Tricking Diseases into Synthesizing Their Own Worst Enemies:
A Revolutionary Strategy for Drug Discovery Succeeds on an Enzyme
By Jason Socrates Bardi
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 March 15 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 strategyclick
chemistrymobilizes 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
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,"
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
"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
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 triazolethe
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 Chemical Biology, the W.M. Keck Foundation, and
the J.S. Guggenheim Memorial Foundation.
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