Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Abstract

The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-A resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.

Scheme 1.

Structures of the anti1 and syn1 TZ2PA6 regioisomers formed by 1,3-dipolar cycloaddition (7). The phenylphenanthridinium, triazole, and tacrine moieties are shown from top to bottom.

Fig. 1.

Overall fold and structural quality of the TZ2PA6-mAChE complexes. (A) Overall view of the mAChE molecule (cyan ribbon) showing the syn1 isomer (orange bonds; transparent molecular surface) bound within the enzyme active-site gorge; the long Ω loop Cys-69-Cys-96 is displayed in yellow. (B and C) Determined structures of the bound anti1 and syn1 isomers (yellow and orange bonds, respectively; blue nitrogens; numbered triazole atoms; same orientation as in Scheme 1), with the respective 2.45- and 2.65-Å resolution final 2F_o-F_c electron density maps contoured at 1 σ (cyan).

Fig. 2.

Close-up views of the TZ2PA6-mAChE complexes. (A and B) Bound anti1 and syn1 isomers (colored as in Fig. 1) with interacting mAChE side chains colored white, yellow, and green/magenta (blue, nitrogens; red, oxygens) for those that respectively interact with the tacrine, triazole, and phenanthridinium moieties of the isomers. The isomer molecular surfaces are displayed in transparency. The side chains of the catalytic residues Ser-203, Glu-334, and His-447 are shown as white bonds, and hydrogen bonds between mAChE residues and the isomers are shown as white dotted lines. (C) Stereo superimposition of the anti1 and syn1 complexes (colored as in A and B) according to all Cα atoms of mAChE. The side chains of residues Trp-286 and Tyr-337 and of dipeptide Tyr-341-Gly-342, which adopt distinctive positions in the complexes, are shown in magenta and green, respectively. The χ values for the Trp-286 side chain are (anti χ₁ =-73°, χ₂ = 100°; syn χ₁ =-158°, χ₂ = 50°). (D) Stereo superimposition of the anti1 complex with the decidium-mAChE complex (Protein Data Bank ID code 1J07; ref. 10) according to all Cα atoms of mAChE in the two complexes. The anti1 and decidium phenylphenanthridinium moieties (yellow and white bonds, respectively; blue, nitrogens) adopt distinct positions and orientations relative to Trp-286 in the PAS, whereas their alkyl chains diverge. The side chains of the PAS residues are highlighted in green (blue, nitrogens; red, oxygens). The mAChE molecular surface buried at the anti1 complex interface is displayed in transparency.

Fig. 3.

Distinctive topographies of the PAS regions in the anti1 and syn1 complexes. Views of the PAS region of mAChE bound to the phenanthridinium moiety present in the anti1 (A) and syn1 (B) isomers (colored as in Figs. 1 and 2). The mAChE molecular surfaces buried at the complex interfaces are shown in yellow, with the Tyr-72 and Trp-286 side chains highlighted in green and magenta, respectively. The mAChE surface areas (Connolly's surfaces) buried to a 1.6-Šradius probe at the anti1 and syn1 complex interfaces by the phenylphenanthridinium and linker first carbon are 256 and 313 Ų (the double-face burying of the syn1 phenanthridinium being counterbalanced by the deeper burying of the anti1 phenyl). The gorge mouth openings (Richards' surface) for the anti1 and syn1 complexes are 14 and 29 Ų, respectively.