Choline Esterase inhibition assay
Acetyl and butyryl choline esterases (AChE and BChE), are related enzymes found across the animal kingdom. AChE role as a neurotransmitter is well known several years ago, however, the role of BChE is newly emerging. Choline esterases are widely targeted for their possible therapeutic value and toxicity, inhibitors of choline esterase are used for a variety of disorders such as Alzheimer’s disease (AD) and conversely for their toxic potential as pesticides. BChE exhibits broader substrate specificity, that can act on both acetyl and butyryl choline, while AChE only acts on the acetylcholine. However, it is not acting as a neurotransmitter, recently the effect of BChE on the hydrolysis of ghrelin is reported. Ghrelin serves as a stimulant for hedonic feeding, promoting food intake and fat storage. Therefore, it can regulate feeding behavior and food intake.
In addition to its central actions, AChE inhibitors exhibited several peripheral functions including skeletal and smooth muscle contraction, parasympathetic end-organ activation, and regulation of cardiac activity.
Alzheimer’s disease (AD) is the most common reason for dementia in elderly individuals all over the world. AD was reported to be associated with cholinergic neuron degradation leading to a decrease in ACh release. ACh deficiency results in impaired cognitive abilities, memory functions, and emotional responses. One of the effective therapeutic strategies for AD is the use of AChE inhibitors which results in the improvement of cholinergic function.
Both AChE and BChE are exhibiting non-catalytic function in the morphogenic roles in the neurodevelopment of vertebrates. In addition, AChE and BChE were found to deposit in amyloid plaques associated with dying neurons in AD. BChE is present in glial cells, hippocampus, and the temporal nerve cortex, and is involved in cognitive function. Despite the minor role of BChE in the hydrolysis of ACh in healthy brains, several studies revealed that in the case of AD, BChE activity remarkably increases, while that of AChE decreases Consequently, both enzymes play a prominent role in the regulation of ACh and they are considered as essential curative targets to increase cholinergic levels.
1- Acetylcholine esterase inhibition
AChE catalyzes the hydrolysis of AChE to choline and acetic acid. In this assay, Acetyl thiocholine (ASCH) is used as a substrate for the enzyme. ASCH is hydrolyzed by AChE to produce thiocholine, which produces a strong yellow color after reaction with DTNB reagent, that can be measured calorimetrically indicating the enzyme activity.
2- Butyryl choline esterase inhibition
Similar to acetylcholine esterase, BChE can hydrolyze butyryl thiocholine producing thiocholine which reacts with the DTNB to produce yellow color indicating its activity.
Results (end point measurement) of each assay can be represented either as
1- Initial screening at two concentration levels (100 and 10 ug/mL or uM)
2- IC50 (5 concentrations).
N.B. kinetic measurement is not available right now