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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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