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.