title: Determination of affinity and efficacy of acetylcholinesterase inhibitors using isothermal titration calorimetry.
abstract: BACKGROUND
  Acetylcholinesterase (AChE), an enzyme rapidly terminating nerve signals at synapses of cholinergic neurons is an important drug target in treatment of Alzheimer's disease and related memory loss conditions. Here we present comprehensive use of isothermal titration calorimetry (ITC) for investigation of AChE kinetics and AChE-inhibitor interactions.
  
  
  METHODS
  Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus was assayed for interactions with five well known AChE inhibitors, galanthamine, tacrine, donepezil, edrophonium and ambenonium. In ITC experiments the inhibitors were injected to the enzyme solution solely (for thermodynamic characterization of binding) or in presence of the substrate, acetylcholine (for determination of inhibitors potency).
  
  
  RESULTS
  Detailed description of various experimental protocols is presented, allowing evaluation of inhibitors potency (in terms of IC50 and Ki) and thermodynamic parameters of the binding. The potency of tested inhibitors was in nano to micromolar range which corresponded to activities determined in conventional method. Binding of all inhibitors showed to be enthalpy driven and obtained Ka values demonstrated good correlation with the data from standard Ellman's assay.
  
  
  CONCLUSIONS
  Obtained results confirmed the usability of the ITC technique for comprehensive characterization of AChE-inhibitor interactions and AChE kinetics. The method reduced the complexity of reaction mixture and interference problems with the advantage of using natural substrates.
  
  
  CONCLUSIONS
  The work reports complete thermodynamic characteristics of the AChE - inhibitor complexes. Due to the universal character of ITC measurements, described protocols can be easily adapted to other enzymatic systems.
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Draczkowski
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Tomaszuk
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Pawel
authors.initials: P
authors.lastname: Halczuk
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Strzemski
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Matosiuk
authors.affiliation: Faculty of Pharmacy, Medical University of Lublin, ul. Chodzki 4a, 20-093 Lublin, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Jozwiak
mesh_terms: D000109:Acetylcholine / Q000737:chemistry*; D000110:Acetylcholinesterase / Q000737:chemistry*; D000549:Ambenonium Chloride / Q000737:chemistry; D000818:Animals; D002151:Calorimetry / Q000379:methods; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D000077265:Donepezil; D004491:Edrophonium / Q000737:chemistry; D004593:Electrophorus / Q000378:metabolism; D029941:Fish Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005702:Galantamine / Q000737:chemistry*; D007189:Indans / Q000737:chemistry; D007700:Kinetics; D010880:Piperidines / Q000737:chemistry; D013619:Tacrine / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D002800:Cholinesterase Inhibitors; D029941:Fish Proteins; D007189:Indans; D010880:Piperidines; D005702:Galantamine; D013619:Tacrine; D000549:Ambenonium Chloride; D004491:Edrophonium; D000077265:Donepezil; D000110:Acetylcholinesterase; D000109:Acetylcholine
keywords: Acetylcholinesterase; Enzyme activity assay; ITC; Inhibitor; Interactions; Isothermal titration calorimetry