One of the leading neurodivergent diseases ailing the elderly today is Alzheimer's disease (AD). Despite being so common among the older demographic, AD has no definitive treatment. This research study's purpose is to find treatments for this disease by inhibiting cholinesterase enzymes. Prior research discovered that levels of acetylcholine, a neurotransmitter, were unusually low in patients with AD. Other studies also found that the enzyme activity of butyrylcholinesterase (BChE) increased in individuals with Alzheimer's disease while the activity for acetylcholinesterase (AChE) remained the same. Because cholinesterases hydrolyze acetylcholine, a potential approach to restoring acetylcholine levels would be the inhibition of cholinesterases. We previously reported that amino acids bearing the 9-fluorenylmethyloxycarbonyl (Fmoc) group selectively inhibited BChE over AChE. One of the most potent Fmoc-amino acids in the initial study was Fmoc-Lys-O^-. It was postulated that the cationic ammonium group mimicked the cationic group of acetylcholine and facilitated similar binding interactions with the enzyme. Although both Fmoc-Lys-O^- and acetylcholine bear a positively charged group, they differ in that Fmoc-Lys-O^- ammonium group lacks the methyl (CH₃) substituents found in acetylcholine. One possibility is that the methyl groups facilitate van der Waals interactions with the enzyme leading to more favorable binding. To test this model, we are investigating the effect of adding methyl groups to the side-chain amino group. Enzyme inhibition is determined using kinetics assays using Ellman’s reagent and monitored by UV-Vis absorbance. Initial results comparing Fmoc-Lys-O^- and its trimethylated analog, Fmoc-Lys(CH₃)₃-O^-, suggest a modest <five-fold improvement in inhibition, but also a weak inhibition of AChE. The results highlight the challenge of developing potent and selective inhibitors. We are currently conducting computational experiments to evaluate the potential interactions of the molecules with the enzymes. We have also moved forward in evaluating the inhibitory effects of the Fmoc-Lys-O^- dimethylated and monomethylated analogs to test the relative importance of adding methyl groups to the original Fmoc-Lys compound. Together, the studies are aimed at identifying the features important for BChE inhibitors with an overall aim of developing more effective inhibitors to aid in the treatment of Alzheimer’s Disease.

Jason Schwans

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