How small molecules can be used to monitor cell death
by Alicia B. Berger
Apoptosis is a normal physiological process of programmed cell death or "cell suicide" that eliminates damaged or stressed cells. Errors in apoptosis are involved in approximately 70% of human diseases including cancer, ParkinsonÕs, Alzheimer's, and the massive cell death that occurs after a heart attack or stroke. The caspases, a 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.
Caspases in Apoptosis
Caspases are proteolytic enzymes (or proteases) that catalyze the breakdown of large proteins to smaller peptide fragments. By propagating death signals through the activation of other caspases and by acting as a cellular demolition crew by chewing up proteins, this family of enzymes orchestrates the apoptosis program. This program can be executed via two separate pathways: the intrinsic pathway that is activated by stress events, and the extrinsic pathway that is activated by the detection of death signals. Each pathway initially causes single units of initiating caspases (caspase 8 or 9) to bind together (dimerize) and become active. Both pathways merge when the initiator caspases cleave and activate the executioner caspases 3 and 7 (Figure 1).
Caspase-Targeted "Activity Based Inhibitors and Probes"
In order to study how caspase activity-gone-haywire contributes to disease, tools are needed to track the activity of caspases. The tools currently available are unreliable because they can not differentiate between active and inactive caspases, nor can they reliably distinguish between close caspase family members.
The Bogyo laboratory is focused on developing reagents that do not have the same pitfalls as current tools. These reagents are small chemical tools called Activity Based Inhibitors and Probes (ABIs and ABPs) that can be engineered to inhibit the activity of specific caspases. Why inhibit caspases? By examining the result of their inhibition, researchers can see what important roles they play.
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
There are strong incentives for open sourcing drug research. Western markets today are prohibitive to drug development for neglected diseases. Currently, 10% of global R&D is focused on 90% of the global health burden for neglected diseases. Patent incentives, heavy competition, and skyhigh clinical trial costs have deterred investors in the biopharmaceutical industry to fund research initiatives focused on developing treatments for neglected diseases that affect only developing nations.
The first and foremost disincentive is the absence of pharmaceutical markets in most developing nations. In many cases, the general public cannot afford drugs from pharmaceutical providers. In Ethiopia, for example, where the mean household income of village dwellers is roughly $140 a year, only $36 annually can be afforded to pay for treatment costs.
The low numbers of drugs to treat tuberculosis (TB) demonstrate an example of the pharmaceutical industry's oversight of third-world R&D efforts. Only 22 active TB drugs are in development by pharmaceutical companies worldwide, "a startlingly low figure for a disease with such heavy global burden," claims an article published in 2004 by Pharmaprojects, a pharmaceutical R&D database. The World Health Organization declared TB a global emergency in 1993. The disease affects two billion people - one-third of the world' s population - and is the largest cause of death of any single infectious disease.
The Bogyo laboratory recently published the development 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). 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 that caspase-7 does not require full cleavage for activation. In showing this, the lab was able to identify a novel active form of caspase-7 that was called a Òhalf-cleavedÓ intermediate. Furthermore, the team showed that Òhalf-cleavedÓ 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.
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 ÒrescueÓ 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.