(page 2 of 2)

Click Chemistry

Together with TSRI Assistant Professor Valery Fokin, Professor K. Barry Sharpless, who is the W.M. Keck Professor of Chemistry, and Associate Professor M.G. Finn lead the second of the two chemically oriented projects on the grant.

"Our role is to develop and synthesize molecules that could potentially be inhibitors of HIV protease, and, using chemical tools, to learn more about the mutations of the protease," says Fokin. The technique they use is in situ click chemistry.

Click chemistry, a modular protocol for organic synthesis that Sharpless developed, is a powerful and original approach to drug design. In short, it relies on using energetic yet stable building blocks that will react with each other in a highly efficient and irreversible spring-loaded reaction. In its in situ variant, click chemistry uses the target enzyme itself to bring these building blocks together and to direct the formation of the desired inhibitor.

The idea is to use the HIV protease itself to design its own inhibitor by providing it with various building blocks. Only those building blocks that can form an inhibitor will be selected by the enzyme to "click" together. This technique has great potential to cut through a frenzy of possible inhibitors to demonstrate the best.

"We want to let the enzyme teach us what inhibitors [it prefers]," says Finn. "Those, in general, should be the better inhibitors."

The idea seems almost fantastic, but Sharpless and his colleagues have already had success with in situ click chemistry.

A few years ago, they published a paper in the scientific journal Angewandte Chemie describing the use of this technique to make a powerful inhibitor to acetylcholinesterase, a brain enzyme that 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. They have since expanded on this work using this method in several other systems.

As part of the program project grant, Sharpless and Finn want to see if they can apply the techniques of click chemistry to designing inhibitors of HIV protease.

"That enzyme," says Finn, "should, in principle, be amenable to the same kind of [click chemistry] strategy as acetylcholinesterase."

The strategy, Finn explains, is best applied to enzymes that have regions of protein-protein interfaces, such as proteins like HIV, where a dimer is formed by two identical protease monomers. Acetylcholinesterase itself is not a dimer, but it does have two binding regions adjacent to each other.

These protein-protein interfaces often have multiple potential binding sites for small molecules, and the trick with in situ click chemistry is to find classes of compounds that will bind tightly enough in the two faces of HIV that their proximity will allow their natural reactivity to take over.

Looking for a Short Cut

Employing the enzyme to make its own inhibitor could provide a great short cut. For instance, to take a simple case where inhibitors are made by combining two chemical structures—say one of 10 "A" structures and one of 10 "B" structures—then the possible number of structures multiplies.

With 10 possible "A" structures and 10 possible "B" structures, there would be 100 possible compounds. But with 100 possible "A" structures and 100 possible "B" structures, there would be 10,000 possible compounds.

"We don't have to make and screen all those," says Fokin. "We just have to allow the enzyme to select which ones fit best."

Inside the enzyme's binding pocket, the components should click together into a potent inhibitor of HIV protease, and once Sharpless, Fokin, and Finn recover the inhibitor, they can determine its structure and produce it in much larger quantities so that their colleagues on the program project grant can study the interaction of this inhibitor with the protease in structural and tissue culture experiments.

As the cycle continues, the way that the TSRI researchers envision it, other members of the project will provide mutant and wild type protease, and the Sharpless, Fokin and Finn laboratories will then repeat the process.

Dynamic Therapy

At the moment, this project is still in the early stages.

Together with the computational team, Sharpless, Finn, and Fokin are deciding on the best building blocks to start with. The Sharpless group has also been developing a benign copper catalyst and a methodology for copper-catalyzed "stitching" of azide and alkyne building blocks that will allow them to make a variety of the inhibitor analogs they are interested in. In addition to generating libraries of the analogs, the technique could also be used to produce large amounts of click inhibitors for further studies.

"Everything changed when we discovered the copper-catalyzed process for the synthesis of triazoles," says Fokin. "It makes the whole process go a lot smoother."

Another intriguing question they are asking is whether they can possibly apply the technique of in situ click chemistry in vivo.

In vivo means literally "in life," which in biology is generally understood to apply to experiments that take place in a living organism. In this case, in vivo in situ click chemistry suggests the development of a new type of cocktail—one that is made inside the target enzymes inside a living organism.

The idea is to give a cocktail of building blocks to a patient from which the final structure of the best inhibitor will be made. Only those building blocks that are effective against the protease enzymes encoded by the particular strain of HIV that infects that one patient would react and make inhibitors. Another intriguing alternative is to provide a pharmacist with a collection of building blocks, or "pre-drugs", which can be dispensed to each patient based on the specific information about the mutation.

"We asked, 'Can we actually apply click chemistry to treatment?'" says Fokin. "'Can we use our bodies to decide what kind of inhibitors to make?'"

The idea is that the therapy would be flexible enough so that it would work no matter which strains of HIV infect a person. By giving patients the subunits, the particular drug needed at that moment would be selected by whichever strain of HIV infected them.

While these ideas are tantalizing, they are a long way from becoming a reality. The investigators are still doing the first in situ studies with HIV, and any eventual in vivo studies would have to be done in cells first and then in model systems, before extensive human trials for safety and efficacy could even begin.

Still, says Fokin, the concept as they envisioned it offers a new way to approach what is now the long-standing problem of HIV drug resistance.


1 | 2 |




An inhibitor docked in the HIV protease using Professor Art Olson's AutoDock program. Image courtesy of Valery Fokin.