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Tricking Diseases into Synthesizing Their Own Worst Enemies: A Revolutionary Strategy for Drug Discovery at The Scripps Research Institute (TSRI) Succeeds on an Enzyme

La Jolla, CA. March 15, 2002 - In a first attempt to test a new general strategy for drug discovery, chemists at The Scripps Research Institute (TSRI) and TSRI's Skaggs Institute for Chemical Biology created the most potent blocking agent known against an enzyme implicated in Alzheimer's disease.

In the current issue of the journal Angewandte Chemie, 2001 Nobel laureate K. Barry Sharpless, W.M. Keck Professor of Chemistry at TSRI, and colleagues at TSRI and the University of California at San Diego, describe how click chemistry, a modular protocol for organic synthesis that Sharpless developed, was used to make a drug-like molecule that powerfully blocks the neurotransmitter destruction caused by the brain enzyme, acetylcholinesterase.

Unlike existing methods, this new drug-discovery strategy - click chemistry - mobilizes the target itself, acetylcholinesterase in this case, to play a decisive role and select the final synthetic step. The acetylcholinesterase enzyme actually catalyzed the click reaction that created that enzyme's own inhibitor, and, remarkably, the result is by far the most potent inhibitor ever discovered for this important, widely studied brain enzyme.

"Think of this as a Trojan Horse approach for battling disease, but this horse goes the Greeks one better," says Sharpless. "We create the pieces that can be clicked together to make the horse, then we leave them outside the gates of, for example, a bacterium. If the pieces look right, it goes to work, constructing its own worst enemy, and doing so within its own defensive walls."

"This is a breakthrough typical of Barry Sharpless," says TSRI President Richard Lerner. "For the first time, you are eliciting a contribution from the dynamic enzyme, asking it to make the inhibitor it prefers."

Troy's Homemade Horse

Finding inhibitors, molecules that fit snuggly into the active sites of a particular target and modulate its activities, is the basis for molecular medicine. Essentially all diseases operate by inducing unnatural function in enzymes. Many of those diseases, including cancer, not to mention a whole alphabet of ailments starting with AIDS, Alzheimer's, anthrax, and arthritis, can be treated by inhibiting enzymes.

The enzyme selected for click chemistry's proof-in-practice was one of the first brain enzymes to be identified. Acetylchonlinesterase breaks down acetylcholine, the neurotransmitter that propagates nerve signals. Inhibitors of acetylcholinesterase are used to treat the dementia associated with Alzheimer's disease, increasing the amount of acetylcholine in the brain, in turn enhancing brain activity.

In the current study, Sharpless and his team synthesized specialized molecules, which are stable as they are but which also possess a built-in programmed desire to be incorporated whole into a larger molecule. When several such components in this molecular construction set are brought together in specific ensembles, their pre-programming causes them to react by cycloaddition, predictably and irreversibly clicking together to create a single larger molecule with no by-products.

Under normal circumstances, with the click chemistry components randomly circulating in a reaction vessel, it might take years to line up properly for a click reaction to take place. However, when the target enzyme was introduced into the picture, active spots on the enzyme's surface acted like hands that grabbed and oriented the click components, snapping them together.

Some pairs of click chemistry components will fit together snugly inside the acetylcholinesterase and some will not. The pairs that do fit snugly together are much more likely to snap together in the presence of acetylcholinesterase. In orienting and initiating the reaction, and cutting the reaction time from years to minutes, the enzyme functions as a chemical catalyst.

Sharpless calls this variation of click chemistry "in situ," which is Latin for "in the natural position." In this case, the reaction is in situ because the enzyme directs which way the pieces come together. "Once together in the correct orientation, they will click," he says.

More than the sum of the two parts, the triazole acetylcholinesterase inhibitor the team found has powerful "femtomolar" (10-15) activity against the enzyme. This exceeds by several hundred times the potency of the hundreds of previously known acetylcholinesterase inhibitors.

"I think it is one of the most fascinating ideas I have ever heard," says Professor Samuel J. Danishefsky of Memorial Sloan-Kettering Cancer Center and Columbia University, who heard Sharpless lecture on this research at Columbia University about a week after the announcement of his Nobel Prize. "The very enzyme that you are trying to inhibit was used to assemble the inhibitor."

"It works a lot better than we ever anticipated," says Sharpless.

A Simple and Fascinating New Approach

The reaction is an example of what Sharpless calls "click chemistry," a methodology for chemical synthesis he invented a few years ago.

"The idea [of click chemistry] is a very simple one," says TSRI Associate Professor M.G. Finn of the Department of Chemistry and The Skaggs Institute for Chemical Biology, who is an author on the report. "If you are going to make a drug (or anything), why do it with techniques that are difficult when you can do it with techniques that are easy?"

In click chemistry, chemicals (like acetylcholinesterase inhibitors) are made from modular chemical "blocks" that can be joined together in various combinations in very few steps. Reactions are chosen from readily available starting materials that react with high reliability and form easily isolated products in high yield without additional reagents.

Sharpless calls the reactions that join these blocks together "spring-loaded" because the blocks are designed to have a higher energy content than the product, which enables them to react together and form larger structures reliably.

The azides and acetylenes that were used to make the acetylcholinesterase inhibitors are, according to Sharpless, "cream of the crop" building blocks for click chemistry, because they will not react with other molecules but instead fuse irreversibly into various product structures (triazoles) when brought together.

Selecting the one triazole that is the best inhibitor of acetylcholinesterase was the job of the acetylcholinesterase enzyme itself.

This enzyme has a large binding pocket with separate places for the azides and acetlyenes to bind. When the two separate building blocks both bind to the acetylcholinesterase, they can react and form a triazole - the one that fits best inside acetylcholinesterase.

The best inhibitors thus formed will be those that bind tighter than the azides and acetylenes from which they are formed.

The research article "Click Chemistry In Situ: Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a Femtomolar Inhibitor from an Array of Building Blocks" is authored by Warren G. Lewis, Luke G. Green, Flavio Grynszpan, Zoran Radic, Paul R.Carlier, Palmer Taylor, M.G.Finn, and K. Barry Sharpless and appears in the March 15, 2002 issue of Angewandte Chemie.

The research was funded by the National Institute for General Medical Sciences, the National Institutes of Health, the National Science Foundation, The Skaggs Institute for Research, the W.M. Keck Foundation, and the J.S. Guggenheim Memorial Foundation.

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