biology


medicine


Spying
on the
Grim Reaper
How small molecule inhibitors and probes can be used
to monitor cell death and possibly stop it in its tracks
by Alicia B. Berger

A
poptosis is a normal physiological process of pathway that is activated by the detection of death signals.
programmed cell death or “cell suicide” that eliminates Each pathway initially causes single units of initiating
damaged or stressed cells. Errors in apoptosis are caspases (caspase 8 or 9) to bind together (dimerize) and
involved in approximately 70% of human diseases including become active. Both pathways merge when the initiator
cancer, Parkinson’s, Alzheimer’s, and the massive cell death caspases cleave and activate the executioner caspases 3 and
that occurs after a heart attack or stroke. The caspases, a 7 (Figure 1).
family of enzymes that break down proteins, are key players
in the apoptosis program, but few tools are available to
study caspase activity. However, in the August 18, 2006
issue of Molecular Cell, a team of researchers, including the
author of this article, from the lab of Dr. Matthew Bogyo
in the Pathology Department of the School of Medicine
described the synthesis of novel tools called Activity Based
Inhibitors and Probes (ABIs and ABPs) that can be used to
inhibit and track caspase activity. ABIs and ABPs are helping
researchers understand apoptosis, and may help to diagnose
and develop therapeutics to treat diseases that result from
aberrant apoptosis. Figure 1: Caspase involvement in the intrinsic and extrin-
sic pathways of apoptosis.
Caspases in Apoptosis
Caspases are proteolytic enzymes (or proteases) that Caspase-Targeted “Activity Based Inhibitors and
catalyze the breakdown of large proteins to smaller Probes”
peptide fragments. By propagating death signals through In order to study how caspase activity-gone-haywire
the activation of other caspases and by acting as a cellular contributes to disease, tools are needed to track the activity
demolition crew by chewing up proteins, this family of of caspases. The tools currently available are unreliable
enzymes orchestrates the apoptosis program. This program because they can not differentiate between active and
can be executed via two separate pathways: the intrinsic inactive caspases, nor can they reliably distinguish between
pathway that is activated by stress events, and the extrinsic close caspase family members.
In order to study how caspase activity-gone- The Bogyo laboratory is focused on developing reagents
that do not have the same pitfalls as current tools. These
haywire contributes to disease, we need reagents are small chemical tools called Activity Based
Inhibitors and Probes (ABIs and ABPs) that can be
tools that can be used to track the activity of engineered to inhibit the activity of specific caspases. Why
inhibit caspases? By examining the result of their inhibition,
caspases.
12 stanford scientific

To understand how ABIs and ABPs work, picture a cell
that contains many different active and inactive caspases.
If this cell is treated with a caspase-3 targeted ABI, it will
only inhibit active caspase-3, leaving all other enzymes
untouched. The same logic would apply if a cell is treated
with a caspase-3 targeted ABP; only active caspase-3 would
be inhibited and labeled with the probe (Figure 3).
Developing Highly Selective Activity Based
Inhibitors and Probes for the Caspases
The Bogyo laboratory recently published the development
Figure 2 Figure 3
of highly selective ABIs and ABPs specific for caspases 3,
7, 8 and 9. These ABIs and ABPs are composed of three
components: 1) a warhead that irreversibly binds to the
caspase active site, 2) a specificity region, and 3) a cap (ABI)
or tag for visualization and identification (ABP) (Figure 2).
If our inhibitors and probes prevent cell
death, they could potentially be used as a
therapeutic in conditions in where there
is massive cell death such as after heart
attack, stroke, and organ transplantation.
The specificity region is important for the development of
inhibitors and probes that target the different caspases. To
make these uniquely different inhibitors and probes, the
researchers synthesized a “library” of small molecules with
different combinations of amino acids in the region between
the warhead and the cap/tag. This library was then screened
to determine which combinations of amino acids more
effectively inhibited certain caspases. While both ABPs and
ABIs inhibit caspase activity, ABPs are unique in that they
allow the visual tracking of caspase activity. Both reagents
can be used in many systems ranging from simple protein
mixtures in a test tube to more complex living cells and
organisms.
The ABIs and ABPs developed by the Bogyo team can
selectively inhibit and label individual caspases. Interestingly,
they can also be used to study the activation of caspases.
Using a general caspase probe and a caspase-3/7 selective
inhibitor developed in this study called AB06, it was shown
layout design: Mac Sriyanong
biology
that caspase-7 does not require full cleavage for +
activation. In showing this, the lab was able to medicine
identify a novel active form of caspase-7 that was
called a “half-cleaved” intermediate. Furthermore, the
team showed that “half-cleaved” caspase-7 is first activated
by caspase-9 and then further processed by caspases 3 and 7,
thus uncovering a previously unknown processing pathway
for this caspase. Based on this information and contrary to
previous assumptions, caspase-7 might play a role distinct
from that of caspase-3 in the cell. The old and new model of
caspase-7 activation is shown in Figure 4.
Figure 4
The Future of Caspase-Targeted Activity Based
Inhibitors and Probes
Studies are currently underway to test if caspase ABIs
and ABPs can be used to “rescue” apoptotic cells from death.
If so, they could potentially be used as a therapeutic against
massive cell death. For example, the compounds could be
used to treat patients after a heart attack, stroke, or organ
transplant.
Researchers in the Bogyo group are also in the process
of developing caspase ABPs with tags that can be detected
by special instruments from inside the body for imaging
studies. ABPs like these have many applications including
visualization of the amount of cell death after heart attack or
the amount of tumor cell death during chemotherapy.
The potential applications of caspase-targeted ABIs and
ABPs are only beginning to be realized. Future work with
these tools will be incredibly valuable for researchers. These
compounds could facilitate basic scientific research, provide
a way to image caspase activity in live animals, and serve as
novel therapeutic agents for trauma and disease. S
ALICIA BERGER is a PhD student in the Cancer Biology Program. In
addition to science writing she enjoys singing in the Stanford
Symphonic Chorus and downhill skiing.
To Learn More:
For more information, visit Dr. Bogyo’s lab website at
http://bogyolab.stanford.edu/
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