Summary: For the first time, chemists have completely synthesized verticillin A, a notoriously complex fungal molecule with surprising anticancer potential. The molecule’s fragile structure required a complete rethinking of its synthetic sequence, allowing researchers to not only recreate it but design more stable and potent derivatives.
Early tests in human cancer cells are particularly promising against diffuse midline glioma, a devastating pediatric brain tumor with limited treatment options. With the synthesis resolved, researchers can now systematically explore verticillin-based therapies in broader cancer models.
Key facts
Breakthrough synthesis: Verticillin A has been recreated for the first time since its discovery more than 50 years ago. Therapeutic potential: A stabilized derivative showed strong effects against pediatric diffuse midline glioma cells. Structural information: Rearranging critical bond-forming steps was essential to controlling the molecule’s delicate stereochemistry.
Source: MIT
For the first time, MIT chemists have synthesized a fungal compound known as verticillin A, which was discovered more than 50 years ago and has shown potential as an anticancer agent.
The compound has a complex structure that made it more difficult to synthesize than related compounds, although it only differed by a couple of atoms.
“We have a much better appreciation of how such subtle structural changes can significantly increase the synthetic challenge,” says Mohammad Movassaghi, a chemistry professor at MIT.
“We now have the technology that allows us not only to access them for the first time, more than 50 years after they were isolated, but we can also create many engineered variants, which can allow for more detailed studies.”
In tests on human cancer cells, a verticillin A derivative showed particular promise against a type of pediatric brain cancer called diffuse midline glioma. More testing will be needed to evaluate its potential for clinical use, the researchers say.
Movassaghi and Jun Qi, associate professor of medicine at Dana-Farber Cancer Institute/Boston Children’s Cancer and Blood Disorders Center and Harvard Medical School, are senior authors of the study, which appears today in the Journal of the American Chemical Society. Walker Knauss PhD ’24 is the lead author of the paper. Xiuqi Wang, a medicinal chemist and chemical biologist at Dana-Farber, and Mariella Filbin, research director of the Pediatric Neurology-Oncology Program at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, are also authors of the study.
A complex synthesis
Researchers first reported the isolation of verticillin A from fungi, which use it to protect against pathogens, in 1970. Verticillin A and related fungal compounds have attracted interest for their potential anticancer and antimicrobial activity, but their complexity has made their synthesis difficult.
In 2009, Movassaghi’s lab reported the synthesis of (+)-11,11′-dideoxyverticillin A, a fungal compound similar to verticillin A. That molecule has 10 rings and eight stereogenic centers, or carbon atoms that have four different chemical groups attached to them. These groups must be linked in a way that ensures that they have the correct orientation or stereochemistry with respect to the rest of the molecule.
However, once that synthesis was achieved, the synthesis of verticillin A remained challenging, even though the only difference between verticillin A and (+)-11,11′-dideoxyverticillin A is the presence of two oxygen atoms.
“Those two oxygens greatly limit the window of opportunity you have in terms of doing chemical transformations,” Movassaghi says. “It makes the compound much more fragile, much more sensitive, so even though we had had years of methodological advances, the compound still posed a challenge for us.”
Both verticillin A compounds consist of two identical fragments that must join together to form a molecule called a dimer. To create (+)-11,11′-dideoxyverticillin A, the researchers performed the dimerization reaction near the end of the synthesis and then added four critical carbon-sulfur bonds.
However, when trying to synthesize verticillin A, the researchers discovered that waiting to add those carbon-sulfur bonds at the end did not result in the correct stereochemistry. As a result, the researchers had to rethink their approach and ended up creating a very different synthetic sequence.
“What we learned was that the timing of events is absolutely critical. We had to significantly change the order of events that formed the links,” Movassaghi says.
The synthesis of verticillin A begins with an amino acid derivative known as beta-hydroxytryptophan and then, step by step, researchers add a variety of chemical functional groups, including alcohols, ketones and amides, in a way that ensures correct stereochemistry.
A functional group containing two carbon-sulfur bonds and one disulfide bond was introduced early on to help control the stereochemistry of the molecule, but the sensitive disulfides had to be “masked” and protected as a pair of sulfides to prevent them from breaking down in subsequent chemical reactions. After the dimerization reaction, the disulfide-containing groups were regenerated.
“This particular dimerization really stands out in terms of the complexity of the substrates we are putting together, which have a very dense range of functional groups and stereochemistry,” says Movassaghi.
The overall synthesis requires 16 steps from the starting material beta-hydroxytryptophan to verticillin A.
Kill cancer cells
Once the researchers successfully completed the synthesis, they were also able to modify it to generate verticillin A derivatives. Dana-Farber researchers then tested these compounds against several types of diffuse midline glioma (DMG), a rare brain tumor that has few treatment options.
The researchers found that the DMG cell lines most susceptible to these compounds were those that had high levels of a protein called EZHIP. This protein, which plays a role in DNA methylation, has previously been identified as a potential drug target for GDM.
“Identifying the potential targets of these compounds will play a critical role in better understanding their mechanism of action and, more importantly, will help optimize the Movassaghi laboratory’s compounds to be more specific for the development of new therapies,” says Qi.
Verticillin derivatives appear to interact with EZHIP in a way that increases DNA methylation, inducing cancer cells into subprogrammed cell death. The compounds that were most successful in killing these cells were N-sulfonylated (+)-11,11′-dideoxyverticillin A and N-sulfonylated verticillin A. N-sulfonylation, the addition of a functional group containing sulfur and oxygen, makes the molecules more stable.
“The natural product itself is not the most potent, but it is the synthesis of the natural product that brought us to a point where we can produce these derivatives and study them,” Movassaghi says.
The Dana-Farber team is now working to further validate the mechanism of action of verticillin derivatives and also hope to begin testing the compounds in animal models of pediatric brain cancers.
“Natural compounds have been valuable resources for drug discovery, and we will fully evaluate the therapeutic potential of these molecules by integrating our expertise in chemistry, chemical biology, cancer biology and patient care. We have also profiled our lead molecules in more than 800 cancer cell lines and will be able to understand their functions more broadly in other cancers,” Qi says.
Funds:
The research was funded by the National Institute of General Medical Sciences, the Ependymoma Research Foundation and the Curing Kids Cancer Foundation.
Key questions answered:
A: Verticillin A’s fragile, densely functionalized structure made it inaccessible for more than five decades, and solving its synthesis unlocks controlled production, structural variants, and systematic therapeutic exploration.
A: Derivatives interact with EZHIP, a protein linked to DNA methylation, triggering increased methylation that pushes cancer cells toward programmed cell death.
A: The researchers are validating the mechanism in detail and plan to move to animal studies, while profiling the derivatives in hundreds of cancer cell lines to identify broader clinical potential.
Editorial notes:
This article was edited by a Neuroscience News editor. Magazine article reviewed in its entirety. Additional context added by our staff.
About this news about brain cancer research
Author: Sarah McDonnell
Source: MIT
Contact: Sarah McDonnell – MIT
Image: Image is credited to Neuroscience News.
Original Research: Closed access.
“Total synthesis and anticancer study of (+)-verticillin A” by Mohammad Movassaghi et al. Journal of the American Chemical Society
Abstract
Total synthesis and anticancer study of (+)-verticillin A
We report the first total synthesis of (+)-verticillin A, more than 50 years after the fungal metabolite was first isolated.
Our initial strategy for the sulfidation of a dimeric diketopiperazine (DKP) produced the undesired stereochemistry for the epidithiodiketopiperazine (ETP) substructures of the (+)-verticillin A alkaloid.
We subsequently developed a protocol to directly introduce the disulfide with the correct relative stereochemistry into a complex DKP using benzydryl hydrodisulfide prior to dimerization.
Given the sensitivity of ETPs to carbon-centered radicals and UV irradiation, we developed a strategy to mask the disulfide as an alkyl sulfide pair before ambitious radical dimerization, fusing two disulfide DKPs at the C3-C3′ bond, followed by photochemical desulfonylation of N1.
A late-stage revelation of ETP substructures provided (+)-verticillin A, the first dimeric ETP natural product containing C12 oxygenation that can be accessed by total synthesis. (+)-Verticillin A and its N1-sulfonylated derivatives demonstrated potent biological activity in cancer cell lines and effectively regulated histone lysine 27 trimethylation (H3K27me3) levels in the cell, leading to apoptosis.
Treatment of cell lines expressing high levels of EZH inhibitory protein (EZHIP) with (+)-verticillin A led to upregulation of H3K27me3, suggesting that (+)-verticillin A and its N1-sulfonylated derivatives interact with EZHIP.
A thermal shift assay using cell lysates confirmed that N1-sulfonylated (+)-dideoxyverticillin A binds to EZHIP, while the structurally related ETP (+)-chaetocin A did not show any intracellular engagement with EZHIP.
The interaction between (+)-verticillin A and its derivatives with EZHIP can be exploited to treat pediatric cancers that are sensitive to H3K27me3 alteration.
