Incorporating Substrate Features in Fmoc Amino Acids to Develop Potent and Specific Butyrylcholinesterase Inhibitors for the Potential Treatment of Alzheimer's Disease

Abstract

Alzheimer's Disease (AD) is a chronic neurodegenerative disease leading to irreversible memory loss and is the most common form of dementia. While a cure for AD is not available, inhibitors of the two major classes of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are under investigation to mitigate the effects of this disease. In individuals with AD, studies showed that BChE activity increased while AChE activity was similar. The increased BChE activity is suggested to deplete acetylcholine, a neurotransmitter, and contribute to dementia. We identified Fmoc-amino acids as BChE inhibitors, and a lysine-based compound that shares structural similarities with the substrate was one of the most potent inhibitors. Although the original inhibitor had the same positive charge as acetylcholine, the inhibitor lacked methyl groups found in the actual substrate. We hypothesized that methylation of the lysine side chain better mimics leading to better inhibitors. Due to challenges in synthesizing a trimethylated product, we first synthesized a dimethyl amino analog of lysine using 34% formaldehyde, acetic acid, NaBH4, and 1,4-dioxane. The product was purified by chromatography and characterized by NMR. Enzyme kinetics assays monitored by UV-Vis spectroscopy were then used to determine inhibition constants (KI values). The KI values for the dimethylated product Fmoc-Lys(CH3)2-O- was four-fold lower than Fmoc-Lys-O- (40 μM versus 150 μM, respectively), indicating the dimethylammonium compound is a better inhibitor. Studies comparing BChE and AChE inhibition indicated that the compound selectively targets BChE relative to AChE. Computational docking was then used to visualize possible binding sites of the inhibitor. The results suggested that the cationic amino group was binding in the BChE active site similar to the substrate and supports the hypothesis that adding substrate features lead to a more potent BChE inhibitor. Currently, we are testing different synthesis approaches to obtain the trimethyl ammonium compound to then further test the effect of introducing substrate features. Together, the results are aimed at systematically evaluating the contributions of introducing substrate features in enzyme inhibitors to develop more potent inhibitors.