In the fairy tale Cinderella, the prince has to go through the entire kingdom, trying to find his princess by testing people's feet to see if they fit a glass shoe. Time consuming, but not too bad compared with conventional searches for the 'feet' in drug discovery — that is, the drugs — which have often been performed without any knowledge of the protein 'shoe'. To stretch an analogy, structure-based drug design addresses this issue to some extent by using knowledge of the shoe to preassemble feet more likely to fit, but better still, what if the shoe itself could be used to make the ideal foot? This is the idea behind in situ 'click chemistry', a strategy developed by Barry Sharpless and colleagues, which could be thought of as testing a range of foot parts designed to link together in the shoe only if they can form a foot that fits perfectly. The first example of the application of this technique to the discovery of a femtomolar inhibitor of acetylcholinesterase (AChE) is described in a recent issue of Angewandte Chemie.

Click chemistry uses chemical building blocks containing functional groups that are thermodynamically 'spring-loaded' to react only with appropriate complementary functional groups in other blocks. The present study exploited the Huisgen 1,3-dipolar cycloaddition, which links azides and acetylenes — functional groups that are generally compatible with enzymes under physiological conditions, and that can easily be incorporated into a wide range of building blocks. The Huisgen cycloaddition can be carried out in water, and — ideally for the current purpose — the reaction depends on the proximity and appropriate alignment of the two components.

AChE, a target of drugs for Alzheimer's disease, was used as a template for the assembly of the building blocks — in this case, small molecules known to bind with affinities in the micromolar to nanomolar range to either the active site or an adjacent site of AchE. These were linked to azide or acetylene groups by alkyl chains of varying lengths. Each of a possible 49 pairs of small molecules was incubated with AChE, and subsequent examination of the reaction mixtures by mass spectrometry indicated that only one azide–acetylene pair, TZ2–PA6, had combined. No such reaction occurs in the absence of enzyme, and blocking the active site inhibited the formation of TZ2–PA6, showing that the active site is acting as the template.

TZ2–PA6 is the strongest non-covalent inhibitor of AchE discovered so far by two orders of magnitude. It seems likely that broader applicability of the in situ click chemistry approach will be the subject of much future research.