.
With this guidance, the team succeeded
in adding extra oxygen atoms to phorbol’s B ring, first via a so-called ene
(pronounced “een”) reaction conducted under flow conditions, where reactants
mix as they run together through tubing. The team then introduced other B ring
groups in a stepwise, controlled manner to obtain the desired spatial
arrangements of the atoms. In total, only four to six steps were required to
obtain analogs of EBC-46 and a dozen steps to reach EBC-46 itself.
Wender hopes that the far broader
availability of EBC-46 and its PKC-influencing cousin compounds afforded by
this breakthrough approach will accelerate research into potentially
revolutionary new treatments.
“As we learn more and more about how cells function, we’re learning more about how we can control that functionality,” said Wender. “That control of functionality is particularly important in dealing with cells that go rogue in diseases ranging from cancer to Alzheimer’s.”
Source: https://news.stanford.edu/2022/10/03/breakthrough-production-acclaimed-cancer-treating-drug/
Journal article: https://www.nature.com/articles/s41557-022-01048-2
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