Biological and Medicinal Chemistry

Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, 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

Beside reversible butyrylcholinesterse inhibitors (BChEIs), a plethora of covalent ones, typically pseudo-irreversible carbamates, have been reported in literature. For the latter, however, in most cases the proper confirmation of their covalent mode of action is lacking. Additionally, the available reports on 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 (hBChEIs) and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead is presented. 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 evidence. The computational studies provided valuable insights into the steric constraints and identified problematic, bulky carbamate warheads that could not reach and carbamoylate the catalytic Ser198. QM calculations lent further evidence that the steric effects seemed to be a key factor in determining the covalent binding behaviour of these carbamate ChEIs and their duration of action. Furthermore, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatization with an azide-containing fluorophore enabled fluorescent labelling of plasma hBChE. This proof-of-concept study highlighted the potential of this novel approach and these compounds to be further developed as clickable molecular probes for investigating tissue localization and activity of ChEs.

Content

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Supplementary material

Thumbnail image of ChemRxiv 08082022 Supporting.docx
Supporting Information for Pseudo-irreversible butyrylcholinesterase inhibitors: SAR, kinetic, computational, and crystallographic study of the N-dialkyl O-arylcarbamate warhead
Contains enzyme kinetics, X-ray crystalography data and NMR spectra.