University Times

“Playing around with snails leads to unexpected things,” University of Utah biochemist Baldomero M. Olivera told an audience last week during Pitt’s Science 2006 symposium. Olivera’s decades of work with venomous sea snails not only has led to the discovery of drugs to treat pain and epilepsy but also to some useful insights for drug development itself.

His talk, “Animal Biodiversity and Drug Discovery: Cone Snail Venoms, A Case Study,” was the seventh in the annual Klaus Hofmann lecture series, established in memory of the late Pitt biochemistry professor to recognize a basic scientist whose work has clear clinical relevance.

The talk was one of four featured lectures in the annual two-day celebration of science held at Pitt Oct. 5 and 6.

“The way this story developed may be instructive to some of you, particularly to those students who, when they leave Pittsburgh, no longer have access to the great facilities you have at the University of Pittsburgh. All is not lost,” Olivera said, adding that the science they learn is more important than the instruments they have.

His story begins in 1968, when he returned to his native Philippines from his postdoctoral work at Stanford University.

“Many years after you start working with something, people ask you why you started working on it,” he said. “The truth of the matter is we began working with cone snails because we had nothing else to do,” he said, describing how he left Stanford’s modern labs for the less-well-equipped University of the Philippines College of Medicine. “It was pretty clear I wasn’t going to be competitive in DNA replication,” he said with a laugh. Faced with the dilemma of what to do, he decided to pick a project that required what he had: essentially no equipment.

He looked to the beautiful but deadly cone snail, which is prevalent in the waters of the Philippines. Cone snail species make up about 700 of the 10,000 known varieties of venomous marine snails.

“They are so striking, they’ve always attracted people,” he said, noting that their intricately patterned shells have been prized and collected for centuries.

“Our interest in cone snails was stimulated not by the fact they are beautiful … but from the fact that this particular snail can kill people,” Olivera said, adding that, untreated, stings from the most dangerous variety, conus geographus, kill 70 percent of their human victims.

Olivera decided he would work to purify the components of the venom to determine exactly why the snail is deadly. That required only two things: enough starting material from which to derive the components, and an assay. “If what you’re interested in is the components that kill people, it’s not entirely obvious what kind of assay you should use,” he said.

Medical literature showed that people who were stung died from paralyzed diaphragms, rendering victims unable to breathe. So Olivera’s team decided to assay muscle paralysis by injecting mice with various fractions of snail venom and allowing them to cling upside down on a screen. When a paralytic fraction of the venom was injected, the mouse would lose its grip and fall into a bin below.

“All we did was inject various fractions of the venom into a mouse and simply measured how long it took for the mouse to fall,” he said. “That really is the starting point for all this work.”

Using the “falling diagnostic,” Olivera found that the venom is based on two peptides (small groups of amino acids similar to proteins). One was akin to cobra venom, he said. “The second acts very much like what kills you when you go to the wrong restaurant that prepares fugu,” he said, equating the sting from the four-inch long snail as “like being bitten by a cobra and eating a lethal dose of puffer fish at the same time.”

And, while the question of exactly what the snails use to kill was answered in analysis from that long-ago experiment, much more has resulted from continued study of their venom.

Olivera described what he called “a brilliant idea, in retrospect,” when, 25 years ago, his young undergraduate researcher thought to inject the venom directly into the central nervous system rather than the chest cavity of the mice.

“I thought this wasn’t such a good idea and tried to discourage him,” Olivera admitted, adding, “I think the power of American universities and the reason why the most creative research is done in these universities is because students do what they want, not what their professors tell them.”

Surprisingly, various fractions of the venom created a variety of unexpected responses: some put mice to sleep, others made them run, climb or drag their back legs, while still others made them tremble or kick.

That led to the discovery that instead of being strictly for paralyzing prey, the venom is a very complex mixture of diverse pharmacologically active compounds. That discovery, in turn, led to the development of medical treatments from those sea snail peptides.

The lesson: “Keep your eyes open when you see something you don’t expect,” he advised his audience, urging them to be “opportunistic.”

Doing the math, he noted that each conus variety has more than 100 peptides in its venom, and there are 700 types of conus, each with a different set of peptides. “Almost no overlap,” he observed. That means there are about 100,000 pharmacologically active peptides that could be studied for possible medical uses.

One already approved in 2004 by the Food and Drug Administration is being marketed as PriAlt — so named because it is the primary alternative to morphine.

“You can give this peptide and it will continue working even after morphine no longer works,” he said, touting its usefulness in managing ongoing pain in cancer or AIDS victims.

Some conus hunt by using their proboscis like a lure to attract fish, then delivering the poison with a harpoon-like disposable tooth. The fish bites, jerks and is immobilized almost immediately, then is reeled into the snail’s mouth. But, in the lab, the injected toxins took longer to work after traveling through the nervous system than the instantaneous paralysis observed in nature, prompting a closer look. “It’s always good to check back on what the real biological system is doing,” he advised.

Olivera found that “snails have discovered combination drug therapy” — using groups of toxins that act together. (He calls them ‘cabals’ because “they’re out to overthrow the normal physiology of the fish.”)

His group discovered the paralyzing “lightning strike” cabal that acts by utilizing separate peptides to block sodium and potassium channels. “This is a very effective strategy,” he said, noting that the venom causes axons to fire uncontrollably near the site of the sting. “It’s equivalent to a massive electric shock right at the site of the injection,” he explained.

Other snails have developed the “nirvana cabal,” which facilitates their style of hunting: to simply open wide and swallow fish that have been lulled into lethargy. “It makes fish act like they’re in an opium den.”

The nirvana cabal is made of components that make neuronal circuitry less active. “So we’ve looked at these to see if they can be used pharmacologically for different conditions where you have overactive neuronal circuits,” Olivera said. Two of these peptides have been found useful for treating intractable pain and epilepsy and now are in clinical trials.

Olivera said that while 70 percent of successful drugs are designed to mimic a natural product, animals are rarely a source of leads for new medications. Most of those products are from microorganisms, “because that’s what the drug industry knows how to do,” he said. Other discoveries are based on deriving medications from plant-based traditional ethnic treatments.

“So, how do you look for a drug if you’re a natural products guy? You go into a marine environment and collect a huge bunch of organisms and you grind them up – the whole organism. If we had done this to cone snails, we would not have found this drug.”

Key to the breakthroughs in conus-based drug discovery is the emphasis on understanding the biology that underlies the peptides’ purpose in nature. Researchers would be wise to keep in mind that animals produce their compounds in highly specialized structures for highly specialized purposes, he said.

“I think it’s necessary to change the paradigm and really look at the specialized tissue where these compounds are being produced,” he said.

“You always read about the drug pipeline being empty,” Olivera said. “And here we are; we weren’t looking for drugs at all and it’s almost like we stumbled over them just by trying to understand what these cone snails do.”

—Kimberly K. Barlow