Screening for Alzheimer's drugs in tandem

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By combining enzymes, a macrocyclic sensor and a dye, scientists may be able to find new drugs for Alzheimer's disease

A simple assay based on sequential enzymatic reactions and a fluorescent sensor could help scientists to discover new Alzheimer’s disease drugs.

Some Alzheimer’s drugs work by blocking the activity of acetylcholinesterase, an enzyme that degrades the neurotransmitter acetylcholine to choline. To find new enzyme inhibitors, researchers need to identify choline formation, or the loss of acetylcholine, so they can tell whether the enzymatic reaction has stopped. But, acetylcholine and choline are both quaternary ammonium ions with very similar structures, making it difficult to distinguish between them.

To overcome this problem, teams led by Werner Nau at Jacobs University Bremen, Germany, and Yu Liu at Nankai University, China, have combined two sequential enzymatic reactions with a calixarene macrocycle that binds to a fluorescent dye to make a tandem assay that can screen for new inhibitors. The enzymes are highly specific and only work on one substrate.

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The tandem reaction involves a fluorescence ’’switch-on’’ displacement assay as a sensor for specific analytes

In their assay, they use acetylcholinesterase to turn acetylcholine to choline. A second enzyme - choline oxidase - turns the choline into betaine. While choline and betaine are similar, they have different affinities for binding within the calixarene. Because of this difference, the dye can replace the betaine inside the calixarene. This turns off the dye’s fluorescence, which is easy to detect. If the enzymatic reactions are inhibited, no betaine will be produced and so the dye’s fluorescence stays on.

Pablo Ballester, an expert in supramolecular chemistry at the Institute of Chemical Research of Catalonia, Spain, welcomes the research. He says it ’represents a clever implementation of the use of enzymes to overcome the reduced binding selectivity exhibited by the negatively charged synthetic receptors of the calixarene family in the recognition of quaternary ammonium ions.’

Nau’s team demonstrated their assay with acetylcholinesterase and choline oxidase but the principle could be adapted to other enzymatic reactions. In the future, Nau hopes to ’set up a large library of calixarene and dye pairs that are useful for a large range of analytes and biochemical reactions.’

Russell Johnson