The Philadelphia Chromosome

by Jessica Wapner

  The team members needed a way to screen potential compounds for activity against those other kinases because that, they believed, was where the real promise—and profits—lay. They wanted to keep Abl in the mix because its connection to CML was still the best defined in kinase research, but the E. coli test alone was far too narrow. They needed to test drug candidates against many more kinases. They’d made a butterfly net when what they needed was a fishing trawl. To make any headway in the search for “hits”—that is, compounds that reached their intended target, accomplishing the goal they were after—they would need a far more powerful assay, one that could screen their experimental molecules against PDGFR, EGFR, and PKC. They needed a way to produce those more appealing enzymes outside of the cell, but no one had any idea of how to do that. They needed help.

  Seeking out experts in kinases led Lydon and Matter to Chuck Stiles, a pioneer in the study of PDGFR, at Dana-Farber. Stiles in turn introduced them to Tom Roberts, who had found a way to use baculovirus, a rod-shaped DNA virus, to study tyrosine kinases outside of the cell. By inserting the kinase-encoding gene into the baculovirus genome, Roberts induced the virus to produce copious amounts of kinase. Essentially, he’d created a machine with a virus for a motor that churned out kinase, whatever kind of kinase they wanted. It was the ideal system for studying kinase inhibitors.

  The Ciba-Geigy team began collaborating with Stiles and Roberts, and the baculovirus system became their fundamental tool to identify hits. The team also consulted with Robert Weinberg, who had, early on, supported the concept of inhibiting signaling pathways with drugs. Now, with method and advisors in place, the team could move forward with the task at hand: finding a molecule that inhibited the kinase. “And that was when we got seriously going,” said Matter.

  17

  _______

  PLUCKING THE LOW-HANGING FRUIT

  By the time Lydon and Matter found a way to screen new compounds in the late 1980s, Druker had become a competent scientist. “I was less of a burden in the lab,” he said. “Now, instead of asking every single stupid question, I was a little bit more on my own.”

  He knew about the interactions between his mentor, Tom Roberts, and the industry people from Switzerland, though they were happening only in his peripheral view. In 1987, Ciba-Geigy had sent Elisabeth Buchdunger, the cell biologist Matter had hired, to learn about tyrosine kinase signaling from Chuck Stiles. In the meantime, Druker was continuing his work unpacking the signaling pathway triggered by the polyomavirus that caused cells to become cancerous, a stream of events that also included kinases.

  It was right around this time that Druker finally had the 4G10 antibody he’d spent two years trying to make. 4G10 would allow him to measure the amount of phosphotyrosine—that is, the amount of tyrosine onto which phosphates had been bound by kinase—in a sample of cells. The amount of phosphotyrosine revealed by the antibody reflected the amount of active kinase. It was like counting the number of sand castles to estimate how many children had been at the beach.

  The antibody intrigued the Ciba-Geigy team, and they wanted to know more about it. Could they use it in their search for drug candidates? Where could they get it? An antibody to phosphorylated tyrosine could be a valuable research tool for someone making a tyrosine kinase inhibitor. It was an ideal way to test the power of potential drug candidates. If a candidate was effective, then there would be less phosphorylated protein. Cells could be exposed to an experimental compound, and then 4G10 could be used to measure the amount of phosphotyrosine in those cells. If the inhibitor was working, the amount would be far lower than in unexposed cells. It was the same thinking that had, in part, led Druker to make the antibody in the first place. The Ciba-Geigy team wanted 4G10. All eyes turned to its creator.

  In 1988, after Buchdunger’s stay at Dana-Farber, it was Lydon’s turn. He loved the collaboration with academia. Lydon thrilled at the freedom of academic research to pursue an interest without a profit-oriented goal, to move about the lab without the lingering shadow of company bureaucracy, and to push experiments forward without waiting for approval. He had chosen to work in industry because he wanted to make new medications, but he relished a chance for a holiday. “What you miss in industrial research is the excitement and fast-moving pace of academic research,” said Lydon. “Without that, you just get out of touch in industry because it’s very isolated. You can’t talk about your work [because] it’s all proprietary.”

  He was also eager to learn about 4G10 from Druker, the pleasant postdoc in the Roberts lab who, Lydon noted, had a medical degree, still a rarity in an academic research lab. Druker’s qualifications stood out because Lydon knew that if they found a drug candidate, eventually they’d need a clinician to test it out on cells and, if they were lucky, on actual patients. From that vantage point, an MD who understood kinase science was rare and appealing. Knowing the potential value of the antibody, Druker was happy to share it. Industry-academia collaborations were still relatively simple at the time, the process not yet fraught with material transfer agreements—the legal process guiding the movement of substances in and out of the academic laboratory—and other administrative tasks that inevitably slow the work and cause tension. “Of course you’re going to supply it to them,” Druker, who has earned a small amount of money from 4G10 over several decades, said of his attitude at the time. This antibody, which he’d created for the sole purpose of learning a new skill that might be useful, now brought him face to face with Nick Lydon.

  Two kinase enthusiasts, they had plenty to talk about. Their conversations revolved around the intricate details of what it would take to develop a kinase-blocking drug: what the chemists at Ciba needed to do, what qualities the compound had to have, how it could be tested in cancer cells.

  During their talks, they kept coming around to the Bcr/Abl tyrosine kinase, the one implicated in CML. At Ciba-Geigy, the chemists were screening compounds, looking for hits against PKC, PDGFR, EGFR, Abl, and other kinases that had been found to be overexpressed in various types of cancer. PKC, PDGFR, and EGFR attracted the most attention. They were the headliners, whereas Abl was more of a sideshow, a curiosity. Abl—and it was only the Abl enzyme being screened, not Bcr/Abl, which existed only in CML cells—was still the least promising in terms of the target population size.

  But by 1988, Druker and Lydon kept circling back around to Abl when they talked about kinase inhibitors. They were convinced that the Bcr/Abl tyrosine kinase led directly to CML. The final proof showing that Bcr/Abl caused CML, that the link wasn’t just a loose association, would not come until 1990. But the reports from Witte, Canaani, Heisterkamp, Grosveld, and Groffen were enough evidence for them that the fusion kinase caused the leukemia. And the more they talked, the more they started to think that Bcr/Abl was their best shot to test the idea of tyrosine kinase inhibition. At this unproven stage of research, the certainty of the kinase/cancer connection seemed far more important than the commonness of the disease. And the concrete connection between Bcr/Abl and CML did not exist with any other kinase. As Lydon and Druker mulled over the science, Abl moved from an opening act to the main event. By 1989, Druker was adamant that the drug development effort should focus on Bcr/Abl. “CML is going to be the first to fall to this kinase inhibitor,” Druker told Lydon.

  Lydon concurred. Bcr/Abl was “the low-hanging fruit of the oncogene era,” he says. He knew that, as a rare disease, CML was less appealing from a marketing perspective. Compared with the number of people who would buy a breast cancer or heart disease drug, the number of people who would buy a CML drug was tiny. But the clarity of the connection meant this kinase could be the perfect target for proving the principle behind the strategy.

  Not everyone involved with the kinase program was so convinced of the importance of focusing on Bcr/Abl, least of all the executives at the company. The marketing projections combined with the skepticism that still clung to the entire effort negated the possibility of focusing exclusively on Bcr/Abl. Matter’s
team was instructed to keep testing molecules against any kinase, paying the most attention to PKC and PDGFR.

  18

  _______

  A DRUG IN SEARCH OF A DISEASE

  The chemists at Ciba-Geigy began churning out compound after compound. They weren’t drugs yet; that title could be awarded only after the compound was known to effect some change in the body, not just in a culture of enzymes. During this stage of searching for hits, the chemicals were compounds, candidates, or, if they showed hints of anti-kinase activity, agents. And Matter zealously pushed the chemists to come up with more and more of them, a pressure that Jürg Zimmermann welcomed.

  Zimmermann had grown up on a small farm in the Swiss Alps with plenty of time to be lost in his thoughts while he minded the cows and goats in the field. “It was a beautiful life,” he said. “Then I started to feel bored.” At the age of ten, he began showing an interest in science, and his teacher offered him extra lessons during lunch break. The teacher introduced Zimmermann to the lab, showing him how to do experiments, how to make plastic, how to change the colors of things using chemicals. Zimmermann was hooked. “I saw an opportunity to get away from farming stuff,” he said, “and it was fascinating, trying to understand what was happening in the world.”

  In 1974, at age sixteen, he finished school and took a three-year apprenticeship at Ciba-Geigy, where his work focused on coming up with new types of plastic. Though he loved the work, he didn’t like having to follow someone else’s instructions all the time. “I was already very interested in inventing something, in designing experiments by myself,” he recalled.

  Zimmermann left the company in 1977 to obtain a degree in chemistry in Zurich. Academia fed his hunger for knowledge. After studying chemical engineering for three years, he studied organic chemistry for four years, followed by another four years obtaining his doctoral degree in Australia and then Canada. “I still think this was the best time of my life,” he said. Although he would try to fit in skiing and mountain climbing when he could, his best weekends, he said, were the ones that left him time to read.

  At the end of those eleven years, Zimmermann faced the decision of whether to become a professor or join a pharmaceutical company. He’d grown frustrated doing experiments simply as an academic exercise. Molecules that he and his fellow researchers had synthesized would be thrown away without being tested for any potential application, the sole goal being to prove that the structure they’d created was the one they’d intended to create. “I always protested,” he recalled. “Wouldn’t it be nice to synthesize something that could be [useful]?” After all, that was what he’d loved about chemistry in the first place: You make something. Just as Lydon had gravitated toward the practicality of kinase research, Zimmermann wanted to be in a place where he could put that urge for application to good use. He opted to return to Ciba-Geigy, joining the oncology group.

  Alex Matter opened Zimmermann’s eyes to the world of cancer outside the lab. He told the chemists of patients’ suffering, motivating them to create the new drugs that were so desperately needed. The understanding that Matter brought to Zimmermann, isolated in the lab as he was, were enough to sustain his determination to do what Matter was asking them to do. In fact, it was a dream come true. “Imagine how it is for a young scientist who wants to make something, who wants to achieve something, who wants to make a difference, [to be told] ‘That’s exactly the reason why I hired you, we want to make a difference,’” said Zimmermann.

  • • •

  AN ISRAELI SCIENTIST named Alexander Levitzki gave the field of anti-kinase drug research its next push forward. Levitzki had shown that staurosporine, a naturally occurring antifungal agent made by bacteria, blocked PKC, a kinase known to be involved in several cancers. Levitzki’s 1986 report became a turning point. “It was the discovery that staurosporine [inhibited] PKC that really made the pharmaceutical industry sit up and take notice,” wrote Sir Philip Cohen in a 2002 review of kinase drug development.

  The only problem was that staurosporine wasn’t specific enough. That is, it blocked PKC, but it also blocked other kinases. To treat cancer, a kinase inhibitor would have to block a single kinase and only that kinase. Because so many bodily processes involve kinases, the drug had to target only the dangerous one in order to avoid serious toxicities like liver, kidney, or even heart failure. This feature of the staurosporine work was fuel for the fire of the skeptics who said it couldn’t be done. Kinases were too similar to one another, the thinking went, and they were all competing with each other for ATP, the keeper of energy inside each bodily cell, rendering futile any attempt to take aim at one single enzyme. “A myth therefore began to permeate the field that it was ‘impossible’ to develop protein-kinase inhibitors with the requisite potency and specificity,” wrote Cohen. The inspiration spurred by staurosporine quickly began to deflate. Its trajectory followed the same path as so many other early pharmaceutical bloomers: hype in the wake of encouraging results in the lab, followed by shattered hopes and cynicism when the compound’s flaws emerged. Under this lingering cloud of nay-saying, the Ciba-Geigy chemists continued creating molecules that they thought might block PKC, EGFR, PDGFR, or Bcr/Abl.

  Their strategy was to focus on the exact place on the kinase that bound phosphate on its way from ATP to the protein. The theory behind kinase inhibition was that each kinase had a particular notch or groove that made a tight fit with ATP, and that the shape of these notches varied from kinase to kinase. That variation was what made kinases viable drug targets. Zimmermann believed that this binding site—the exact spot where the kinase bound ATP to capture the phosphate that would be used to switch on another protein—seemed like the most likely location of each kinase’s unique fingerprint. That binding site was what allowed the kinase to serve its function on the cell, so it made sense to Zimmermann that this area would be distinct both from the binding site of other kinases and from other areas on the same kinase. There were no actual images of the molecule inside the cell, but Zimmermann knew the chemistry and could make an educated guess about the chemical makeup of that binding site notch. And knowing this molecular structure enabled him and the other chemists to think about what kind of chemical could prevent it from working. If they knew the shape of the gears, they could throw the right wrench into the clockwork.

  The first step in creating a possible drug was to design a molecule that might fit into that slot. If that molecule adhered to the binding site on the kinase, then ATP wouldn’t be able to attach there, and the kinase would not get its phosphate. Minus the arrival of a phosphate, the next protein would never be switched on to perform its function, and the cascade of signals that led to the development of cancer would not occur. The kinase would never get a chance to set cancer in motion if it couldn’t get to the phosphate in the first place.

  Once they had an idea of the shape they were going for, the team could think about how to make a molecule that fit the bill. Zimmermann, Traxler, and the other chemists explored all manner of chemicals that were already known to inhibit certain cellular processes. The art of Zimmermann’s chemistry lay in figuring out the combination of elements—carbon, oxygen, hydrogen, nitrogen, and less common ones like fluorine—that would block the site at which the kinase bound to ATP, and how to make that compound. Starting with substances that had already shown anti-kinase tendencies and sketching out their ideal molecular structures on paper, Zimmermann and the other chemists began introducing other atoms into the mix.

  Several molecules had already shown some anti-kinase activity (albeit with the coarse profiling tools available at the time). There was the isoquinolinesulfonamide from Hidaka’s work. A group from Japan found that a molecule they named erbstatin inhibited EGFR. A group in Israel, led by Yosef Graziani, showed that quercetin, part of a naturally occurring group of chemicals known as flavones, also affected kinase activity inside some tumor cells. The same had been seen with some isoflavones, which also occurred naturally. And there was staurosporine, t
he antifungal agent that Levitzki had been exploring as an inhibitor of PKC. The fatal flaw of that compound had been its lack of specificity. Could it be adjusted in a way that led it to one kinase, and one kinase only, inside the cell?

  The next task was to actually make the chemical, which might require fifteen steps. If the chemists wanted to engineer an interaction between carbon and fluorine, they could follow the rules of chemistry to make that happen. Mix chemical A with chemical B to make chemical C. Mix chemical C with some commercially available reagent to create chemical D. They were creating recipes for new compounds. “Then hopefully at the end you have synthesized a molecule that resembles the one you had drawn on your piece of paper,” Zimmermann said.

  Zimmermann and Mayer had been asked to focus specifically on PKC, while others on the team homed in on other targets. “People said it’s hopeless, forget it,” Zimmermann recalled. In the cafeteria during lunch, his colleagues would goad him on, incredulous that he would spend his time on such a task. But Zimmermann didn’t see things that way. He’d been given a task, and so he would give it a try. Along the way, he rarely stopped to wonder whether such specificity was feasible; he had just assumed it was. “I just didn’t know that it was extremely difficult,” he said.

  plate 14

  For Zimmermann, the properties of the elements—the explosive nature of hydrogen, the extraordinary versatility of carbon, the adherence of water—had always been more than titillating knowledge. They were ways to make things happen. The rings of weightless, negatively charged electrons surrounding the core of an atom of one element often had space for electrons of an atom of another element. A molecule—a cohesive mix of atoms with its own unique properties—might contain a hydrogen atom, which another atom with an abundance of electrons would stick to as if with glue. Some molecules dissolved in fat rather than water. Inside an oily environment, two chemicals might join together, though such bonds were usually weak. For Zimmermann, these hidden worlds held endless possibilities. Combining molecules together in various ways had led to the creation of plastic, of countless medications, of every synthetic substance. The manmade world was made of chemistry. So was the natural world, for that matter. Surely, Zimmermann thought, there was a way to manipulate some molecules into a kinase-inhibiting drug.