The formal mechanism of linear mixed and non-competitive enzyme inhibition implies the binding of inhibitors to both the active site of the free enzyme in competition with the substrate, and to an allosteric site on the enzyme-substrate complex. However, it is evident from a review of the scientific literature that the two-site mechanism is frequently mistaken as the actual underlying mechanism of mixed inhibition. In this study, we conducted a comprehensive assessment of the mechanistic relevance of this type of inhibition using a statistical approach. By combining a statistical analysis of the inhibition cases documented in the BRENDA database with a theoretical investigation of inhibition models, we conclude that mixed inhibitors exclusively bind to the active site of enzymes. Hence ruling out any implication of allosteric sites and depriving the two-site model of any mechanistic relevance.

Acetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer’s disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with K_i values of 11.12 ± 2.88 and 29.86 ± 1.12 nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of β-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.

The Michaelis–Menten model of enzyme kinetic assumes the free ligand approximation, the steady-state approximation and the rapid equilibrium approximation. Analytical methods to model slow-binding inhibitors by the analysis of initial velocities have been developed but, due to their inherent complexity, they are seldom employed. In order to circumvent the complications that arise from the violation of the rapid equilibrium assumption, inhibition is commonly evaluated by pre-incubating the enzyme and the inhibitors so that, even for slow inhibitors, the binding equilibrium is established before the reaction is started. Here, we show that for long drug-target residence time inhibitors, the conventional analysis of initial velocities by the linear regression of double-reciprocal plots fails to provide a correct description of the inhibition mechanism. As a case study, the inhibition of acetylcholinesterase by galantamine, a drug approved for the symptomatic treatment of Alzheimer’s disease, is reported. For over 50 years, analysis based on the conventional steady-state model has overlooked the time-dependent nature of galantamine inhibition, leading to an erroneous assessment of the drug potency and, hence, to discrepancies between biochemical data and the pharmacological evidence. Re-examination of acetylcholinesterase inhibition by pre-steady state analysis of the reaction progress curves showed that the potency of galantamine has indeed been underestimated by a factor of ~100.

Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate‐leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible catalytic poisons bearing sulfur instead of phosphorus convert ChEs in its aged form only by covalently binding to the key catalytic serine. Kinetic and crystallographic studies of the interaction between Torpedo californica acetylcholinesterase ( Tc AChE) and a small organosulfonate, methanesulfonyl fluoride (MSF), indeed revealed irreversibly methylsulfonylated serine 200, to be isosteric with the bound aged sarin/soman analogues. The potent bulky reversible inhibitor 7‐bis‐tacrine (BTA) adopts, in the active site of the crystal structure of the MSF‐enzyme adduct, a location and an orientation that closely resemble the one being found in the crystal structure of the BTA‐enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulfonylation by a factor of two. This unexpected result can be explained on the basis of two facts: i) the steric hindrance exerted by BTA to MSF in accessing the active site and ii) the acceleration of the MSF‐enzyme adduct formation as a consequence of the lowering of the rotational and translational degrees of freedom in the proximity of the catalytic serine. It is well known that pralidoxime (2‐Pyridine Aldoxime Methyl chloride, 2‐PAM) alone or in the presence of the substrate acetylcholine cannot reactivate the active site serine of the Tc AChE‐MSF adduct. We show that the simultaneous presence of 2‐PAM and the additional neutral oxime, 2‐[(hydroxyimino)methyl]‐l‐methylimidazol (2‐HAM), triggers the reactivation process of Tc AChE within the hour timescale. Overall, our results pave the way toward the likely use of a cocktail of distinctive oximes as a promising recipe for an effective and fast reactivation of aged cholinesterases.