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Serendipity and Sweat in Science
'Frog Man' Daly Follows Curiosity To Ends of the Earth

By Anna Maria Gillis

On the Front Page...

When he first contacted NIDDK's Dr. John W. Daly in 1990, John Dumbacher says he was afraid that the senior scientist would think "I was just a nutty kid." But Dumbacher, then a graduate student in ornithology at the University of Chicago, needed the help of one of the world's leading natural product chemists to test what seemed a far-fetched idea.

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Although there was nothing in the scientific literature proving that birds could store noxious compounds for chemical defense, Dumbacher was certain the flesh and feathers of the Pitohui contained toxins. While handling the bird in the field in Papua New Guinea, Dumbacher had cut his hand and licked the wound. His mouth numbed. He also knew that the locals wouldn't eat what they called the "rubbish bird" unless it was skinned and specially prepared to make it safe.

Dumbacher sent samples to Daly and called every 3 months to track progress. "Around Thanksgiving, Daly called and said, 'We need more tissue. This looks like it's poisonous.'"

"No other chemists rose to the bait," says Daly of the bird project. The chief of NIDDK's section on pharmacodynamics didn't expect the birds to be especially toxic, but curiosity made him prepare and test the extracts anyway. "I injected them into a mouse, and within minutes it had convulsions and died," adds Daly.

Soon Daly began mentoring Dumbacher, and he worked on the project with chemists Thomas Spande and Martin Garraffo, two of his long-term NIDDK collaborators.

After Daly isolated the toxin, Garraffo did a mass spectrum and called Daly that weekend with the toxin's molecular weight and spectral analysis. The pattern of chemical fragments Garraffo described was one that Daly recognized right away — he had seen it before during analysis of compounds in extracts from skins of poison-dart frogs that he'd collected nearly 30 years before. "I knew this had to be a batrachotoxin," says Daly, still excited about making that connection. Finding the potent neurotoxic alkaloids in birds had defied his expectations.

Scientists (from l) Bernhard Witkop, John Daly and Takashi Tokuyama study the structure of batrachotoxins, potent chemicals that affect nerve and muscle cells. (Photo circa 1969)

"You don't know how serendipitous this was," adds Dumbacher. "Of all the natural product chemists in the world who could have looked at it, John did. Another lab might have taken a couple of years to figure out what it was." Since the Pitohui finding, the group has also found a range of batrachotoxins in another bird called Ifrita kowaldi.

Daly, who has been at NIH since 1958, hadn't planned on becoming an expert on bioactive alkaloids. But he got started on this research path in 1963 when Bernhard Witkop, then his lab chief, asked him to go to western Colombia to work on toxins from poison-dart frogs. Daly thinks he was "an ideal person for this job because I'd always been interested in biology." As a child in Oregon, he collected frogs, snakes and lizards and kept terrariums in his basement so he could breed what he'd found.

Witkop wanted to study the frogs' chemistry, but the cost of getting the frogs was prohibitive until Daly got involved. "NIH was paying me $16 a day per diem — far, far less than what was being asked by professionals to collect in the dangerous rain forests of Colombia," says Daly.

The frogs collected near Río San Juan yielded several batrachotoxins, which were featured in a 1966 article in Medical World News. Charles Myers, then a herpetology graduate student in Panama, chanced upon that story. He wrote to Daly and proposed they collaborate on a study of related poisonous Panamanian frogs to determine whether bright coloration and toxicity are linked. The two have worked together ever since.

Daly (r) poses with former postdoctoral fellow Kenneth Seamon in 1983, shortly after they discovered that forskolin was a powerful research tool.

In fact, if Myers had never contacted him, Daly muses that his nearly 40 years of frog alkaloid work may have ended with his initial Colombian forays. "Without Chuck Myers, this program would never have happened. He came up with a great deal of the money for the field work, planned the logistics and worked on requisite permits."

Myers, now a world-renowned herpetologist based at the American Museum of Natural History in New York, sees his collaboration with Daly as one where a taxonomist and chemist taught each other. "I was interested in the taxonomic and evolutionary implications of the toxins, which also have novel pharmacological properties," says Myers. "Scientists talk about doing this kind of interdisciplinary work, but we don't often see it."

Myers taught Daly how to use museum collection records, some dating back to explorations in the 1800s, to pick field sites. "You see someplace from where 20 or 30 frogs were preserved and you know where to go," says Daly.

To get to their gathering sites, the researchers have traveled by jeep, pack animal and dugout canoe and on foot. "Our field work was not to biological research stations, but to quite remote areas," says Myers. Initially, this concerned Myers, who feared Daly would get lost. It was an unfounded worry: "John has a built-in compass."

Once in the field, the two had a simple test to decide whether to take a particular frog. "It involved touching the frog, then sampling it on the tongue. If you got a burning sensation, then you knew this was a frog you ought to collect," says Daly. A healthy sense of self-preservation did prevent them from tasting Phyllobates terribilis, a Colombian frog the locals handle with caution because a single one contains enough batrachotoxins to kill a dozen or more people.

The frogs Daly, Myers and their colleagues eventually collected cover a wide territory — Panama, Colombia, Ecuador, Peru, Venezuela, Brazil, Argentina, Madagascar, Australia and Thailand — and their skins have yielded more than 500 natural substances, mainly alkaloids that the animals picked up from their diet of ants, millipedes, beetles and other arthropods. "The frogs are much better bioprospectors than I am," says Daly. "They're the ones that found the chemicals in the arthropods."

"When we got started, there was only one alkaloid known in a vertebrate and that was samandarine from the European fire salamander," says Daly. Initially, he thought that the frogs made their own alkaloids because scientists who grew the salamanders in captivity said the salamanders produced the alkaloids on their own, a belief that threw Daly off track for a while. "One frog can have as many as 70 different alkaloids," says Daly. "Certainly, they wouldn't have the separate biosynthetic machinery for all of them."

Myers says Daly was "dogged" in his determination to learn the source of the alkaloids. Daly says he, like most scientists, was initially skeptical that the alkaloids came from an environmental source. To test whether animals acquired or made their own chemical defenses, Daly and colleagues took captive-raised frogs that had no alkaloids in their skin and fed them crickets dusted with alkaloids from wild frogs or with leaf-litter insects. The frogs fed alkaloid-laced crickets did indeed sequester the alkaloids in their skin. More recently, Daly and his colleagues have shown that one group of Australian frogs make one type of alkaloid and sequester another type that they get from their environment. That various unrelated frogs and toads have developed the ability to sequester alkaloids indicates the importance of chemical defense in their evolution, says Daly.

In 1973, Daly collected toxin-containing frogs near Río Saija in western Colombia. Skinning the poison dart frog Phyllobates terribilis required protective gear.

For all his interest in field biology, Daly considers himself a pharmacologist with a strong background in chemistry. He trained as an organic chemist at Stanford University and as a pharmacologist with Julius Axelrod. Early in Daly's NIH career, he worked with Axelrod on catecholamines and many aspects of drug metabolism, which was useful training for Daly's future work on animal-derived alkaloids. "I learned pharmacology and physiology from a real master," says Daly. "He instilled in me an appreciation for designing simple experiments to probe complex questions."

Four classes of alkaloids — the batrachotoxins, histrionicotoxins, pumiliotoxins and epibatidine — discovered by Daly have remarkable biological activity on specific ion channels essential to nerve and muscle function.

Daly and his colleagues demonstrated that batrachotoxins selectively opened sodium channels that control nerve and muscle cells. They then modified a batrachotoxin to make a radioactive analog. Scientists now use this radioactive probe to study whether and how local anesthetics, anticonvulsive drugs and other medicines attach to sodium channels.

Daly's group showed that pumiliotoxins have potential as heart stimulants because of their effects on the ion channels in that organ. They also demonstrated that epibatidine, a trace alkaloid from an Ecuadorean frog, was 200 times more potent than morphine as a painkiller, and that it acts not through morphine-sensitive targets, but through receptors for nicotine. Many syntheses of this potent alkaloid have been developed in labs around the world, and one analog made it into clinical trials for the treatment of chronic pain. "I'm a firm believer that if you find a compound that will target one macromolecule, then you'll be able to learn something important," he says.

Daly, a National Academy of Sciences member, has received many honors for his work. Most recently, the American Chemical Society gave him the 2002 Ernest Guenther Award in the Chemistry of Natural Products. The award cites his "pioneering contributions to natural product chemistry, organic chemistry, enzymology, neuropharmacology, membrane biology, and evolutionary herpetology, which are truly without equal in contemporary science."

Intellectual curiosity is the driving force behind Daly's approach, says his colleague Spande. He likes "research that answers some nagging question that may first appear to be tangential or even inconsequential to the main mission."

For instance, "John did not dismiss out of hand, as many would have, the possibility that any bird could be toxic, but was willing to take the time to check this unlikely hypothesis, and bingo, the world now had a major, fascinating (but initially controversial) problem in chemical ecology to wrestle with," says Spande.

This tendency of Daly's to challenge more popular hypotheses is what makes "him true to the scientific way," says Dr. Kenneth Seamon, vice president of drug development for Immunex Corp. and a former postdoctoral fellow in Daly's lab. Seamon worked with Daly on what some scientists consider his most far-reaching work. Together, they discovered that forskolin, a cardioactive chemical from the Coleus plant, could activate one of the most important enzymes in the body. The enzyme adenylyl cyclase stimulates production of cyclic AMP, which, in turn, controls many biochemical reactions in cells. Their discovery that forskolin could be used to increase cyclic AMP levels gave scientists a much-needed way to determine the physiological and pharmacological role of cyclic AMP in organs, tissues and cells.

Seamon learned three things from his mentor that he practices today: Don't prejudge what a scientific outcome will be, collect a lot of information before drawing a conclusion and integrate disciplines. "It is astounding how [Daly] combines biology, chemistry, biochemistry and pharmacology. His ability to integrate is unique."

Besides intellectual breadth, Daly has other abilities that contribute to his success. His colleagues universally comment on his capacity for long hours, his open door policy, and, as NIDDK's Martin Garraffo says, "his willingness to explain things to 'mere mortals.'"

Daly's work ethic was greatly influenced by Dr. Joseph E. Rall, who was NIDDK's scientific director when Daly was learning to be a scientist. "He told me 'Any good scientist spends 60 hours a week on it because he loves it,'" says Daly.

And there's more to research than the bench or field. "I feel strongly about the obligations of being a scientist," says Daly. Reviewing papers and giving feedback are high on the list. Rall gave him feedback regularly, and "I try to do the same with my group, maybe not always successfully, but I try."

"He takes being a mentor seriously," adds Dumbacher, now a Smithsonian Institution researcher. "He was always over my shoulder and made sure I didn't screw up the chemistry," says the ornithologist. Daly also guided Dumbacher to all the right papers, gave him advice on negotiating collection permit issues, encouraged him to learn what local people knew about an animal and helped him get his first major paper published. "There are lots of skills that students have to learn. John taught me what I needed to be professionally competent, not just a scientist."

Daly plans to stay at the bench and go into the field for as long as he can. He would have liked another chance to collect Phyllobates terribilis and find the source of its batrachotoxins, he says, "But it's too hard to get a collecting permit in Colombia now." He's shifted his collecting efforts to Madagascar and Thailand, where he has new frogs to find and new students to mentor.

Daly has no career regrets. He's liked NIH, and, even when other offers came he never thought seriously about them. "I love research so much that disrupting it to move someplace else has never appealed to me," he says. "NIH has been a blessing to me. I don't think I would have prospered in an academic world where I'd have to defend my research and lie about where it was going, because, in many cases, I didn't know where it was going to go."


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