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  2. Pseudo-irreversible butyrylcholinesterase inhibitors: Structure-activity relationships, computational and crystallographic study of the N-dialkyl O-arylcarbamate warhead

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

  • Eur J Med Chem. 2023 Feb 5;247:115048. doi: 10.1016/j.ejmech.2022.115048.
Anže Meden 1 Damijan Knez 1 Xavier Brazzolotto 2 Fabrice Modeste 3 Andrej Perdih 4 Anja Pišlar 5 Maša Zorman 1 Maja Zorović 6 Milica Denic 2 Stane Pajk 1 Marko Živin 6 Florian Nachon 2 Stanislav Gobec 7
Affiliations

Affiliations

  • 1 University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Aškerčeva 7, SI-1000, Ljubljana, Slovenia.
  • 2 Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, Unité Neurotoxiques, 91223, Brétigny sur Orge, France.
  • 3 Institut de Recherche Biomédicale des Armées, Département des Plateformes et Recherches Technologiques, Unité Développements Analytiques et Bioanalyse, 91223, Brétigny sur Orge, France.
  • 4 University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Aškerčeva 7, SI-1000, Ljubljana, Slovenia; National Institute of Chemistry, Hajdrihova ulica 19, SI-1000, Ljubljana, Slovenia.
  • 5 University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Biology, Aškerčeva 7, SI-1000, Ljubljana, Slovenia.
  • 6 Faculty of Medicine, Institute of Pathological Physiology, University of Ljubljana, SI-1000, Ljubljana, Slovenia.
  • 7 University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Aškerčeva 7, SI-1000, Ljubljana, Slovenia. Electronic address: stanislav.gobec@ffa.uni-lj.si.
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.

Keywords

Butyrylcholinesterase; Carbamate; Cholinesterase inhibitors; Clickable fluorescent probe; Pseudo-irreversible inhibition.

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