Twelve years ago, when Stuart Schreiber was 28, he earned the questionable distinction of being the first chemist to synthesize a naturally occurring compound called periplanon-b, the sex attractant of the American cockroach. Schreiber says he was drawn to periplanon-b because of its geometric beauty. Having synthesized the molecule in his Yale laboratory, though, he decided he might as well pursue the obvious experiment, so he descended into the chemistry building basement.
I went down with one of my graduate students, a flashlight, a shoe box--it was all pretty primitive--and I had a book showing pictures of different kinds of cockroaches, he explains. But I had trouble distinguishing the male from the female. I was reading that the markings on the leg would distinguish them, but I could never do that. So I found some American cockroaches--the big ones--and I didn’t know if they were male or female, but only the male responds to this periplanon-b. It was very exciting: you could take extraordinarily tiny quantities of the stuff, picograms, and puff them into the air, and these cockroaches would flap their wings and stand up. Only half of them would do it, and that’s how you knew which were the males.
The news reports of Schreiber’s synthesis were less than adulatory--Esquire magazine gave him a Dubious Achievement Award for creating a cockroach dating service. Still, Schreiber’s experience with cockroach sexuality was the spiritual beginning of what may become a revolution in modern medicine and biology. It led Schreiber unwittingly from his mundane life as an organic chemist into the world of cell biology and the study of how signals are passed to living cells from the outside world. Little was known about that process, called signal transduction, when Schreiber sent his roaches into a tizzy. Researchers saw that chemicals floating in the body outside the cells somehow caused activity among the cells’ genes; in the early 1980s, though, they had only just begun to ask why.
They knew that though DNA acts like an encyclopedic how-to manual for a cell, it is kept squirreled away in the nucleus, surrounded by material called cytoplasm, and sealed up tightly in the cell membrane. Tens of thousands of receptors stud the membrane, waiting for messages from the world outside the cell. The messages and receptors take the form of proteins, and their union is the first step in a pathway that acts like an electric circuit, carrying signals to the nucleus.
First a single molecule (periplanon-b, for instance) sidles up to a receptor on the external membrane of a cell. By contacting and binding to the receptor, it sets off a Rube Goldberg chain reaction of events inside the cell. There another molecule combines with a neighboring compound to trigger the next connection, and the signal is passed from chemical to chemical until it reaches the nucleus, activating the genetic mechanisms and the DNA inside. As a result, the cell might divide and replicate, or, if it’s already busy dividing, it might stop. It might die or differentiate into some other kind of cell. It might pour out molecular messengers to activate more signal pathways in other cells.
But beyond knowing that chemicals had to be alerting one another within the cell, researchers had no idea what was going on, in detail. Not until 1993 were they able to describe even a single pathway completely, from membrane to nucleus. And that description was the work of researchers at a dozen labs.
Now Schreiber, along with Jerry Crabtree of Stanford and researchers at their two labs, thinks he has discovered a systematic way to reveal all the interactions that make up any pathway. Better still, the group’s method may allow them to co-opt the signaling machinery inside cells, turning it on and off at will. The incipient technology holds promise as a new weapon for fighting a range of diseases, from cancer to cystic fibrosis and other developmental disorders, and Schreiber and Crabtree have become two of the most widely read and cited researchers in all of science.
The story of their discovery is one of those medical mysteries in which the attempt to answer a single biological question leads to a discovery of revolutionary importance. It starts with Schreiber and his aesthetic obsession with molecules. Although in recent years he has been called off-scale brilliant and clearly the leading chemist of his generation, Schreiber was far from a child prodigy. He spent his high school days working at a pizza parlor 60 hours a week and, by some accounts, partying the rest of the time. He went to college, at the University of Virginia, only because his sister talked him into it. His understanding of education was so limited that when he walked into a required freshman chemistry course and saw his classmates diligently taking notes in notebooks, he wondered how they knew to buy notebooks and how they knew to take notes.
Then he experienced a revelation. I discovered chemistry, he says. It was my first learning experience: it was just beautiful. I was attracted to the aesthetics of chemistry, the shapes of molecules, the orbitals, the geometry. I latched onto organic chemistry, read everything I could get my hands on, took every graduate school course there was. He went on to get his Ph.D. at Harvard, where he studied with Nobel Prize- winning chemist Robert Burns Woodward, famous for synthesizing extraordinarily complex molecules. Woodward passed on his passion for that scientific art.
By the time he was 25, Schreiber was an assistant professor at Yale, where he did his cockroach work and started asking questions more relevant to biologists than chemists. At first they were about periplanon-b and male cockroaches: How could such tiny quantities of a single molecule elicit such a dramatic response from an organism without ever entering its cells? Or as Schreiber puts it, How on earth does that work?
Before Schreiber got an answer to this question, though, he was sidetracked further into hard-core biology. One of his Yale colleagues, Robert Handschumacher, identified the exact spot on human cell membranes where a compound known as cyclosporine would attach itself. Cyclosporine, which had been found in Norwegian dirt samples near the Arctic Circle, was considered extraordinarily useful for controlling the immune system. Not only could it help prevent the body from rejecting transplanted organs, but it seemed to work miracles on autoimmune diseases--diseases in which the immune system attacks the body’s own cells. But because cyclosporine often required toxic doses to function, researchers were on the lookout for similar but nontoxic agents.
Handschumacher found the cyclosporine receptor--the one cellular protein to which cyclosporine would bind--by fishing with cyclosporine in a soup of proteins made from the thymus gland and seeing what stuck. The process was like taking one piece of a 1,000-piece jigsaw puzzle and dragging it through the other 999 pieces until one of them hooked on. He named the receptor cyclophilin, after its affinity for cyclosporine. When Handschumacher told Schreiber about the two chemicals, Schreiber found himself with a new obsession. He realized for the first time that the organic molecules he studied as a chemist were not so different from the proteins, like cyclophilin, that form much of the working components of human cells. Proteins, he says, are long strings of amino acids, but with side chains that can rotate and adopt different structures. As an organic chemist, someone who’s used to studying the shapes of things, I thought I might really understand how these proteins take on shapes, and therefore how they can interact with one another.
Schreiber started a cyclosporine research program in his laboratory but was interrupted in 1986 by the announcement from a Japanese pharmaceutical company of a new chemical compound, called FK506, that also appeared to be a powerful immune-system suppressor. Schreiber liked the idea of studying a new drug without cyclosporine’s long history. With his students and colleagues, he quickly synthesized FK506 and then went fishing, as Handschumacher had, for whatever protein it might be binding to in the cells. When they found such a protein, they named it FKBP, for FK506 binding protein. Though they didn’t know FKBP’s natural function in the body, it could clearly interfere with the immune response, at least when FK506 was around. But how exactly were the pair accomplishing that feat?
With this question, Schreiber took a giant step into cell biology--and into signal transduction in particular. Early studies of cyclosporine and FK506 suggested that both proteins somehow managed to interfere with an important signal pathway called the T cell receptor pathway. T cells are the primary defense mechanisms of the immune system. When chemicals known as antigens, found on the surfaces of foreign invaders, bind to the molecular receptors on the membrane of a T cell, the T cell receptor pathway is turned on, signaling the DNA in the cell nucleus to create and release a protein called interleukin-2. Interleukin-2 stimulates T cells all over the body to replicate; in essence it’s an alarm call, telling the immune system to prepare for danger. With cyclosporine or FK506 in the neighborhood, though, T cell replication ceases, antigens or no antigens.
When Schreiber began probing it, the T cell receptor pathway was an unknown. But Schreiber wanted to understand how FK506 and cyclosporine shorted out this pathway, and to do that he and his colleagues had to trace its wiring from beginning to end. You literally do it by looking at one molecular interaction after another, Schreiber says. You establish one, and then you pull out the thing it interacts with and ask what that one binds to. It is actually that simple. The complexity comes in when you realize that there are a lot of things binding to a lot of other things simultaneously, and you have to dissect the system to get at one interaction at a time.
Schreiber was not alone in studying the T cell pathway. At Stanford, Jerry Crabtree was approaching the same problem from a different point of view. Crabtree, who grew up on a West Virginia farm, studied medicine in Philadelphia before moving on to research. He learned the nuances of cloning and DNA technology at the National Institutes of Health during the 1970s--at the time, says Crabtree, about as exciting a place as anywhere could possibly be.
Crabtree’s interest was in how genes are turned on and off during an organism’s development. By the end of the 1980s he was studying T cell activation. Simply put, he says, when T cells are activated by an antigen, for the next two weeks they will proceed step by step through a very precise preprogrammed sequence of events. Some 200 to 500 genes will activate, one after another, ticking off like soldiers standing up to be counted, and will do so the exact same way each time, each gene producing its own particular protein. To make all these proteins, the immune system needs about two weeks--the same amount of time it takes the body to handle most infections. That’s why serious infections such as pneumonia seem to last just that long. The question was, says Crabtree, How are those genes turned on at the right time?
Crabtree and his researchers had two pieces of information to work with. Once a cell had passed a critical point in the activation process, which happened within an hour, it was committed to it. Take away the stimulus--the antigen--and the cell would still tick along through its two-week sequence. Furthermore, other researchers had established that T cells were sensitive to cyclosporine and FK506. Add either to the cell, and the activation process never starts.
Because the production of interleukin-2 (IL-2) is an important first step in this sequence, Crabtree started his research there. The activation of that IL-2 gene, he explains, would commit a cell to this process. That was the molecular core of the commitment. So the question boils down to: What turns on the IL-2 gene? He discovered that a molecule known as nuclear factor of activated T leucocytes (NF-AT) made the DNA in the nucleus produce IL-2. NF-AT was made from two components, one in the cytoplasm outside the nucleus, the other in the nucleus. When an antigen attached itself to a T cell receptor, something in the signal pathway sent the component in the cytoplasm, NF-ATC, swooping down into the nucleus to join up with its counterpart, NF-ATN, and turn the gene on. Moreover, it seemed to be this particular liaison--NF-ATC with NF-ATN--that cyclosporine and FK506 shut down.
Crabtree published his findings in 1991. By then Schreiber had moved to Harvard; after reading the paper, he called Crabtree, and the two agreed to collaborate. They set out to discover exactly what FK506 and cyclosporine were doing inside the cell. Schreiber speculated that it may not have been FK506 alone that created the therapeutic effect, or even FKBP, but somehow the two together. He knew from his studies that when FK506 bound to FKBP, the pair created a nice bumpy surface that looked as if it would bind neatly to some other molecule with complementary indentations. That surface, says Crabtree, could be exerting the therapeutic effect.
But if so, what was sticking to the surface? Schreiber and Crabtree got the hint they needed from Jeff Friedman, a graduate student working down the hall from Crabtree on a different project involving cyclosporine. Friedman found that when cyclosporine and cyclophilin combined, they bound themselves to yet another protein. Then, when one of Schreiber’s postdocs at Harvard, Jun Liu, showed that the new protein was something called calcineurin, they knew they’d hit the jackpot, because calcineurin also bound to the combination of FK506 and FKBP.
Calcineurin was a key molecule in the T cell pathway. When it performed properly, it was responsible for sending NF-AT to work on the DNA. Take calcineurin out of the circuit--the combination of either FK506 and FKBP, or cyclosporine and cyclophilin did the trick nicely--and the wiring diagram was missing a crucial connection. In the T cells, FK506 allows two proteins that normally have nothing to do with each other-- calcineurin and FKBP--to bind together, so cyclosporine and FK506, says Schreiber, act like a kind of molecular glue.
Natural versions of those gluelike molecules are vital for signal pathways to function. While the proteins that make up these signal pathways are always present in the cell, they will not normally interact until signaled to do so. That signal can take one of two forms: proteins, for instance, can change their shape, an event initiated by other proteins adding or snatching away a few small charged molecules. The new configurations allow the proteins to dock in the docking sites of others and pass their signals along. At one time, biologists thought most cellular interactions proceeded this way.
But researchers have now demonstrated that proteins can also pass along molecular signals just by getting close enough to latch onto each other (dimerizing, in the lingo of biologists), or by latching onto two other molecules--receptors, for instance--and pulling them close together. This is the proximity effect, and it’s how FK506 does its magic. Now, says Schreiber, researchers realize how prevalent the proximity effect is within the cell. Because signal pathways employ this mechanism so extensively, he says, it struck him, as it struck Crabtree, that if they could create molecules to emulate FK506 and work as molecular glue, they would gain control over the pathways.
Crabtree says, We thought maybe we could control the way proteins work by making some small organic molecule and just physically proximating two proteins. They would build a molecule called a dimerizer, shaped like a little barbell or a two-headed key. One end would latch onto one protein target and the other to another, bringing the two proteins close enough to do whatever they would do naturally.
It took Schreiber’s and Crabtree’s labs some 18 months to pull it off; they tried different drugs and compounds, putting pounds, putting molecules together every which way. Finally they concocted a dumbbell- shaped molecule by sticking two FK506s together. The chemical, which they called FK1012, was so similar to the components of the cell membrane that it would melt into the membrane and emerge on the other side. Once inside the cell, it would latch onto two proteins and tie the things together, says Crabtree. The physical proximity of that signaling unit would lead to some biological response.
This simple approach offered the researchers what looked like limitless power. Now, says Crabtree, there’s almost no level of biological control within the cell that we can’t approach, beginning at the cell membrane and going all the way down to the nucleus. They have used their molecular dimerizers to turn on the signal pathways below the membrane rather than relying on hormones to do it outside the cell. They’ve turned on signal pathways from the middle of the wiring diagram and even headed straight to the DNA in the cell nucleus and activated the genes directly, all of which used variations on their tiny dumbbell to induce a proximity effect. The variations are found by trial and error, but Schreiber’s lab has developed several mechanisms to accelerate the hit-and-miss process, allowing the researchers to come up with dimerizers that work wherever they choose on the signal pathways.
Controlling signal pathways has remarkable potential. Schreiber and Crabtree say they can use their technology to activate or deactivate genes at will, by switching on or off the pathways that control them. They can also pull proteins out of pathways at any time, to see how that affects development. The technique is simple. They create molecules that act like FK1012, binding to the protein in which they’re interested; these molecules, in Schreiber’s words, cause it to no longer function, and gum up its activity site. Then the researchers can learn the protein’s purpose by triggering cell development or replication and seeing what the cell does without it.
They’re learning to create molecules that will drag a protein into a proteus zone, the cell’s internal garbage incinerator, in which it is rapidly destroyed. If this works in animals as well as it does in the laboratory, it should allow the researchers to study what a protein does on a particular day, or even in a particular hour, of development. We could say what’s happening at, say, day 14 after conception for a particular mouse protein, explains Crabtree, by adding the dimerizer, making the protein disappear for a short time, and determining the consequences for development.
Eventually, Schreiber and Crabtree hope to be able to use their methods on human cells. The human body is prey to a host of diseases caused by missing or defective genes--sickle-cell anemia, for instance, and cystic fibrosis. Genetic engineers would like to insert genes into cells or replace defective genes with new ones. There are also infectious diseases that can be fought by prompting cells to create proteins, such as interferon, that stimulate the immune system.
While genetic engineers have been learning how to insert new genes into human cells and then get them back into the body, they have yet to conquer the problem of turning the genes on when they’re needed, or turning off genes that are producing harmful proteins. With their specifically engineered dimerizers, Crabtree and Schreiber may have found a way to do just that: FK1012 will permeate cell membranes and can be designed to turn on or off the pertinent signal pathways, and do it in dosages that can sit quite comfortably in a tiny pill. If you give a tiny, tiny bit of FK1012, says Crabtree, much less than you would give in aspirin, for example, it will go all over the body and get to the right places.
How far their revolution will go is anyone’s guess. Schreiber and Crabtree have licensed the technology to a Boston-based biotech company, Ariad, that’s working on everything from turning on proteins that will dissolve blood clots to gaining control over the signal pathways that lead to growth inhibition or programmed cell death. The reason for the latter is cancer. Using gene therapy to allow an organic molecule to kill only tumor cells would obviously be very significant, says Schreiber. The new challenge is to get DNA into tumor cells, and then target those genes to kill the cells. If you can solve that problem once, then the organic molecule will work continuously.
Schreiber and Crabtree admit that their approach entails a host of challenges. But with the new approaches that open up as cell biology converges--in great part through their work--with fields like structural biology and Schreiber’s own chemistry, the vision of overcoming those difficulties becomes ever clearer and sharpe.
