It’s not exactly perpetual motion, but last year’s Nobel prize-winning chemist has since announced a drug discovery strategy in which the target enzyme automatically catalyzes its own inhibitor.

Nobelist K. Barry Sharpless at the Scripps Research Institute in La Jolla, Calif., calls his invention “click chemistry.” His report of which he is senior author appears in the international English-language journal Angewandte Chemie (Applied Chemistry), dated March 15, 2002, and published in Weinheim, Germany. The paper’s title: “Click chemistry in situ: Acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks.” Its lead author is bioorganic chemist Warren Lewis, a candidate graduate student at Scripps.

“One thing we did,” Lewis told BioWorld Today, “was synthesize the most potent blocker of acetylcholinesterase known. We found that we could use the enzyme itself to generate its inhibitor. Normally what people do,” he observed, “is make a bunch of compounds, test them on high-throughput bioassays, then synthesize all the individual compounds. That would be pretty difficult to do, but doable.

“What we did instead,” Lewis recounted, “was a synthesis where we used the enzyme to put its two pieces carbon and nitrogen atoms together. These two binders are at different sites in the acetylcholinesterase enzyme, of which the inhibitors are Alzheimer’s disease [AD] therapeutics. Acetylcholinesterase,” he explained, “breaks down acetylcholine, the neurotransmitter that propagates nerve signals. Inhibitors of the enzyme treat AD dementia, increasing acetylcholine and enhancing brain activity. Our drug-like enzyme inhibitor powerfully blocks the acetylcholine neurotransmitter destruction caused by that brain enzyme.”

Sharpless pointed out, “Finding inhibitors molecules that fit snugly 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 functions in enzymes. Many of these ailments,” he added, “including cancer not to mention a whole alphabet of ills, starting with AIDS, Alzheimer’s, anthrax and arthritis can be treated by inhibiting enzymes.”

Lewis described the atomic structure: “It has an active-site binding region at the base of the structural gorge or pocket. That gorge leads down to the bottom of the enzyme, where acetylcholine sits. We took one piece, which binds to the bottom of the gorge, and another piece that binds to the top. Then we put complementary reactive groups on those pieces. They’re designed to react with each other to connect up the two pieces. The groups carry their own energy into the reaction.”

Enzyme Blocker Surpasses All Prior Inhibitors

“We showed that this click chemistry technique,” Lewis went on, “can find in a small combinatorial library one of the potent enzyme inhibitors, using the target itself rather than making the compounds individually first, then screening them. We used the target to generate its own inhibitor, and were able to make a blocker that was far better than anything anybody knew before.

“People have tried to block the acetylcholinesterase enzyme,” Lewis noted, “to generate therapies for brain diseases like Alzheimer’s and myasthenia gravis. So basically it’s a drug target. They have made inhibitors, but these are generally in the nanomolar down to the picomolar range. That is, they’re moderately tight binders. We made one with femtomolar activity against the enzyme. It’s hundreds of times tighter than the stuff that’s already out there. Specifically, it associates with the enzyme, and stays there. It doesn’t come off for a long time. The others come loose much more rapidly.

“Think of this click chemistry as a Trojan horse approach for battling disease,” Sharpless analogized, “but this horse does the Greeks one better. We create the pieces that can be clicked together to make the horse, then leave it outside the gates of for example a bacterium. If the pieces look right, it goes to work, constructing its own worst enemy the inhibitor.”

“Applying the process of cycloaddition,” Lewis related, “we took these two pieces that have all the energy and put them close to one another. These are carbon-carbon triple bonds and three nitrogens in a row or chain. You can imagine that these three atoms come together with the other two, flip around and wake up to give you a five-member ring.”

The click chemistry team, Lewis pointed out, is not at present focusing on designing a better drug for Alzheimer’s disease. “The academic lab at this point doesn’t do the drug stuff,” he observed. “Our click chemistry work is more of a proof-of-principle. It’s possible,” he allowed, “that we will come out with an Alzheimer’s drug in the future, using these techniques, with what we discovered about the acetylcholinesterase enzyme.”

Refining The Click Chemistry Technique

“Meanwhile,” he said, “for one thing, I’m working on improving the analytical techniques, to find where the enzyme is able to bring the two pieces together. That’s quite a challenge,” he explained, “because as soon as we make an inhibitor, it doesn’t come off the enzyme anymore. It just stays there, so we’re not able to generate more of it. We’re left with a limited amount of stuff, which makes this a really general technique. We have to come up with a way of detecting very small amounts of the product.

“At the same time,” he went on, “we’ll start applying it to other systems to other disease targets. We have metalloproteinases for one thing, against cancer and arthritis. And we’re interested in adding proteases in general. Also a lot of motifs that we can use, such as inhibitor scaffolds, we can call upon to build large and very potent enzyme inhibitors.

“We’re always looking for new systems to work on,” Lewis observed. “If any pharma or biotech company has an expression system and is trying to do structure-activity relationships to develop more potent inhibitors, we’re interested in talking to them. They can certainly use our click chemistry techniques. We’ve talked to people from Novartis [AG], but nothing has really materialized there yet. We’ve stimulated some interest,” he went on. “There are some people talking to us, but there are a lot of practical challenges that we have to meet in these collaborations: taking this academic example and bringing it into real-world application.”