Nick Cox is a chemist and postdoctoral scholar in the Stanford ChEM-H Medicinal Chemistry Knowledge Center. For the past two years, he has helped the Jennifer Cochran lab develop a novel molecular warhead to deliver drugs to cancer cells without harming healthy cells. Graduate student Anna Koster spoke with Cox about this work, which he published in a recent paper in Angewandte Chemie.
What was the key finding of this work?
We demonstrated that a protein called knottin, which comes from the exploding cucumber plant, can deliver a deadly payload to tumor cells. In nature, knottin protects the seeds of the cucumber plant from digestion by rodents and birds by affixing itself to key digestive enzymes. In our system, we’ve genetically engineered knottin to bind to a different protein target called integrin, which is found on the surface of tumor cells. Once knottin adheres to integrin, the drug-protein complex gets internalized, releasing the drug warhead directly into tumor cells without harming surrounding healthy tissue.
Antibodies are typically the choice messenger proteins for carrying cancer-fighting drugs to tumors, but are not always ideal because of their large size and tendency to elicit an immune response. Knottin is 40 times smaller than an antibody. Because of its small size, knottin can more easily penetrate into tissues and even cross the blood-brain barrier, meaning it can get to tumors that larger antibody messengers can’t reach, such as brain tumors. Proteins this tiny usually break down quickly, but the knottin protein backbone is basically tied up in a knot (thus, the name), making it unexpectedly stable for its size. This is one of very few examples where a protein this small has been used as the scaffold for targeted drug delivery.
What are some of the chemistry challenges in creating a protein-drug conjugate like this?
The problem with “bioconjugation”—the attachment of an organic molecule to a biological molecule, like a protein—is that large biological molecules really like hanging out in water, and the small, greasy organic molecules that you’re trying to attach don’t. So, it can be a little bit tricky to join the molecules without causing your protein to misfold or crash out of solution. Another big challenge for us was finding the right chemical linker to connect the knottin to the drug payload. The linker must be stable in the environment outside the cell but then must be broken once inside the cell to release the toxic cargo. From a chemistry standpoint, it’s challenging to design chemical bonds that are likely to break under certain circumstances but not others. It took a lot of trial-and-error on our part.
What are some challenges that remain before this treatment can reach patients?
This potential therapy is a long way off from ever being tested in humans, but I think that what it does (at least I hope) is to encourage more investment in developing technology like this. Even if this one particular molecule doesn’t end up making it to the clinic, it does justify investigating other types of drug conjugates. There are already modified knottin proteins that have gone into the clinic for a variety of applications, including in vivo tumor imaging and tumor visualization during surgery. The difference in our study is that we attached knottin to a drug instead of a fluorescent imaging molecule. Given these precedents, I think it’s only a matter of time before a protein like this makes it into the clinic for targeted drug delivery, whether or not it’s the one we published.
What’s next for this project?
Future directions for this project are going to involve, most immediately, trying to test out the drug conjugate in an animal disease model. In the current paper, we showed a very good response from a variety of different cancer cell lines, in particular brain cancer and pancreatic cancer cells. So, we’re hoping that we’ll be able to find some collaborators, both at Stanford and elsewhere, who will be interested in taking this molecule into animal models of these cancers.