Introducing Substrate Features in Fmoc‐Amino Acid‐Based Compounds Increases the Potency of Butyrylcholinesterase Inhibitors: A Potential Therapeutic for Alzheimer’s Disease

Abstract

Butyrylcholinesterase (BChE) and Acetylcholinesterase (AChE) are enzymes responsible for terminating neurotransmission by hydrolyzing acetylcholine. Low levels of acetylcholine in the brain is correlated to the reduced cognitive function in individuals suffering from neurodegenerative disorders such as Alzheimer’s Disease (AD). Relative to healthy individuals, those suffering from AD exhibit a significant increase in BChE activity, and little change in AChE activity. We previously found that 9‐fluorenylmethoxycarbonyl (Fmoc) amino acid‐based inhibitors have been effective to inhibit BChE. As enzymes are specific for their substrate, esterification of the carboxy‐terminus with a trimethylammonium moiety to mimic the cationic group of the substrate may lead to increased potency and specificity. To test this model, a series of inhibitors bearing a cationic trimethylammonium group were synthesized, purified by silica gel chromatography, and characterized by NMR. Enzyme assays monitored by UV‐Vis spectrometry suggested the compounds were potent inhibitors. However, high‐performance liquid chromatography (HPLC) experiments suggest that the Fmoc‐ester derivatives were acting as a substrate and being hydrolyzed by BChE, complicating analysis and minimizing their potential to act as inhibitors. To combat this limitation, a more stable amide was substituted for the ester. HPLC results indicate this substitution prevents enzymatic hydrolysis. Initial biochemical results for the Fmoc‐amide derivatives suggest the compounds inhibit BChE with KI values in the 0.06–10.0 μM range. Overall, the results suggest that introducing substrate‐like characteristics within the Fmoc‐amino acid‐based background increases their potency and may help guide the generation of future inhibitors.Support or Funding InformationThis research was supported by NIH award number R25GM071638