Inflammatory pain is relatively common and can usually be controlled with anti-inflammatory medications such as ibuprofen. But prolonged use of these drugs can lead to ulcers and intestinal bleeding. Fewer treatments exist for neuropathic pain, a type of pain that results from nerve damage. Even the most sophisticated treatments for neuropathic pain provide only temporary relief and have dangerous side effects.

When he chose to work on the protein fluoride-resistant acid phosphatase (FRAP), Zylka hoped to find more targets for pain treatment. He was interested in the fact that the protein was found in nociceptors, a type of neuron that senses pain.

A long line of investigators before Zylka had studied FRAP. Several attempted to isolate the protein so that they could determine its function. But they made little progress and the gene for FRAP remained unknown.

“The mystery has persisted for decades,” Zylka says. “Basically people dropped it because other pain-sensing neuron markers came along that were easier to use.”

When Zylka came to UNC in 2006 he directed his lab toward identifying the FRAP gene. He knew that FRAP was an acid phosphatase and that it could be found in pain-sensing neurons. So his lab looked for acid phosphatase genes in these neurons. Their search turned up an enzyme called prostatic acid phosphatase (PAP). Typically found in the prostate, the PAP enzyme is used as a diagnostic marker for prostate cancer. Wondering why a prostate protein was found in neurons, Zylka’s lab conducted new experiments with PAP. Their results revealed that FRAP and PAP were the same protein.

Click to read photo caption. Confocal microscopy images by Bonnie Taylor-Blake. ©2009 Endeavors magazine.

Since PAP is commonly used in tests for prostate cancer, purified PAP was readily available. This allowed Meg Twomey, a technician in Zylka’s lab, to test the protein in mice relatively quickly. In her first experiment, Twomey injected mice with PAP. A day later, she applied heat or pressure to each mouse’s paw and measured the time it took for the animal to withdraw its paw. Mice that were not injected with PAP had short reaction times, indicating a normal pain response. The injected mice, on the other hand, tolerated the pain stimulus much longer. And the effect lasted for several days.

“We were blown away,” Zylka says, “but we were also incredibly skeptical.” He thought that maybe the effect was an anomaly. But Twomey repeated the experiments and confirmed that the effect of PAP was real. “This was the eureka moment,” Zylka says.

Further testing revealed that PAP could relieve different types of pain, including heat-induced pain, inflammatory pain, and neuropathic pain. One dose of PAP relieved pain for days. Morphine, one of the most powerful pain-relieving drugs on the market, lasts for only a few hours. And since PAP is an enzyme naturally found in the body, the mice experienced no side effects even when receiving the highest doses. Equivalent doses of morphine caused paralysis.

While the thought of developing PAP into a pain treatment was exciting, Zylka says, a key piece of information was missing. “No one actually knew what it was doing physiologically,” he says. In order for PAP to be used in pain treatment, Zylka’s group would have to figure out exactly how the protein was relieving pain.

Graduate student Nate Sowa helped uncover PAP’s pain-relieving secret. Enzymes, Sowa says, are like the body’s recycling centers. They have the ability to break apart, or degrade, molecules into their basic components so that they can be reused. Some enzymes can degrade more than one molecule, but often there is one molecule the enzyme prefers.

Early on, Sowa suspected that adenosine monophosphate (AMP) was PAP’s favored target. Adenosine, a molecule known to suppress pain, is the main component of AMP. If PAP was degrading AMP, then the extra adenosine from the reaction could be responsible for the pain relief. Sowa tested PAP activity with many different molecules, including other adenosine-containing molecules such as adenosine triphosphate. He found that PAP favored AMP over other molecules.

Sowa and Zylka suspected PAP was transforming AMP into adenosine, which activates adenosine receptors found at the surface of cells. To confirm their hypothesis, they would have to demonstrate that the pain-relieving effect was lost in mice that lacked adenosine receptors.

At this point, Sowa says, they were faced with putting the brakes on a fast-moving project. Due to strict quarantine regulations, it can take several months, or even years, to acquire transgenic mice. But Stephen Tilley, a professor in the School of Medicine’s pulmonary medicine department, helped them keep up the breakneck pace.

“Steve Tilley was the only person in the world who had the mice that we needed and his lab was at UNC,” Zylka says. Within a matter of days, Sowa was able to test PAP on mice that lacked adenosine A1 receptors. The pain-relieving effect disappeared, demonstrating that the A1 receptors were required for PAP to relieve pain. “We got the results right before I left for Christmas break,” Sowa says. “It was a great Christmas gift.”

Click to read photo caption. Confocal microscopy image by Bonnie Taylor-Blake. ©2009 Endeavors magazine.

Zylka now has a direction for developing PAP as a medical treatment. It will be several years before PAP is available to patients, but Zylka envisions it will become a standard treatment for pain. Many patients suffering from intractable pain receive spinal cord injections through an automatic pump filled with morphine. One day, Zylka says, the pumps could be filled with PAP instead. But the majority of chronic-pain sufferers do not require treatment as drastic as a morphine pump. Zylka hopes his work will lead to a less invasive treatment, such as a pill, that will boost PAP’s pain-relieving activity.

Zylka has a provisional patent for PAP and his lab is working to find molecules that interact with the enzyme. There’s still a lot of work to be done before PAP can be developed commercially, but according to Scott Forrest at the Office of Technology Development at UNC, PAP has great potential. “PAP is a new tool for discovering traditional oral drugs for pain treatment,” Forrest says.

Zylka, who is still a relatively young investigator, never expected to dig up the mother lode with an abandoned protein. But, he says, “it’s nice to start with a bang.”

Meagen Voss received a master’s degree in neurobiology in spring 2010.

Mark Zylka is an assistant professor in the UNC Neuroscience Center and the department of cell and molecular physiology in the School of Medicine. Nate Sowa, one of the primary authors of the research, is a doctoral student in neuroscience in the School of Medicine. Lab technician Meg Twomey and research analyst Bonnie Taylor-Blake were coauthors of the study. This work was the cover story for the October 9, 2008, issue of Neuron. Funding came from the National Institutes of Health.