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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Chem Biol Interact. Author manuscript; available in PMC 2009 September 25.
Published in final edited form as:
PMCID: PMC2585062

Probing Gorge Dimensions of Cholinesterases by Freeze-Frame Click Chemistry


Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a ~50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChE’s. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti- regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene + azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species specific AChE ligands that conform to the determinants in the gorge.

Keywords: acetylcholinesterase, click chemistry, species specific inhibitors, Drosophila acetylcholinesterase, active center gorge, femtomolar inhibitors


Click chemistry has recently being defined as a ” chemical philosophy introduced by K. Barry Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. This is inspired by the fact that nature also generates substances by joining small modular units”(1). In recent years freeze-frame click chemistry has been tested on number of macromolecular biological templates evolving into an established approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks. Representative examples of successful applications of click chemistry include in situ design of a 200 pM carbonic anhydrase (EC inhibitor (2), 1.7 nM HIV-1 protease (EC inhibitor (3), 62 nM α- 1,3-fucosyltransferase (EC inhibitor (4), and as a chronologically first and most noteworthy design of femtomolar acetylcholinesterase (AChE; EC tight binding reversible inhibitors (5,6,7).

Joining acetylene and azide derivative building blocks in the course of a cycloaddition “click” reaction can inherently produce two different isomeric substitutions of a resulting 1,2,3-triazole heterocyclic ring, syn- and anti- (Scheme I).

In the absence of a macromolecular template or a small molecular metal catalyst both substitutions appear as equally likely reaction product. The influence of the binding site geometry of a macromolecular template or interaction with metal catalyst (8) results in preferential formation of one of the products.

In this communication we analyze products of cycloaddition “click” reaction formed in AChEs and their potency for inhibition of AChE from different species, with aim of designing species specific AChE inhibitors.

Results and Discussion

Comparison of active center gorge geometries of various AChEs reveals high level of similarity between mammalian (mouse) and fish (Torpedo californica) AChEs, while larger differences in both shape and volume (Figure 1) are observed for insect (Drosophila melanogaster) AChE.

Figure 1
Solvent accessible Connolly surfaces generated (using 1.4 Å probe radius) for Drosophila melanogaster, Torpedo californica and mouse AChEs. Cross-section through the center of each molecule reveals different geometries of the centrally located ...

It is therefore not surprising that in situ “click” chemistry screening using the same libraries of acetylene and azide building blocks yields identical products when mammalian and fish AChEs are used as reaction templates (57). All “in situ” generated triazole inhibitors of mammalian or fish AChE were “syn- substituted” reflecting the curvature of their long and narrow active center gorges. In contrast “anti- substituted” triazoles, inherently more elongated in shape, appeared as two to three orders of magnitude weaker inhibitors (Table 1). This very large difference in inhibitory potency was entirely due to a markedly compromised fit of compounds within the active center gorge shape resulting in reduction of their mean residence times in the gorge and acceleration of their dissociation rates from the enzyme (Table 1).

Table 1
Kinetic and equilibrium interaction constants of tightest triazole inhibitors generated “in situ” using fish (eel Electrophorus electricus) and mammalian (mouse) AChE as cycloaddition reaction templates. Given are structures of syn- substituted ...

This similarity in substitution preference is not, however, preserved throughout the entire cholinesterase family of enzymes. Table 2 indicates that insect (Drosophila) AChE as well as mammalian (mouse) butyrylcholinesterase (BuChE) are preferentially inhibited with “anti- substituted” triazoles, and thus show an inverted preference in comparison with mammalian and fish AChEs. This is not surprising and could be predicted to result from the significant difference in geometry of the active center gorge of insect AChE and the gorge of BuChE relative to gorges of other AChEs, as illustrated in Figure 1.

Table 2
Interaction constants of syn- and anti- substituted TZ2/PA6 triazole inhibitors with AChEs from several different species and with BuChE.

The crystal structure of mouse AChE in complex with one of the tightest binding inhibitors syn-TZ2PA6 (9,10) reveals a snug fit of a triazole inhibitor within the active center gorge of AChE captured in an unique, previously unseen conformation (Figure 2B). Weaker binding of this triazole to Drosophila AChE and to mouse BuChE (Table 2) is consistent with a less complementary fit into their active center gorges inferred from molecular modeling (Figure 2A and 2C). Binding of syn-TZ2PA6 to BuChE appears compromised largely because of slower association rate, likely due to the extensively solvated BuChE gorge, where water molecules need to be displaced from the gorge in the course of binding. The interaction with Drosophila AChE is obviously imperfect due to severe steric clashes in the peripheral site area and the lack of triazole ring support near the base of the active center gorge.

Figure 2
Syn-TZ2PA6 bound in the active center gorges of A) Drosophila AChE, B) mouse AChE and C) human BuChE. Panel B represents actual crystal structure of the complex (9; PDB id 1q83) while in panels A and C syn-TZ2PA6 is placed based on a protein overlays ...

An additional and very good indicator of specific differences in the active center gorge conformations of various AChEs is achieved by overlaying a series of tacrine based inhibitors crystallized in complex with fish (Torpedo californica), mammalian (mouse) or insect (Drosophila melanogaster) AChE (Figure 3).

Figure 3
Overlay of nine tacrine based AChE inhibitors crystalized in complex with Torpedo AChE (five inhibitors in red sticks, PDB ids 1acj, 1adc,1ut6,1zgb,1zgc) mouse AChE (two inhibitors in blue sticks PDB ids, 1q83, 1q84) and Drosophila AChE (two inhibitors ...

These structures were overlaid by finding smallest deviations of corresponding AChE protein backbone atom positions in different AChE structures. Inhibitors were not involved in the overlay process. Positions of tricyclic tacrine portions of all nine overlaid inhibitors, however, superimpose remarkably closely indicating strong intermolecular stabilization at the base of all AChE gorges and a high degree of structural similarity between AChEs in this region. Subsequent inhibitor atoms extending towards AChE peripheral site progressively diverge in the overlay, with two points of maximal deviation. One is around the intervening chain and formed triazole of the inhibitory ligands in the narrow passage of the AChE gorge defined by the sidechain of the peripheral site Tyr (121 in fish and 124 in mammalian AChEs), known as a “choke point”. The other divergence is at the end of inhibitory molecules interacting with the AChE peripheral site. Divergence in this region at the peripheral site could be compensated, but only in part, by conformational flexibility of Trp 286 (mammalian numbering) observed for tight binding complexes at the peripheral site (8,9 and Figure 2B and Figure 3B). The two sites of divergence in positions of the three groups of inhibitors (crystallized in fish, mammalian and insect AChEs) clearly indicate differences in shape of the corresponding active center gorges. The congested “choke point” located deep into the active center gorge in fish and mammalian AChEs but is shifted closer to the gorge opening and the peripheral site in the insect AChE. Thus, more space exists around the intervening chain of inhibitors. Effectively the gorge becomes less “channel like” and tortuous. The small gain in gorge volume is however compensated by severe restriction of available binding space in the area around peripheral binding site.

With details of these differences in mind, we are now designing a library of compounds aimed at species specific interactions with different AChEs to produce species selective AChE inhibitors. It is clear that a high affinity Drosophila AChE specific triazole inhibitor will have “anti-“ substitution at the central triazole ring, and that the moiety interacting with the peripheral site will be both smaller in volume and extend over a shorter distance compared to the corresponding specific inhibitors of mammalian or fish AChEs. Additionally, a greater accessible volume creating a wider gorge at the very base of the Drosophila active center gorge should allow for introduction of specific tricyclic ring substitutions of tetrahydroamino acridine that would sterically preclude a tight fit of the inhibitor within the mammalian and fish AChEs, while allowing it to be accommodated in the gorge of Drosophila AChE.

We demonstrate here that “anti-“substituted triazoles have a preference for inhibition of Drosophila AChE. Also, inhibitors containing a peripheral site binding moiety smaller than the phenylphenanthridinium exhibit an order of magnitude greater “syn-“/”anti-“ preference for mouse AChE inhibition (Table 1). Introducing additional steric bulk at the tacrine moiety and combining appropriate building blocks by the “in situ” click chemistry approach, we expect to identify potent ligands that will specifically probe AChE active center gorges in a species specific manner.


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