Institute of Crystallography - CNR

Kinetic Modeling of Time-Dependent Enzyme Inhibition by Pre-Steady-State Analysis of Progress Curves: The Case Study of the Anti-Alzheimer’s Drug Galantamine

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.

International journal of molecular sciences (Online)
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