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Using the serine hydrolase probes, Cravatt and his student Nadim Jessani looked for proteins that could uniquely identify different cell lines, like a fingerprint or an eye scan. They chose well-characterized cell lines to start with, so that they would have a baseline from which to judge the effectiveness of the technology.

What Jessani and Cravatt found were clusters of proteases, lipases, and esterases that they could use to distinguish human breast cancer cells from other types of cancer cells. They also found that they could use these markers to distinguish invasive tumor cells—those that can migrate to new tissues after metastasis—from non-invasive ones.

More importantly, they also found that invasive cells from different cell lines looked more like each other than they do like the non-invasive cells that they derived from.

"It's not just the upregulation of new enzymes," says Cravatt. "The cells shut off the original markers that would lead you to believe that they are, for example, a melanoma."

Furthermore, they identified an enzyme, the membrane-associated hydrolase KIAA1363, that had previously not been associated with cancer. Based on the fact that KIAA1363 is up-regulated in invasive cancer cell lines, Cravatt and Jessani suggest that this enzyme may represent a new marker of tumor progression.

They were even able to predict invasiveness in other cells based on the readout of their activity-based assays and the upregulation of KIAA1363.

Significantly, many of the proteins that they were able to profile using the technology are secreted, found in the membrane, or are expressed in low abundance—difficult to study using traditional proteomics methods.

"[The paper] shows that we can go into cell lines that have been characterized for a decade and see new stuff, such as enzymes that correlate with invasiveness that have never been seen before," says Cravatt.

As insightful as these studies are, they are nevertheless limited by the breadth of the probe, which has the ability to label all the active serine hydrolases but none of the active proteins in other families. And while the serine hydrolases as an incredibly large family, comprising fully one percent of all predicted gene products, that still leaves the glass 99 percent empty.

There are many other important families of enzymes that would be interesting to study using the method, but for which suitable probes are lacking. The kinases, the metalloproteinases, the phosphatases, for instance, are all large families with many enzymes relevant to numerous disease pathologies waiting to be interrogated.

"Can we generalize the concept to address other important enzyme families?" asks Sorensen. "That is the question."

A More Generalized Approach

It's a question that's simple to pronounce but hard to solve.

Indeed, any chemical that can attach itself to a residue in the active site of a protein could be a potential probe. The problem is finding ones that react broadly.

In order to address this, Cravatt, Sorensen, and their student Greg Adam took what they call a non-directed strategy to come up with more candidate probes.

They made combinatorial libraries of probes based on a common "chemotype"—sulfonyl esters, which are commercially available in hundreds of variations, linked to a variable alkyl/aryl binding group. By attaching different binding groups to various sulfonyl esters, the nature of the protein activity profile can be changed. By varying these binding groups, the researchers constructed a library of compounds and then screened these against complex proteomes, looking for activity-dependent protein reactivity.

In a Nature Biotechnology article appearing this month, Sorensen and Cravatt describe how one probe in their library can be used to detect six or seven mechanistically distinct, known enzyme families.

"This is the first evidence that this technology can be expanded to cover a significant portion of the proteome," says Cravatt. "The challenge [now] is to cover the maximum amount."

Sorensen is pursuing projects in total synthesis to produce additional probes, looking towards bioactive natural products for hints on how to design them.

"There are a lot of natural products that nature designed to interact with biological molecules," he says.

One of these, a compound called fumagillin, was discovered in a fungus that inhibits a virus that infects the bacterium Staphylococcus aureus. Fumagillin, as it turns out, reacts with certain metalloproteinases and is a powerful anti-angiogenic and anti-tumor agent.

Sorensen reasoned that the reactivity of the compound might be a good scaffold upon which to build a more broadly reactive probe. By building these molecules from commercially available precursors, Sorensen and his group can design precursors and suitable structural alternatives.

By varying the chemical nature of one of its chains, Sorensen has been able to redirect fumagillin-like compounds to other proteins in the metalloproteinase family. Now they are seeing if they can address metalloproteinases in a global fashion.

Working with Students

Cravatt says projects like these are perfect for students at TSRI because they offer a twofold education. Students learn both how to make molecules and test them in living cells to see how they work—a truly interdisciplinary approach that is rewarding for the students and the instructors alike.

"That's what really drew me—the ability to do both chemistry and biology," says Adam, who is beginning his fifth year in the Chemistry Program.

Jessani, who starts his fifth year in the Macromolecular and Cellular Structure and Chemistry Program this fall, was immediately drawn to the biological applications of the work. "The serine hydrolases are such a huge family," he says. "They had developed the probes, and it was obvious they would be good at looking at biological systems."

"He has really done tremendous interface work," Cravatt adds. And, he says, TSRI is perfect for investigators like Cravatt and Sorensen who design such projects because there are students who legitimately want to work in both fields.

"It's a lot of fun to work with these students," notes Sorensen.


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Gel demonstrating the ability to combine (or multiplex) proteomics probes capable of labeling different classes of enzymes allowing for larger fractions of the proteome to be profiled in one experiment--in this case, human breast cancer cell lines were labeled with a combination of rhodamine-tagged phenyl sulfonate (red) and fluorescein-tagged fluorophosphonate (green). Image courtesy Gregory Adam.









Activity-Based Proteomic Profiling. Click to enlarge