MIT chemists have produced verticillin A in the lab for the first time. This fungal molecule was identified more than 50 years ago and has drawn attention for its potential as an anticancer agent.
Verticillin A is notoriously hard to build because of its intricate chemical architecture. Even compared with closely related compounds, it proved far more challenging to synthesize, despite differing by only a couple of atoms.
“We have a much better appreciation for how those subtle structural changes can significantly increase the synthetic challenge,” says Mohammad Movassaghi, an MIT professor of chemistry. “Now we have the technology where we can not only access them for the first time, more than 50 years after they were isolated, but also we can make many designed variants, which can enable further detailed studies.”
In lab tests using human cancer cells, one verticillin A derivative stood out against a pediatric brain cancer known as diffuse midline glioma. The researchers emphasize that additional testing is needed to assess whether it could eventually be useful in the clinic.
Movassaghi and Jun Qi, an associate professor of medicine at Dana-Farber Cancer Institute/Boston Children’s Cancer and Blood Disorders Center and Harvard Medical School, are the senior authors of the study, published in the Journal of the American Chemical Society. Walker Knauss PhD ’24 is the paper’s lead author. Xiuqi Wang, a medicinal chemist and chemical biologist at Dana-Farber, and Mariella Filbin, research director in the Pediatric Neurology-Oncology Program at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, are also authors.
Why This Fungal Molecule Was So Hard to Make
Researchers first reported isolating verticillin A from fungi in 1970. Fungi use the compound to help defend themselves from pathogens. Verticillin A and similar fungal molecules have been explored for possible anticancer and antimicrobial activity, but their complexity has made them difficult to synthesize.
In 2009, Movassaghi’s lab reported the synthesis of (+)-11,11′-dideoxyverticillin A, a compound closely related to verticillin A. That molecule contains 10 rings and eight stereogenic centers, meaning carbon atoms that each connect to four different chemical groups. Those groups must be positioned with the correct orientation, or stereochemistry, relative to the rest of the molecule.
Even after that earlier success, verticillin A itself remained out of reach. The key difference between verticillin A and (+)-11,11′-dideoxyverticillin A is two oxygen atoms, but those additions made a major difference in how the molecule behaves during synthesis.
“Those two oxygens greatly limit the window of opportunity that you have in terms of doing chemical transformations,” Movassaghi says. “It makes the compound so much more fragile, so much more sensitive, so that even though we had had years of methodological advances, the compound continued to pose a challenge for us.”
Rethinking the Chemistry Step by Step
Both versions of the verticillin molecule are built from two identical halves that must be connected into a structure called a dimer. In the earlier synthesis of (+)-11,11′-dideoxyverticillin A, the team carried out the dimerization near the end of the process and then formed four crucial carbon-sulfur bonds.
When they tried to apply that same sequencing to verticillin A, it did not work. Adding the carbon-sulfur bonds late in the process failed to deliver the correct stereochemistry, forcing the team to redesign the entire order of steps.
“What we learned was the timing of the events is absolutely critical. We had to significantly change the order of the bond-forming events,” Movassaghi says.
The new synthesis starts from an amino acid derivative called beta-hydroxytryptophan. From there, the researchers build the structure in stages, adding chemical functional groups, including alcohols, ketones, and amides, while carefully controlling stereochemistry at each step.
To guide that control, the team introduced a group containing two carbon-sulfur bonds and a disulfide bond early in the process. Because disulfides are sensitive, they had to be “masked” by converting them into a protected pair of sulfides so the structure would not break down during later reactions. After dimerization, the disulfide-containing groups were restored.
“This particular dimerization really stands out in terms of the complexity of the substrates that we’re bringing together, which have such a dense array of functional groups and stereochemistry,” Movassaghi says.
In total, the route takes 16 steps from the beta-hydroxytryptophan starting material to reach verticillin A.
Early Tests Against Diffuse Midline Glioma
With verticillin A finally accessible, the researchers could also adjust the approach to create derivates. A Dana-Farber team tested these molecules against several types of diffuse midline glioma (DMG), a rare brain tumor with limited treatment options.
The strongest effects appeared in DMG cell lines that produce high levels of a protein called EZHIP. EZHIP influences DNA methylation and has previously been flagged as a potential drug target for DMG.
“Identifying the potential targets of these compounds will play a critical role in further understanding their mechanism of action, and more importantly, will help optimize the compounds from the Movassaghi lab to be more target specific for novel therapy development,” Qi says.
The verticillin derivatives seem to affect EZHIP in a way that increases DNA methylation, which pushes the cancer cells into programmed cell death. The most effective molecules in these experiments were N-sulfonylated (+)-11,11′-dideoxyverticillin A and N-sulfonylated verticillin A. N-sulfonylation — the addition of a functional group containing sulfur and oxygen — improves molecular stability.
“The natural product itself is not the most potent, but it’s the natural product synthesis that brought us to a point where we can make these derivatives and study them,” Movassaghi says.
Next, the Dana-Farber researchers plan to further confirm how the verticillin derivatives work, and they hope to test 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.
The research was funded by the National Institute of General Medical Sciences, the Ependymoma Research Foundation, and the Curing Kids Cancer Foundation.
