Biological and Medicinal Chemistry

Pseudo-irreversible butyrylcholinesterase inhibitors: structure–activity relationships, and kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead

Authors

  • Anže Meden University of Ljubljana, Faculty of Pharmacy ,
  • Damijan Knez University of Ljubljana, Faculty of Pharmacy ,
  • Xavier Brazzolotto bInstitut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, Unité Neurotoxiques ,
  • Fabrice Modeste Institut de Recherche Biomédicale des Armées, Département des Plateformes et Recherches Technologiques, Unité Développements Analytiques et Bioanalyse ,
  • Andrej Perdih National Institute of Chemistry ,
  • Anja Pišlar University of Ljubljana, Faculty of Pharmacy ,
  • Maša Zorman University of Ljubljana, Faculty of Pharmacy ,
  • Milica Denic Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, Unité Neurotoxiques. ,
  • Stane Pajk University of Ljubljana, Faculty of Pharmacy ,
  • Florian Nachon Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, Unité Neurotoxiques. ,
  • Stanislav Gobec University of Ljubljana, Faculty of Pharmacy

Abstract

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure–activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure–activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

Version notes

The manuscript was corrected by native English speaker.

Content

Thumbnail image of Meden et al Manuscript.pdf

Supplementary material

Thumbnail image of Meden et al. Supporting Information.pdf
Supporting Information for Pseudo-irreversible butyrylcholinesterase inhibitors: structure–activity relationships, and kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead
Contains enzyme kinetics, X-ray crystalography data and NMR spectra.