| STRUCTURE |
TO UNDERSTAND why the inhibitors generated by click chemistry are so powerful--inhibiting at picomolar levels--the Sharpless group turned to several acetylcholinesterase experts: biochemist Pascale Marchot and structural biologist Yves Bourne, working in Marseille for the French National Center for Scientific Research (CNRS), and pharmacologists Zoran Radic and Palmer W. Taylor at the University of California, San Diego. Their recently published structural studies of the inhibitors complexed to acetylcholinesterase explain not only why the click-chemistry inhibitors are so good but also why the enzyme-synthesized syn product is so much better than the chemically synthesized anti product [Proc. Natl. Acad. Sci. USA, 101, 1449 (2004)]. The findings suggest a different approach to drug discovery: harnessing a protein's flexible template to reveal vulnerable conformations that can be accessed by specific chemistries. The structural studies confirm that the click-chemistry inhibitors bind both the enzyme's active and secondary sites. They also show that the triazoles formed are not simply passive linkers but actually interact with the gorge. "Binding interactions within the gorge itself have not been observed with other inhibitors," Marchot tells C&EN. "These interactions may contribute to the exquisite potency of the click-chemistry products." The difference in the potency of the syn and anti products is explained by their binding at the secondary site. A part of the syn product inserts itself between certain enzyme residues near the gorge's rim, whereas the anti product does not. The insertion makes it difficult for the enzyme to make the usual motions for shuttling substrates or inhibitors in and out of the active site. The enzyme is forced into a conformation from which it cannot easily free itself. According to Marchot, the insertion is made possible by a newly found, more open conformation of the enzyme, in which a tryptophan residue at the gorge's rim flips out to the solvent, creating the space that makes insertion possible. Such a conformation had not been seen in other structures--of the free enzyme or its complexes with inhibitors bound at the secondary site--that she and Bourne have analyzed [EMBO J., 22, 1 (2003)]. The new conformation "may have important implications for the enzyme's functioning," she says. "I'm not sure how many people really understand the significance of these results," Sharpless told C&EN. "For the triazole to form in the enzyme, the two pieces--the azide and the acetylene--and the enzyme danced together up the energy surface to a point that people don't see normally," Sharpless explained. "When they got there, the two pieces felt at home and formed the bond. And the product got stuck." Ironically, such simple triazoles are poorly represented in the literature, he noted |
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