Scientists have long struggled to target proteins that lack defined structure and are involved in cancer, neurodegenerative disorders like Parkinson’s disease, and other serious illnesses. Now, a new study from Scripps Research demonstrates a proof of concept for a new strategy: engineering proteases — enzymes that cut proteins at specific sites — to selectively degrade these elusive targets with high precision in the proteome of human cells.
Published on March 24, 2025, in the Proceedings of the National Academy of Sciences, the study shows how to reprogram a protease from botulinum toxin to target α-Synuclein — a protein with unstructured regions used here as a model. The study marks one proof point in a broader approach that could be applied to a wide range of targets across the proteome.
“This work highlights how we can use the power of laboratory evolution to engineer proteases that offer a new way to treat diseases caused by hard-to-target proteins,” says senior author Pete Schultz, the President and CEO of Scripps Research, where he also holds the L.S. “Sam” Skaggs Presidential Chair. “It’s an exciting step toward developing new therapeutic strategies for diseases that lack effective treatments.”
The research builds on botulinum toxin, a bacterial protein best known for its use in Botox, a medication utilized for cosmetic purposes and certain medical conditions. This toxin naturally contains a protease. In its original form, the protease only targets SNAP-25 — a protein essential for transmitting signals between nerve cells. By degrading SNAP-25, botulinum toxin disrupts nerve signaling, leading to the temporary paralysis effect seen after Botox treatments.
To reprogram this precision for α-Synuclein, the research team modified the enzyme using directed evolution, a laboratory process that involves introducing mutations and selecting variants with improved function over multiple cycles. The result: Protease 5. The challenge, however, wasn’t just reprogramming the protease to target α-Synuclein — it was ensuring that it attacked only α-Synuclein and nothing else. Past attempts to evolve proteases for therapeutic use have resulted in enzymes that targeted too broad a range of proteins, cleaving multiple unintended molecules and causing toxicity in cells.
“α-Synuclein is an incredibly hard protein to target because it doesn’t have a stable structure,” says first author Philipp Sondermann, a postdoctoral fellow at Scripps Research. “Most drugs work by latching onto structured proteins, but α-Synuclein is more like a shifting tangle.”
Although α-Synuclein plays a central role in Parkinson’s disease and related disorders, it was used in this study as a model protein representing a broader class known as intrinsically disordered proteins (IDPs) — proteins that lack a defined shape and are notoriously difficult to target with drugs. This instability makes such illnesses challenging to treat because traditional therapies typically work by attaching to stable pockets on proteins, like a key fitting inside a lock. However, α-Synuclein has no such binding site, leaving few viable treatment options. “That’s where proteases come in,” says Sondermann. “Instead of needing a specific binding site, they can be engineered to recognize and cut α-Synuclein directly, preventing it from dangerously accumulating in the brain.”
Using directed evolution, the team stepwise modified the botulinum protease, selecting variants that showed increasing preference for α-Synuclein. “Directed evolution works like selective breeding — just as farmers breed plants for better crops, scientists guide proteins through many small changes, choosing the best version at each step,” explains Sondermann. “Each round of modifications made the enzyme more specialized,” he says, “until it could selectively degrade α-Synuclein while leaving other proteins untouched.”
When tested in human cells, Protease 5 nearly eliminated all α-Synuclein proteins, suggesting it could help prevent the harmful buildup seen in Parkinson’s disease. And because the enzyme was designed to precisely target α-Synuclein, it didn’t cause toxicity or disrupt essential cellular functions.
While the Scripps Research study is an important proof of concept, providing early evidence that this protease strategy could one day become an effective treatment, there are still hurdles to overcome before developing a viable therapy. The greatest challenges include transporting Protease 5 to the brain — since large proteins struggle to cross the blood-brain barrier — and ensuring it doesn’t trigger an immune response.
Yet botulinum toxin itself offers some advantages. In addition to its precision, it has a natural ability to enter neurons, and because the toxin used in Botox treatments is tolerated by the immune system, it may not be rejected in a modified form designed to treat Parkinson’s disease. “Botox has been used for decades with minimal immune reaction, which makes it a promising starting point for a therapeutic enzyme,” notes Sondermann.
The next step will be to develop strategies for delivering the evolved proteases to the target tissues of interest. At the same time, the lab is now applying the methodology developed here to the degradation of key proteins involved in cancer, including c-Myc and K-Ras. “This is just the beginning,” says Schultz. “By developing highly selective protease-based therapies, we hope to create a platform for targeting a wide range of conditions caused by disease-causing proteins.”
This work was supported by funding from the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung and the National Institutes of Health (grant R35GM145323).