The enzyme acetylcholinesterase (AChE)has been used to select the chemicalbuilding blocks to synthesize its ownnovel inhibitor. Researchers at the ScrippsResearch Institute (La Jolla, CA, USA), ledby Nobel laureate K. Barry Sharpless,have exploited ‘click chemistry’ to com-bine azides and acetylenes to give a1,2,3-triazole compound that fits per-fectly into the active site of AChE. Thenew inhibitor has activity in the fem-tomolar range (approximately one-trillionth of a gram per litre), making itseveral hundred times more potent thancurrently known AChE inhibitors used totreat Alzheimer’s disease (AD)-associateddementia.

Click chemistry‘The idea that enzymes can direct the as-sembly of good inhibitors is not new, butit is revolutionary in the sense that it hasyet to reach its full potential in practicalapplications,’ says M.G. Finn, co-authorof the study. Sharpless, Finn and col-leagues selected several potential build-ing blocks that could combine to form98 potential site-specific inhibitors forAChE. The building blocks, based ontacrine and phenanthridinium motifs,were decorated with alkyl azides andalkyl acetylenes of varying chain lengths.Each of the possible combinations wasincubated in the presence of AChE atroom temperature.

MS analysis of the reactions that occurred showed that only one combi-nation had resulted in the formation of atriazole compound. Control experimentsdemonstrated that blocking the activesite of AChE inhibited the formation ofthis triazole, indicating that the enzyme’sbinding cleft was acting as a templatefor the 1,3-dipolar cycloaddition reac-tion that resulted in the novel inhibitor

[1]. ‘The cycloaddition of azides withalkynes is an ideal reaction for clickchemistry,’ explains Finn, ‘because thetwo reactive functional groups are onlyreactive with each other and not withthe protein.’ Finn emphasizes that theScripps group did not discover the reac-tion, but they have exploited its potentialin a new way.

The use of click chemistry has impor-tant advantages in the discovery andprocess aspects of drug discovery. ‘As weillustrated, the in situ approach providesa powerful way to identify promisingstructures. Allowing the target to makeits own inhibitor will often be more effi-cient than making and testing each of the candidates individually,’ explainsFinn. He also points out that if one isoften faced with the problem of improv-ing a complex matrix of properties in-cluding binding affinity, bioavailabilityand details of formulation, click chem-istry makes it easier to synthesize andtest large numbers of analogues for thispurpose.

‘This concept is fascinating,’ com-ments Samuel J. Danishefsky, KetteringChair and Director of the Laboratory forBioorganic Chemistry (Sloan-KetteringInstitute, New York, USA). ‘Sharplessuses relatively straightforward chemistryto present combinatorially a range ofacetylenes to react with a range of azides.Ordinarily, such a set-up would give riseto a diverse mixture of products, but

because the Scripps team conductedsuch a reaction in the context of a pro-tein, each core component of the reac-tion was selected to bind to a differentsite in the protein,’ observes Danishefsky.By bringing the components for the cycloaddition together, the protein se-lects the optimal spacing between eachfunctional group. ‘Since the reaction islikely to be irreversible, it seems that thethree-dimensional structure of the pro-tein is being used to control the playersin the cycloaddition through totally kinetic means,’ he says.

A one-off?What is not yet clear is if the Scrippsgroup were simply lucky in the reactionthey chose to investigate in this study.The click reaction chosen is slow on itsown, so any ‘hits’ that occurred (i.e. any‘clicking together’ of the candidatepieces) were likely to result from thepieces being held together in just theright way. ‘This suggests that the bind-ing of the components to the targetwould be just right in this case, other-wise the required connection would notbe formed,’ says Finn, ‘Still, none of usexpected the resulting molecule to be aspotent as it is, so we might also havestumbled on additional factors thatmake the binding so tight.’ For example,the group looked on the azide andalkyne as ‘benign’ groups, with the jobof only making a connection when theirattached binding groups got into the proper position on the enzyme.However, Finn explained that the result-ing triazole unit itself might have anadded affinity for the enzyme, whichwould make the resulting inhibitor evenstronger than one would be expectedbased on the binding constants of thetwo component pieces.

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Enzyme clicks together its own inhibitorKathryn Senior, freelance writer

Danishefsky thinks that the reason theinhibitors are so potent is because theywere assembled in a fashion where theyhad to be potent. ‘That is what makesthis so brilliant. Sharpless has a devicethat could lead to structures that bindtightly to proteins. In principle, this isone of the goals in the development ofnew drugs,’ he says.

Future applicationsIn principle, the ‘click chemistry’ approachto drug design is applicable to almostany target. If it is possible to use a pureenzyme, the function of which can be

measured easily, then the techniques areeasier to apply. However, Finn stresses,‘the in situ approach has the virtue ofbeing potentially useful when these con-ditions are not met. If everything worksperfectly, one can even imagine admin-istering a set of pieces of a drug to a pa-tient and having the drug assemble itselfat the desired site (e.g. a tumour) in response to the specific nature of the target. I emphasize that we are very, veryfar away from such an application.’

Further development work with otherenzymes is now in progress and Finnconfirms that the group is working

collaboratively with many groups withinand outside of Scripps, on a variety ofdiseases including AIDS, cancer, anthraxand Huntington’s disease. ‘It will be interesting to see how the Scripps scien-tists move this project forward. Minimally,one can be confident that interesting science will accrue. Very possibly, newdrug candidates will ensue,’ concludesDanishefsky.

Reference1 Lewis, W.G. et al. (2002) Click chemistry in

situ: Acetylcholinesterase as a reaction vesselfor the selective assembly of a femtomolarinhibitor from an array of building blocks.Angew. Chem. 41, 1053–1057

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Source plasma for manufacturing plasmaderivative products has been exemptedfrom US Food and Drug Administration(FDA) Guidance that recommends thedeferral of blood donors who have livedfor five years or more in Europe from1980, in an attempt to exclude donorsexposed to bovine spongiform encepha-lopathy (BSE) [1].

The exclusion ruling – due to be im-plemented in May 2002 – follows jointevidence presented to the FDA by AventisBehring (King of Prussia, PA, USA), BayerCorporation (Research Triangle Park, NC,USA) and Baxter Biosciences (Deerfield,IL, USA) on the capacity of their plasma-protein therapy manufacturing processesto remove prions, the infectious proteinsresponsible for BSE and Creutzfeldt–Jakob disease (CJD).

The companies – all members of thePlasma Protein Therapeutics Association

(PPTA) and participants in its Transmis-sible Spongiform Encephalopathy (TSE)Working Group – presented evidence onstudies looking at the removal capacityof prions from manufacturing processesminiaturized to laboratory scale. Thedata demonstrates that many of themanufacturing steps involved in purify-ing proteins also have the ability to remove prions. The studies were made

possible by the development of new,rapid and more practical in vitro assaysfor measuring prions, such as westernblotting and conformation-dependentimmunoassays (CDI).

Plasma is the source of many therapeuticproteins, such as anti-haemophilic clot-ting factor, immunoglobulins, albuminand fibrinogen, which are used in thetreatment of shock, burns, immune defi-ciency disorders, cardiopulmonary bypasssurgery, haemophilia and von Willebranddisease (http://www.plasmatherapeutics.org/plasma_therapeutics/plasma_protein.htm). Each year, more than one millionpeople worldwide receive plasma therapies.

Risk of transmitting CJD‘Although there is only a theoretical riskthat plasma products could transmitCJD, the plasma protein industry feels

New assays for measuring prionsreassure FDAJanet Fricker, freelance writer