New Target for Future Therapeutic Drugs

PASADENA, Calif. - "Sometimes letting nature tell you what's important is the better way to go," says Raymond Deshaies, an associate professor of biology at the California Institute of Technology. Deshaies is referring to new work to come out of his lab and the lab of Randall King at Harvard that defies conventional thinking--they've discovered a chemical that stops a key cell function, but, more importantly, suggests a new possible target within a cell, once thought to be untenable, for future therapeutic drugs.

In a report in this week's issue of the journal Science, lead author Rati Verma, a Howard Hughes Medical Institute (HHMI) Research Specialist in the Deshaies lab, Deshaies, also an assistant investigator for the HHMI, and nine other authors report that a small molecule called ubistatin blocked an important step in the so-called cell cycle, a process fundamental to life where a cell makes duplicate copies of its own DNA for distribution to two daughter cells. Knowing how to stop cell duplication is critical in preventing diseases like cancer, when mutated cells go out of control and proliferate madly. Further, ubistatin blocked the cell cycle by preventing two proteins from interacting together. Prior to this, it was thought unlikely that a compound with low molecular weight like ubistatin--or any future drug--would have much impact on the interaction of proteins with each other.

While ubistatin has other properties that preclude it from being a drug candidate, its stoppage of the cell cycle provides an important clue for future drug development, says Deshaies. "We've found a chemical Achilles' heel in this cell pathway, at least from the viewpoint of these small molecules that comprise most therapeutic drugs."

Because the cell cycle is maddeningly complex, researchers usually pick a single pathway (a pathway is a series of chemical events within a cell that perform some task), then try to make a chemical to block it. They may find such a chemical, but often find it difficult to discover where in the pathway--the target--their drug hit. Finding the target is like finding the proverbial needle in a haystack.

Deshaies's colleague Rati Verma found the needle. Instead of using the typical "top down" approach of starting with a specific target, then looking for a drug to block it, the researchers took a "bottom up" approach of starting with a drug and then searching for the target it blocks. They decided to test a large number of molecules to see if any of them might block any step in one particular pathway called the ubiquitin-proteasome pathway (UP pathway): within the cell cycle, when a protein's job is done, another chain of proteins called ubiquitin attaches to it. That serves as a signal to yet another protein called proteasome. The proteasome, says Deshaies, is the biological equivalent of a Cuisinart. "It attaches to these ubiquitin-marked proteins, then ingests them and chews them up."

The researchers examined an entire cell, specifically that of a frog's egg. The King group decided to screen 110,000 molecules to see if any had an impact on the cell. First, they weeded out those molecules that had no effect on cellular function in the UP pathway. King attached a molecule of luciferase ("the stuff that makes a firefly light up," says Deshaies) to certain proteins that are normally destroyed during cell division. Next, he added this newly created protein (now a readily detectable biological "flashlight") to droplets of cellular material extracted from the frog's egg that had been placed in individual chambers. As the egg extract conducted its normal cell division, the luciferase flashlight was destroyed and the chambers went dark. That meant those proteins had been destroyed as part of the normal progress of the cell cycle. He then separately added the 110,000 small molecules to see if any of them would prevent the loss of the luciferase--essentially looking for a lit-up reaction chamber in a field of darkness.

Using this approach, the researchers eventually narrowed the molecules they were testing down to a few that were operating in a specific part of the pathway--downstream from where ubiquitin attaches to the soon-to-be doomed protein, but before the proteasome ingested and chewed it up. But given that numerous proteins are involved in this process, the question remained--where specifically was the molecule they were testing working? In short, where was the target?

To find out, Deshaies turned to work they had done over the last five years with ubiquitin, which examined how it interacted with various other proteins, including proteasome. Through a process of elimination, says Deshaies, "we figured out that these small molecules called ubistatins were blocking the recognition of the ubiquitin chain by the proteasome." Graphic evidence for how this occurs was provided by a 'picture' taken by David Fushman at the University of Maryland with a nuclear magnetic resonance spectrometer.

This step blocked by ubistatin involves a protein-protein interaction, a surprise to Deshaies. "One interesting thing about our discovery is that it is further evidence that you can affect a protein-protein interaction with a small molecule. The conventional thinking was that if you look at a footprint of a drug binding a protein, the drugs are small, but the footprint that corresponds to one protein binding to another is big. So most people thought that the idea of trying to block the huge footprint of protein-protein interaction with a tiny drug was extremely unlikely. So if I were asked to predict what we would find, I would never have proposed that a drug could prevent the ubiquitin chain from binding to the proteasome, because I was also influenced by this conventional wisdom."