Probing the Proteome

By Mark Schrope

The answers to countless health-related questions we ask about ourselves are written in the amino acid sequences encoded in the genome that give proteins their unique characters. The problem is that for the majority of these proteins, perhaps as many as 70 percent of them, those letters give us answers we can't yet read.

"There are parts of cellular biochemistry that are pretty well understood," says Ben Cravatt, a chemical biologist who came to The Scripps Research Institute as a graduate student in 1992 and recently became the chair of the Department of Chemical Physiology, "but there are parts that are just completely without understanding at all."

Researchers only have a good handle on the functioning of 30 to 40 percent of the human proteome, or the proteins we produce. The rest remains largely uncharacterized, and solving this problem is the focus of Cravatt's lab. He and his team focus especially on enzymes, which facilitate every chemical reaction in the body, with results that have significantly advanced understanding of basic biology, and uncovered a massive array of targets for drug discovery. 

Catching the Real Enzyme Players

The group's impressively broad mission is not only to develop new tools that enable studies of enzymes and their functions, but also to conduct some of those studies themselves.

"The applications are very important," says Cravatt, "because they're not only of interest to us, they also keep our technology efforts honest. If our technologies don't help us make new and significant discoveries, then we're probably not there yet and need to go back to the drawing board."

The Cravatt group searches for specific enzymes involved in healthy cell functions such as signaling in the nervous system or in the spread of cancer, and then works to decipher these enzymes' particular chemical roles. The scientists also conduct broad surveys of enzymes involved in particular cellular functions. Ultimately, both lines of work can and do lead to the identification of promising new targets for drug discovery work.

In 1997, as Cravatt was launching his own research program with an eye toward technology development after completing his Ph.D., most previous work to map enzymes had been limited by tools available to study the abundance of various types of enzymes in cells. One of several complicating factors is that the mere presence of an enzyme does not prove it is active. In fact, in many cases, whether an enzyme ever becomes active is decided based on chemical events that occur after it is produced.

So, as a means of zeroing in on those enzymes most likely to play important roles in normal cellular activities or disease progression, Cravatt and his colleagues wanted to develop tools that would specifically target only active enzymes. The researchers dubbed the chemical strategy they developed, and continue to apply and expand, Activity-Based Protein Profiling (ABPP).

The idea behind ABPP is to develop enzymatic probes that will attach only to activated enzymes. These probes are molecules that carry fluorescent tags and whose structures cause them to attach to the reactive portions of enzymes (which the proteins typically use for catalysis) when performing their functions. With inactive enzymes, these sites are also inactive, so the probes won't attach.

Developing just the right probes for given classes of enzymes is an ongoing challenge, and one that the Cravatt team tackles using two main approaches. The first is to identify broad-spectrum inhibitors that target a wide range of enzymes. Probes can then be created based on these inhibitor structures.

The second approach is employed for enzymes whose active sites have not yet been mapped. The team tests libraries of compounds that, based on what Cravatt calls "chemical intuition," have structures that seem like they would have a shot at properly binding and reacting with these enzymes. In July, a paper on this side of the group's work was the cover story for Nature Chemical Biology, describing classes of compounds the group has shown are effective for labeling a range of enzymes.

"Identifying those groups becomes a base of knowledge that we can build on to develop probes for new enzyme classes," says Cravatt.

Probing the Cells

Once the researchers have the proper probes, these can be used with samples of any type of cell, even inside a living animal, to explore enzyme activities. Addition of probes to cancer cells, for instance, followed by fluorescence scanning and structure analyses will return a view of what enzymes are active in those cells.

Conducting the same experiment with healthy cells, or perhaps related benign cancer cells, and comparing results enables the researchers to spot enzymes that are active only in aggressive tumor cells, strongly suggesting they play a role in cancer progression. Such enzymes can then become the subject of further research to determine their function. If their role is vital, then work can commence using a variation on the ABPP strategy to identify molecules that inhibit a given enzyme as a potential drug treatment. 

One of Cravatt's overriding goals is to find enzymes that play roles in multiple forms of cancer, and the team has already been successful in this endeavor. For instance, they used ABPP to identify an enzyme called KIAA1363, which had never been linked before to cancer, but which they found at elevated levels in a variety of tumor types including pancreatic, ovarian, breast, and melanoma.

In related work, the group has also made important discoveries regarding the nervous system, specifically ways that pain and other signals are communicated there. The work has focused on better understanding the basic signaling mechanisms, but has also revealed areas with pharmaceutical potential. 

Much of the nervous system work has focused on enzymes that break down the body's natural chemical painkillers, such as endocannabinoids, with a special focus on the enzyme fatty acid amide hydrolase, or FAAH. The group has shown that FAAH breaks down these painkillers, and that preventing such activity using inhibitors increases endocannabinoid levels, decreasing pain.

Marijuana contains chemicals called cannabinoids that work in ways similar to the endocannabinoids to reduce pain. Though Cravatt is quick to emphasize that his group does not work with marijuana or its constituent chemicals, the similarities mean that boosting the endocannabinoids can achieve painkilling effects akin to marijuana, but without the chemical (or political and social) side effects. Cravatt's lab has identified a number of promising FAAH inhibitors and some are already under clinical development.

The Big Picture

More recently, Cravatt and his team have developed a system they call the PROtein Topography and Migration Analysis Platform (PROTOMAP). This system has enabled broad surveys of hundreds of endogenous substrates of proteolytic enzymes at a time with surprising results. The PROTOMAP combines the use of cutting-edge technologies for separating and analyzing proteins with a bioinformatics tool the group developed that presents the results of studies in a way that is easily accessible and searchable.

"We're now in the process of applying bells and whistles, but PROTOMAP has proven extremely robust," says Cravatt, "and it's already providing huge amounts of information."

The group's first efforts applying PROTOMAP, which were published in the journal Cell on August 22, focused on the proteases involved in apoptosis, or programmed cell death (see News&Views article "New Protein Survey Upends Understanding of Cell Death Process"). In addition to being a critical cellular activity, abnormal apoptosis is also a key factor in diseases such as stroke, where otherwise healthy brain cells are destroyed. Some of the protease substrates the group has discovered using the PROTOMAP could therefore be promising targets for drug development. Future work using the system will focus on goals such as surveying the proteases involved in cancer growth, which should also lead to new drug targets.

Overall, says Cravatt, "I think we've got a pretty good tool set now." And in addition to keeping research projects going that put these tools to good use, Cravatt also assumed duties as the chair of the Department of Chemical Physiology a year ago.

"I think it's a really exciting opportunity," says Cravatt of the position, "It allows me to help the institute stay strong in an area where it already has tremendous strength, which is enabling chemical technologies to address important biological problems."

Send comments to: mikaono[at]scripps.edu

"If our technologies don't help us make new and significant discoveries, then we're probably not there yet and need to go back to the drawing board," says Ben Cravatt, who is chair of the Department of Chemical Physiology.