In situ assembly of choline acetyltransferase ligands by a hydrothiolation reaction reveals key determinants for inhibitor design.

Wiktelius, Daniel; Allgardsson, Anders; Bergström, Tomas; Hoster, Norman; Akfur, Christine; Forsgren, Nina; Lejon, Christian; Hedenström, Mattias; Linusson, Anna; Ekström, Fredrik

The potential drug target choline acetyltransferase (ChAT) catalyzes the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in-situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.