Shoichet PHOTO BY ELIZABETH WILSON

And it's a bitter pill to swallow when you discover that you've invested months, even years, in a promiscuous inhibitor, only to discover that it's biologically useless.

A new study, however, has identified what may be happening to a significant percentage of these compounds--knowledge that could save pharmaceutical companies much fruitless pursuit.

Some of the molecules, it seems, are collecting in large clumps known as aggregates that inhibit enzymes by gumming them up, rather than "docking" monogamously into an enzyme pocket, report Brian K. Shoichet, chemistry professor at Northwestern University, his graduate student Susan L. McGovern, and their colleagues [J. Med. Chem., 45, 1712 (2002)]. Fortunately, it's simple enough to test for aggregates right off the bat.

"It's important to understand the molecular basis for false positive activity, and this paper makes an important contribution," says Mark Murcko, chief technology officer at Vertex Pharmaceuticals.

The pharmaceutical research community has already pricked up its ears, says Christopher A. Lipinski, a computational chemist with Pfizer Global Research & Development. Since the paper was published online in early March, he notes, three groups at Pfizer have passed it around. "You're talking about the ability, potentially, to save months to years of time," he says.

The fact that some compounds are just bad eggs has been common knowledge among pharmaceutical chemists. But putting a mechanism to the problem has proved difficult. Scientists have had a number of suspicions. For example, it's been thought that some compounds might be acting as covalent inhibitors, chemically reacting with enzymes.

"This work represents an intriguing observation as to why certain hits in high-throughput screening don't make it beyond the screening stage," says Yvonne Martin, a computational chemist at Abbott Laboratories.

Shoichet's group made the discovery while searching for a penicillinase inhibitor. Most of the hits from their screening program were promiscuous.

Several unusual characteristics pointed them toward the problem's source. Most significantly, when they increased the enzyme's concentration, the molecules' ability to inhibit disappeared. The logical explanation, Shoichet says, was aggregates. "Everything clicked when we did that," he says. And with transmission electron microscopy, they indeed saw aggregates 10 to 100 times bigger than the enzyme.

Next, they persuaded the pharmaceutical company Pharmacia to give them 35 compounds from its libraries. Tests soon found that 20 of those compounds formed aggregates.

"People can begin to think more about the problem, now that there's some data," says Gerald M. Maggiora, a senior computational scientist at Pharmacia's lab.

"Both experimental and computational screening teams should be aware of this extensive set of false positives," says Irwin D. (Tack) Kuntz, director of the Molecular Design Institute of the University of California, San Francisco.

The aggregates' structures, as well as why and how often molecules form them, are still mysteries. But ultimately, Lipinski says, the work should begin to address "a fundamental problem that's been plaguing people for a long time."

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Copyright © 2002 American Chemical Society