Potent drugs like chemotherapy can be life-saving, but often with life-threatening side effects. Notably, they can be indiscriminate, killing both cancer cells and healthy cells in one swoop. Increasing a drug's on-target efficiency can reduce side effects and enable healthier outcomes for patients.
A new tool, developed in the lab of University of California San Diego Professor of Chemistry and Biochemistry Neal K. Devaraj, promises to do just that. TRACE (tetrazine release and activation by cellular enzymes) locks tetrazine molecules in a cage that is only released when it comes in contact with a cell-specific enzyme. This work appears in Nature Chemical Biology .
What is bioorthogonal chemistry?
Bioorthogonal chemistry is a process that allows researchers to perform chemical reactions in living systems to track and manipulate cells in real-time without interfering with native biochemical processes. Two designed molecules exclusively seek each other out and "click" together to perform a chemical reaction. Many bioorthogonal reactions belong to a broader class of highly selective reactions often referred to as click chemistry. Scientists use bioorthogonal chemistry for coupling tags, drugs or imaging dyes to biomolecules, even in cells.
A common tool for this is tetrazine, which reacts quickly with partner molecules. In 2008, Devaraj and Joseph M. Fox independently reported the use of tetrazine coupling for bioorthogonal chemistry, introducing one of the fastest bioorthogonal reactions available. Today, tetrazines are found in chemistry and materials science labs around the world, as well as in human clinical trials, where they can be used as a drug-delivery mechanism.
As exciting as this development has been, tetrazine reactions can be indiscriminate, reacting across cell types in complex biological systems. In humans, this means imaging may lack precision or drug therapeutics may act on healthy cells in addition to diseased ones.
To improve efficiency, Devaraj's lab developed molecular cages that encase tetrazine, preventing them from "clicking" with other molecules. The tetrazine only becomes activated when it encounters a particular cellular enzyme that unlocks the cage. Once activated, the tetrazine can quickly trigger a chemical reaction inside target cells.
In order to get really exquisite spatial control, where a reaction is happening in cell A, but not cell B, activation must happen rapidly. The lab studied different tetrazine structures to determine which had the fastest uncaging rates and the quickest reaction times. The researchers also employed a competing tetrazine-reactive scavenger to suppress activation outside target cells, further improving spatial precision and essentially programming the chemistry to work in a single cell type.
"What we've shown is that you can, essentially, program the chemistry in specific cell types," stated Devaraj, who is also the Murray Goodman Endowed Chair in Chemistry and Biochemistry. "You want this to work in a cell type that's over-expressing a particular enzyme, like a cancer cell, but not in other cells — that's what we've figured out."
After proof-of-concept testing, they used real enzymes that are over-expressed in certain diseases in conjunction with doxorubicin (DOX), a potent drug used in cancer therapy with limited clinical applications due to its high cell toxicity. When comparing the tetrazine cages to a control group, DOX was only deployed when the cages came into contact with a specific enzyme.
Beyond drug delivery, the team also built fluorescent probes that only light up after TRACE activation. The lab was able to show that only cells that both expressed the enzyme and the molecular tag fluoresced. Another probe was used to label the surface of cells that have high alkaline phosphatase (ALP) activity, a marker often elevated in certain tumors. The probe attached to a cell‑surface "handle" and turned fluorescent only where ALP was active, allowing precise visualization of enzyme activity on live cells.
Devaraj has been researching tetrazines for nearly 20 years and he shows no signs of stopping. Now that his lab has built the cages, he is looking for ways to improve selectivity which may lead to increased drug efficacy with fewer side effects.
"I am very interested in the idea that you could rethink how you deliver drugs and imaging agents, and that you can do these things in the human body. That's what led us to develop tetrazine reagents a long time ago," stated Devaraj. "It's turned out to be a really rich space, and, all these years later, they're still offering surprises."
Authors are Caroline H. Knittel, Stormi R. Chadwick, Jacob A. Vance, Cedrik Kuehling and Neal K. Devaraj (all UC San Diego).
Funding was provided, in part, by the National Institutes of Health (R35GM141939) and the German Research Foundation (KN 1447/1-1).
