Turning Off Pain's Pathways
Easing pain is practically synonymous with practicing medicine, and since before the days of Hippocrates, doctors have sought the best ways of doing thisto find compounds that not only ease the pain, but do so as fast, effectively, and as long as possibleand without any unwanted side effects.
Every analgesic from opiates to hypnotism to electroshocks to balms have side effects, and therein lies the rub: whether relieving the pain or the side effects is more pressing.
One compound that has been hotly debated in the last 10 years is delta-9-tetrahydrocannabinol (THC), the active ingredient in marijuana. The reason THC works is that it mimics the action of natural cannabinoids that the body produces in signaling cascades in response to a peripheral pain stimulus. THC binds to "CB-1" receptors on cells on the rostral ventromedial medulla, a pain-modulating center of the brain and decreases sensitivity to pain.
Unfortunately, the receptors that THC bind to are also widely expressed in other parts of the brain, such as the memory and information processing centers of the hippocampus. Binding to nerve cells of the hippocampus and other cells elsewhere in the body, THC creates a range of side effects as it activates CB-1 mediated signalingincluding, according to the National Institute of Drug Abuse, distorted perception, difficulty in problem-solving, loss of coordination, and increased heart rate and blood pressure, anxiety, and panic attacks.
The challenge of THC and other cannabinoids is to use them to produce effective, long-lasting relief from pain without the deleterious side effects. Now Ben Cravatt, an investigator in The Scripps Research Institute's (TSRI) Department of Cell Biology and The Skaggs Institute for Chemical Biology, thinks he knows just how to do that.
Regulating the Pathway of Internal Bliss
"When you feel pain, you release endocannabinoids [which provide some natural pain relief]," says Cravatt. "Then the amplitude and duration of their activity are regulated by how fast they are broken down."
In particular, the body releases an endogenous cannabinoid called anandamide, a name which is derived from the Sanskrit word meaning "internal bliss." When the body senses pain, anandamide binds to CB-1 and nullifies pain by blocking the signaling. However, this effect is weak and short-lived as other molecules metabolize the anandamide. The compound has a half-life of only a few minutes in vivo.
In some ways, THC is superior to anadamide as a pain reliever because it is not as readily metabolized. But THC goes on to generally suppress cannabinoid receptor activity all over the body. This, coupled with the fact that it is a controlled substance, makes THC an unattractive target for developing therapeutics.
Even an analogous compoundanother CB-1 receptor agonist like THCwould not be ideal because the cannabinoid receptors are so broadly expressed. "You couldn't possibly control what would happen as the receptor was activated all over the body," says Cravatt.
The solution, as Cravatt sees it, is to increase the efficacy of the natural anadamide the body produces to modulate pain sensations. When anandamide is expressed, it is expressed in a cascade that results from a particular sensation, like pain, and this cascade is tightly controlled and localized.
This localization is crucial, because the pathways that mediate pain do not affect cognition. "How do you selectively inhibit those pathways?" asks Cravatt.
One candidate he has come across in the course of his investigations is fatty acid amide hydrolase (FAAH), a 587-amino acid membrane-bound enzyme that metabolizes endocannabinoids, including anandamide and other small molecules.
FAAH is a target for pain therapy not only because it breaks down the molecules that provide the pain relief but also because it turns out that FAAH seems to be the only enzyme responsible for doing so.
"It is stunning how singular the enzyme is in terms of what it is doing," says Cravatt. "If you could manipulate this enzyme, then you have a good shot at manipulating the endogenous system and get the outcome of a selective effect [of decreased sensitivity to pain]."
Cravatt's hope is that controlling the action of FAAH while the body is sensing pain and releasing anandamide would increase the longevity of anandamide throughout in those pathways that are being stimulated.
"I envision that if someone could make a specific inhibitor to FAAH," he says, "in principal, you could get pain relief without any of the side effects."
To Regulate the Regulators
The work on FAAH came out of Cravatt's graduate research, in which he was identifying another neurologically active fatty acid amide called oleamide. Oleamide appears in the cerebrospinal fluid of tired animals, and the last part of Cravatt's thesis characterizes how it works.
"I was working on identifying proteins associated with the fatty acid amide oleamide," says Cravatt, "I wanted to find enzymes associated with oleamide and get their associated genes and manipulate the system that way. That's how we got FAAH."
"FAAH is a member of a large family of enzymes," says Matthew Patricelli, a former student of Cravatt's who did much of the research on the enzyme while completing his thesis. "But FAAH has a new type of mechanism."
FAAH belongs to a large group of serine hydrolases, a class of enzymes containing active site serine residues that catalyze the hydrolysis of specific substrate molecules. These enzymes arose very early in evolution and are ubiquitous in naturefound even in the earliest single-celled organisms. Almost all mammalian trypsin serine hydrolases cleave their substrates through a reaction involving a Ser HisAsp active site catalytic triad, but FAAH uses a LysSer catalytic dyad. There is no histidine in the active site.
The fact that this mechanism is unique is a boon to possible therapies because the active site of FAAH is also unlike other serine hydrolases. One could block FAAH without worrying about repercussions to other enzymes.
Molecular Probes and Activity-Based Proteomics
In FAAH, Cravatt has been looking to exploit the molecular signaling pathways that the body uses when it senses pain in order to come up with selective targets that can be used to treat clinical problems. He has been studying basic questions like how these neurosignaling molecules function in vivo and what regulates their function.
Another success that the research has spun off is the development of molecular probes to be used as tools for proteomics.
Proteomics is a new field that attempts to link the genomic information coming from the human genome initiative to what is happening inside a cell by looking at which genes are transcribed in that cell. Then insight into how the genome is expressed and how it is controlled can be found by, say, comparing the expression profiles of two different cell typesfrom different tissues, organisms, stages of development, or disease states. Knowing the differential gene expression in healthy and cancerous cells, for instance, shows which enzymes may be the major players in that type of cancer.
In general, expression data from cells is revolutionizing our understanding of how the genome functions in the body. If the human genome is a one-dimensional map, then the expression profiles are like adding a second dimension to the map. The next step is adding another dimension.
"We're trying to create a higher order of proteomics," says Cravatt, "to develop probes that actually read out changes in protein activity directly."
Since much of the protein that is expressed in cells undergoes post-translational modifications, proteinprotein interactions, and other alterations to their activity, not all genes that are expressed are active as proteins. The blood clotting cascade, for instance, relies on over a dozen discrete blood factor enzymes, but these remain in an inactive form in the absence of the signals received during the cascade.
Many, many more proteins are expressed than are actually active at any one time. Attached sugars, phosphates, and other post-translational modifications of these proteins can alter the expression landscape in the cell. The proteins that are most important, for instance, may be the ones that are least expressed.
"All that is invisible to standard proteomic approaches," says Cravatt, who is developing chemical probes that "interrogate" a protein's active site and yield information about whether it is active or not.
He calls this "active site proteomics."
Active site proteomics relies on first identifying broadly active compounds that bind to as many as over a hundred members of an enzyme family. By characterizing the enzymes collectively rather than individually, a large number of enzymes in a cell can be profiled with only a few probes. Then the enzymes bound to each probe can be separated out on gels and identified.
"Our probes will define," says Cravatt, "in a complex proteome of 300 different members of an enzyme family, whether 10, 20, 30 are active and so on." And the resolution of expression levels into activities should add depth and scale to the proteome.
One of the principal uses of these probes will be to generate differential maps of, for instance, a cancerous cell and a healthy cell. These maps should show the differences in activity between enzymes in the two types of cells.
"At some level, that is the end game," says Cravatt.
Much of the versatility in Cravatt's research has come as the result of his attending TSRI's graduate program. Cravatt came to TSRI in the early 1990s after completing his undergraduate degree at Stanford University and completed his Ph.D. under the guidance of Professor Dale Boger, Vice President for Scientific Affairs and Dean of Graduate Studies Norton Gilula, and President Richard Lerner.
"That's one of the few decisions in my life that I have absolutely no
regrets about," Cravatt says. "At that point, there was no other place
that was promoting a chemistrybiology interface. TSRI was preaching
something that was totally unique at that time. I literally did two and
a half years of chemistry and two and a half years of pure biology during
my thesis work."