Bryan G. Fry’s search for unusual venomous creatures has taken him to remote corners of the world, from polar Norway to Antarctica and many places in between. He’s milked venom from more than 20,000 snakes and been bitten by 26 of them. It’s just part of the job for this self-professed adrenaline junkie.
Fry, a professor at Australia’s University of Queensland, is one of a small but growing number of researchers who are using genomic and proteomic tools to delve into the venoms of animals as diverse as cone snails and snakes. Such venomic analyses are improving our understanding of how venomous creatures evolved and aiding both the development of antivenoms and the discovery of new drugs.
Venom-derived drugs have been in medicine cabinets for a while. Captopril, a cardiovascular drug first marketed in 1981, was designed to mimic a peptide found in the venom of the lancehead viper, a South American snake—and it became the first blockbuster drug. More recent success stories include the type 2 diabetes drug Byetta, which is based on a peptide from the Gila monster, a venomous lizard, and the pain medication Prialt, based on a toxin from cone snails. As is often the case, those discoveries owed much to serendipity. Researchers are now hoping systematic analysis of novel venoms will bring more success.
But the molecular complexity of venoms makes analysis challenging. Depending on the species, a venom can contain tens or hundreds of peptides and other molecules. To figure out what those components are, researchers are using next-generation nucleic acid sequencing of the peptide-encoding RNA expressed in venom glands and mass spectrometric analysis of the venom itself.
That’s easier said than done. In conventional proteomic analyses, researchers use peptide mass spectrometric data to search against a database of possible peptide or protein products. But the genomes of most venomous creatures have not yet been sequenced, which means such a database doesn’t exist.
That’s where the RNA sequencing comes in. By sequencing the peptide-encoding RNA expressed in the venom gland, researchers can assemble a species-specific database of possible venom components that can then be searched using mass spectrometric data, says Brian T. Chait, head of the mass spectrometry lab at Rockefeller University.
But not everything in the database will be detectable in the mass spec data. Some RNAs might only be translated into peptides or proteins under certain circumstances, or those peptides or proteins might be expressed at levels that are too low to be detected with current mass spec technology.
For example, Glenn F. King and coworkers at the Institute of Molecular Bioscience at the University of Queensland have identified a peptide in a spider venom that might be used to treat stroke. At least they assume it’s in the venom. They’ve detected the RNA transcript in the spider’s venom gland, but they’ve never actually detected the peptide. Instead, they’ve used the sequence information to synthesize the peptide.
Venoms are attractive as sources of potential therapeutics because the effect they have on prey means “we know they work,” says Mandë Holford, an assistant chemistry professor at Hunter College of the City University of New York who also has a research appointment at the American Museum of Natural History. “We have to figure out where they work and how they work. But we know that the pool we’re starting with has functionally bioactive components in it.”
For example, the venoms of arthropod predators such as spiders and scorpions “have evolved to target ion channels in the nervous systems of their prey,” King says. “These venoms are preoptimized combinatorial libraries of peptides that target ion channels.” Other animals, such as snakes, have evolved venoms to attack the cardiovascular system.
Whatever system they act on, venoms tend to hit similar membrane-protein targets—typically ion channels, receptors, and transporters. But despite those similarities, venom components vary widely among species and even among individuals within a species. Venom composition can change according to the sex of the animal, its habitat, the season, or its diet, says Michel De Waard, a researcher with Grenoble Institute of Neuroscience in France and a cofounder of the start-up Smartox Biotechnology. Overall, the composition of venoms from individual animals of the same species can differ by as much as 10 to 20%, adds Paul Alewood, a venomics researcher at the University of Queensland.
An important use of venomic information is in the development of antivenoms for treating bites or stings from venomous animals. Such medicines are vital in areas where such creatures are a public health threat.
Juan J. Calvete, head of the structural and functional lab at the Institute of Biomedicine of Valencia, in Spain, collaborates with researchers from around the world in the framework of the Global Snakebite Initiative, an international nonprofit organization, to improve access to good quality, robustly tested, safe, and effective antivenoms in the world’s poorest communities. Although knowing the evolutionary history of the animals helps, it’s not enough. Calvete and his collaborators have to analyze the different venoms.
Some snakes have minimalist venoms with only a few proteins, whereas others produce venoms with more than 100. And it’s not obvious which are which before the analysis. “We need to analyze the venoms one by one,” Calvete says.
“Many phylogenetically distant animals can produce similar venoms, and very closely related organisms in the phylogenetic tree can diverge a lot in the venoms they produce,” he says.
The information could help them determine if relatively plentiful antivenoms could stand in for ones that are in short supply in certain parts of the world. In addition, venomic analysis could help scientists develop combination antivenoms that neutralize the venoms of multiple animals in a given region.
Calvete has analyzed many different venoms, and he enjoys each new one as much as the first. “Every venom tells you something different,” he says. “You get surprised by almost every venom you analyze.”
Such variation occurs because both predator and prey are under intense evolutionary selection pressure, Fry says. The most complex venom mixtures are found in the animals who are the least fussy eaters, he says. Such animals are in what Fry calls “an arms race” with prey that have varying physiologies. Specialists—for example, sea snakes that feed only on fish—tend to have simpler venoms, he says.
Fry suggests that as far as venoms go, “you get your greatest return by looking at the weirdest animals.” He himself catches everything his lab works on. The menagerie in his lab includes assassin flies—a predatory insect with enough venom to kill a mouse—and deep-sea venomous octopi he pulled from the abyssal slope of Antarctica. “We are finding all kinds of cool new compounds in these animals.”
That’s what has sent Fry to the ends of the Earth. “If you want to find compounds that are going to be useful as drugs, you’re more likely to find them in a novel venom. And you’re more likely to find a novel venom in a biodiversity hot spot,” Fry says. “The only way you’re going to find your biodiversity hot spots is to have a deep and abiding understanding of the evolution of the animals themselves. Otherwise, you’re just flying blind.”
That means that better understanding the evolution of these animals and their venoms will accelerate the search for new drugs, he suggests.
Other researchers agree. Holford studies cone snails and related snails known as terebrids. All cone snails produce venom, but only some of the terebrids do. The ancestral terebrid had a venom apparatus, but the members of at least eight branches of the modern terebrid family tree no longer have this apparatus.
“We’re using the story of evolution to help us be more targeted in what we go after,” Holford says. “It’s important to do so because not all the snails we work with have a venom apparatus. The evolutionary story is saving us time in terms of identifying what to collect when we go out on expeditions.”
Although evolutionary biology can help researchers focus their efforts, the goal for many remains new drugs and other bioactive molecules. Venom-derived drugs now in development were discovered using conventional screening methods. As venomic strategies expand the catalog of known venom peptides and proteins, they could accelerate the process for finding new drugs.
One drug in clinical trials now is an analog of a peptide from the venom that sedentary sea anemones use to paralyze fast-moving fish long enough to eat them. ShK-186 blocks the Kv1.3 potassium channel, which is activated in a subset of immune cells called effector memory T cells. These cells are involved in autoimmune diseases. The drug, which is in Phase I clinical trials, is being developed by Kineta, a Seattle-based biotech company.
Kv1.3 is a homotetramer that forms a pore on the cell surface. “ShK-186 fits nicely into the pore like a cork,” says Charles Magness, president and chief executive officer of Kineta. The analog “has a small modification that makes it an even more potent and more specific drug” than the peptide found in the anemone.
ShK-186 is in a human clinical trial for psoriasis, but it is effective in animal models of eight autoimmune diseases. “This mechanism of blocking effector memory T cells is applicable across the entire autoimmune space,” Magness says. “The list of diseases where we have animal model validation continues to grow.”
Another venom-inspired peptide is being developed as a treatment for muscular dystrophy. Frederick Sachs and coworkers at the University at Buffalo, SUNY, discovered GsMTx-4 in tarantula venom. They were looking for compounds that block mechanical sensor channels, which are ion channels that respond to mechanical stress.
These ion channels are found in all cells. “The channels usually stay closed unless there’s pathologic stress,” Sachs says. “These channels turn on only when you’re sick, so the inhibitory peptide seems to hunt down sick cells. You could view these peptides like a fire alarm, but nature is warning about membrane fracture instead of fire.”
Sachs is using the synthesized mirror image of the natural peptide, which works as well as the natural peptide. “These peptides are efficacious, nontoxic, nonimmunogenic, and long-lived in the animal, so there is no reason to mutate it. Nature has finished our drug development,” Sachs says.
Sachs started the biotech company Tonus Therapeutics to attract development money from muscular dystrophy charities. Tonus is now collaborating with Akashi, another company funded by muscular dystrophy philanthropies, to develop this peptide, which has been granted orphan drug status by the U.S. Food & Drug Administration. The same drug is effective on other stress-sensitive diseases such as atrial fibrillation and other cardiac arrhythmias.
These and other potential venom-derived drugs still face challenges, particularly image challenges. “Anyone who is in the therapeutics area knows that peptides designed by humans aren’t really long-lasting. They get degraded very quickly in the body,” De Waard says.
But the peptides in venoms are different. They survive in circulation for many hours. They are resistant to enzymatic degradation. Many of them are mini proteins with multiple disulfide bonds that make them compact and highly structured.
“We have to overcome some of the mentality of resistance with regard to peptide toxins as drugs,” De Waard says. “For a long time, peptides were considered bad drugs. We have to educate people that peptides can be good drugs.”
And with a globe-trotter like Fry chasing down venomous creatures, the catalog from which to pull those peptides will just keep expanding.