Failure is Not an Option

By Eric Sauter

As a general rule, Ray Stevens runs every morning between 5:30 and 7:00 AM to clear his head and stay in shape for the ultramarathons he competes in. Ultramarathons are runs that can cover anywhere from 50 to 100 miles over mountaintops and, in some cases, far beyond (several are multi-day events that involve running the length of entire countries).

Approached with a slower pace than other races, ultramarathons take extreme patience, a rare tolerance for risk, and absolute faith in your own ability to survive and ultimately triumph—after all, who knows what might happen over the course of 100 miles?

Or, for that matter, over the course of 15 years determining a single structure of a receptor from the largest protein family in the human genome?

Stevens, a Scripps Research Institute professor in the Department of Molecular Biology, began his search for the structure of the b2-adrenergic G protein-coupled receptor (b2AR) when he started his independent research career as an assistant professor at the University of California, Berkeley in 1992.

"I wanted to focus on structural neurobiology," Stevens said. "These particular receptors are key nervous system molecules in what is the largest superfamily of all protein families."

They are that and more. Adrenergic receptors belong to one of the most diverse protein families in the human genome, with almost 1,000 members. They transduce or convert extracellularstimuli into intracellular signals through a number of pathways involving neurotransmitters, light, hormones, lipids, and proteins. Approximately one third, and perhaps as many as half, of currently marketed drugs are designed to target these receptors.

Stevens stayed with the search through his work at the University of California, Berkeley, and after his arrival at The Scripps Research Institute in 1999. At Scripps Research, he eventually developed some very novel and critical technologies, enlisted some key collaborators from Stanford, and grew his own laboratory to continue this marathon hunting expedition.

Crossing the Finish Line

The team finally succeeded this year; the results were published in a pair of related papers in an October 25 advance online edition of the journal Science (see http://www.sciencemag.org/cgi/content/abstract/1150609 and http://www.sciencemag.org/cgi/content/abstract/1150577).

"Our research into the structure of this receptor is a major step forward in developing a complete picture of how the receptors bind with such specificity and transduce a critical signal in biology," Stevens said. "The study expands our understanding of important signaling properties within the protein's various pathways. Furthermore, the structure can be used to create new and more accurate models of the receptor for structure-based drug design and more effective pharmaceuticals."

Taken together, the studies reveal for the first time a static crystal structure of a human b2AR that provides a high-resolution view of a human G protein-coupled receptor (GPCR) bound to a diffusible ligand. That structure shows similarities to rhodopsin, the well studied receptor needed for vision, as well as very significant differences.

In talking about the new study, Stevens focuses first on the people from Scripps Research who made the long trip with him, Scientific Associates Mike Hanson and Vadim Cherezov who spent, collectively, 13 years on the project. Add Stevens' own 15 years and many others who have helped along the way and you have 30-plus years worth of laboratory time on this single protein structure.

"This structure determination could not have occurred without the multi-year devotion from Mike Hanson and Vadim Cherezov," Stevens said. "There are different types of researchers that one works with, some want conservative projects with anticipated steps forward; others want high risk/high reward. In this case, both Mike and Vadim wanted to do something really significant with their scientific careers. The risk they took in working exclusively on this project was enormous. They put their entire careers on the line for this project and did not give up even when most researchers would have. These guys are heroes in my view."

The greatest impact of the studies, Stevens wrote in a pair of summary articles, is in expanding the knowledge about these key receptors and the possibility that once the various conformational states are understood a bit better, this structural information may aid in the design of more specific drugs with fewer side effects.

The second impact is the technology of the studies, a big part of the challenge. Always frustrated with the glacial pace of protein structure determination—especially after determining that his graduate students were spending 70 percent of their experience conducting routine procedures by rote—Stevens developed some of the first high throughput structural biology tools together with Scripps Research Professors Peter Schultz, Ian Wilson, Peter Kuhn, and Kurt Wüthrich. These included microliter expression/purification, nanovolume crystallization, crystallization/NMR screening, automated collection of images, and synchrotron beam line automation.

The technical innovations that had to occur before the scientists could complete the research were enormous, Stevens pointed out: "Remember, this is a receptor family with almost 1,000 members that is also highly conformationally diverse. Locking into a single state in the membrane on crystals too small to be viewed directly meant lab members had to screen the crystals blindly. They had faith and kept pushing to get it."

Stevens' career has been a balance of technological innovations alternating with focused basic and applied biology. Now that the team has the first structure, Stevens and his colleagues intend to cut back their technical development efforts to focus on the biology of this receptor and how it works.

This is the risk-reward ratio that Stevens has been pursuing all along—large-scale projects that can take years to pay off, but that hit the jackpot when they do.

Miles to Travel

Stevens is a founder of the Joint Center for Structural Genomics (1999), Joint Center for Innovative Membrane Protein Technologies (2003), and Accelerated Technology Center for Gene to 3D Structures (2005), all large-scale projects funded by the National Institutes of Health and focused on the development of technologies to accelerate structural biology.

The Joint Center for Innovative Membrane Protein Technologies was awarded a five-year, $14.5 million grant to develop rapid, efficient, and dependable methods to produce membrane protein samples that can then be used to further investigate physiological functions associated with those three-dimensional structures—a goal that his recent GPCR studies dovetail with nicely.

During all of this, Stevens also managed to start up an interesting biotech company called Syrrx in 1999 that was among the first to use high throughput structural biology tools for drug discovery, including the development of an inhibitor for type 2 diabetes that is now in Phase III clinical trials. The company was sold to a Japanese pharmaceutical company in 2005.

All this activity is due to Stevens' belief in the necessity of risk taking as an essential approach to life. "I'm trying to raise my kids with this idea that you have to get the most out of life and that taking risks is part of that," he said. "You may fail, but you still have to try to do something significant."

Stevens also believes in the power of mentoring, especially since his own interest in science was inspired by an undergraduate chemistry professor. "He was passionate about molecules," Stevens recalls. "I mean, how could somebody love molecules? But, the more that I learned, the more I loved working with molecules, too."

Stevens is now taking the opportunity to pay back those who have helped him along in his career. At the University of Southern Maine, where he did his undergraduate work, Stevens is funding the John S. Ricci Fellowship (named after Stevens' advisor), an internship that will open the doors of Scripps Research to University of Southern Maine undergraduate students. Stevens is also increasing his involvement with the University of Southern California, where he received his Ph.D., trying to increase the connectivity between the university and the broader Los Angeles biotech business world of venture capitalists.

Botulism and PKU

Like the technology-science split, Stevens' projects have always had a foot in both the basic and applied research camps.

In addition to his work with GPCRs, his laboratory at Scripps Research has focused on the structural biology of the neurotoxin that causes botulism. Although botulinum toxin is most known as a potential terrorist weapon (or an alternative to plastic surgery) it has a therapeutic side as well, being used to treat involuntary muscle disorders such as cerebral palsy and neuromuscular dystonias, inherited diseases which often result in painful and abnormal posturing caused by relentless muscle contractions.

Then there is his work in phenylketonuria (PKU), a metabolic disorder that can lead to mental retardation and schizophrenia in children. Stevens is working with the biotechnology firm, BioMarin Pharmaceuticals, to develop a treatment based on his structural discoveries—an enzyme that can metabolize phenylalanine, the malfunctioning metabolic enzyme that is the key to the disorder. A treatment for the mild to moderate form of the disease is currently in Phase III trials, with a followon drug for treating more severe or classical PKU about to enter clinical testing.

In talking about the recent GPCR studies, Stevens pointed out that the very nature of his work demands something of an obsessive personality, particularly if you're going to bet 15 years of your life on it.

"It is a belief that you're on the right path," he said, "but it's also an obsession, because in this case, I just was not going to lose. The really challenging projects require intelligence and creativity plus absolute commitment and faith."

Stevens points to the Gene Kranz book about the American space program and its title, Failure Is Not an Option, as something of a guidepost.

"I had that book sitting on my desk for the longest time," he said.

Send comments to: mikaono[at]scripps.edu

"These guys are heroes in my view," said Professor Ray Stevens of Scientific Associates Vadim Cherezov (left) and Mike Hanson, whose work was key in solving the long-sought receptor structure.