Kinetic and X-Ray Crystallographic Studies of the Binding of ENA-713 to Torpedo Californica Acetylcholinesterase

Compared with other clinically useful carbamates, the anti-Alzheimer drug, (+)S-N-ethyl-3-[(l-dimethyl-amino)ethyl]-N-methylphenylcarbamate (ENA-713; Exelon™) has a longer duration of action in vivo and preferentially inhibits acetylcholinesterase (AChE) of the brain cortex and hippocampus. To understand the basic inhibition mechanism of ENA-713, we studied its reactions with Torpedo californica (Tc) AChE in vitro. The apparent bimolecular rate constant for progressive inhibition was low: ki= 6 M-1min-1 (0.067M Na/K phosphate buffer, pH 7.4, 25°C). After carbamylation (10mM ENA-713) and removal of excess inhibitor by gel filtration, spontaneous reactivation of TcAChE was observed with a rate constant of 0.004 min-1 (pH 7.4, 25°C). Reactivation was base-catalyzed at pH 5.5–8.0 with a maximum at pH 7.5, and displayed an activation energy of 16 kcal/mol. The apparent reversible binding constant for TcAChE with ENA-713 was 200μM, whereas the hydrolysis product, 3-[(l-dimethylamino)ethyl] phenol (NAP), had a Ki of 0.5μM at pH 7.4, 25°C. Thus, the product apparently bound more tightly to TcAChE than did the intact carbamate in the reversible complex. Trigonal crystals of TcAChE were soaked with ENA-713, and the structure was solved and refined to 2.2A resolution. The refinement showed that Ser200 of TcAChE was methylethyl-carbamylated, and NAP was bound non-covalently in the active site. Significant contacts of NAP included hydrophobic interactions with Phe330 and Trp84. We conclude that ENA-713 can inhibit AChE by two mechanisms: (1) covalent carbamylation followed by slow decarbamylation; and (2) action of the carbamate as a vector to deliver the leaving group, NAP, which is itself a good reversible anti-AChE.