Tacrine heterobivalent ligands were designed as novel and reversible inhibitors of cholinesterases. On the basis of the investigation of the active site gorge topology of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) and by using flexible docking procedures, molecular modeling studies formulated the hypothesis of extra interaction sites in the active gorge of hBuChE, namely, a mid-gorge interaction site and a peripheral interaction site. The design strategy led to novel BuChE inhibitors, balancing potency and selectivity. Among the compounds identified, the heterobivalent ligand 4m, containing an amide nitrogen and a sulfur atom at the 8-membered tether level, is one of the most potent and selective BuChE inhibitors described to date. The novel inhibitors, bearing postulated key features, validated the hypothesis of the presence of extra interaction sites within the hBuChE active site gorge.

Saxena, Ashima; Sun, Wei; Fedorko, James M.; Koplovitz, Irwin; and Doctor, Bhupendra P., "Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX" (2011). US Army Research. 151.

E2020 (R,S)‐1‐benzyl‐4‐[(5,6‐dimethoxy‐1‐indanon)‐2‐yl]methyl)piperidine hydrochloride is a piperidine‐based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure‐activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N‐benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site‐specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200‐ to 400‐fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site‐directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE–E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active‐site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild‐type BChE, further confirms our hypothesis.

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