Inhibitor Report for: TBTFA

The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.

Ten meta-substituted aryl trifluoromethyl ketones (m-XC6H4COCF3; X = H, CH3, CF3, C2H5, isopropyl, t-butyl, NH2, NMe2, N+Me3, NO2) have been evaluated as inhibitors of acetylcholinesterases from Electrophorus electricus and Torpedo californica. Trifluoro ketones that have small meta substituents (X = H, CH3, CF3, C2H5, NH2, NO2) are rapid reversible inhibitors, whereas the remaining compounds in this study show time-dependent inhibition. Dissociation constants (Ki values) for these compounds span a range of approximately 10(7)-fold, with trifluoroacetophenone (X = H) being the least potent and m-(N,N,N-trimethylammonio)trifluoroacetophenone (X = Me3N+) being the most potent inhibitor. For the latter compound Ki values are 1.5 and 15 fM for inhibitions of the respective acetylcholinesterases (Nair, H. K., Lee, K., & Quinn, D. M. (1993) J. Am. Chem. Soc. 115, 9939-9941). Linear correlations of log(kcat/Km) for substrate turnover versus pKi of inhibitors have slopes of approximately 0.6, which suggest that aryl trifluoro ketones bind to AChE in a manner that structurally resembles transition states in the acylation stage of catalysis. Substituent variation in the inhibitors allows one to gauge the importance for AChE function of molecular recognition in the quaternary ammonium binding locus of the active site. This locus is frequently termed the "anionic site" and consists of E199, W84, and perhaps Y130 and F330. Correlations of pKi versus hydrophobicity constant are linear for alkyl and trifluoromethyl substituents but fail for nitrogen-containing substituents. However, three-dimensional correlations of pKi versus sigma m and molar refractivity of substituents indicate that dispersion interactions in the anionic locus contribute approximately 10(5)-fold (delta delta G = 7 kcal mol-1) to the above-mentioned 10(7)-fold range of inhibitor potencies. The remaining approximately 100-fold arises from the inductive electronic effects of substituents on the stability of the tetrahedral adduct that forms between the ketone carbonyl of inhibitors and S200 in the esteratic locus of the active site. Values of k(on), the second-order rate constant for binding of time-dependent inhibitors, monitor a diffusion-controlled process. Moreover, k(on) for the quaternary ammonio inhibitor is 20-70-fold higher than for inhibitors that have uncharged meta substituents, which likely reflects the effect of the electrical field of AChE on ligand and substrate binding.

m-(Tert-butyl) trifluoroacetophenone (TFK), a slow-binding inhibitor of acetylcholinesterase (AChE), a transition state analog of acetylcholine, was investigated as a potential neuroprotectant of central and peripheral AChE against organophosphate paraoxon (POX) toxicity. Acute toxicity and pharmacological effects of TFK were investigated on mice and rats. Intraperitoneal administered TFK has low acute toxicity in mice (LD(50) = 19 mg/kg). Effects on motor function as investigated by rotarod and open field tests showed that TFK up to 5 mg/kg did not alter motor coordination and stereotypical exploration behavior of mice. Passive avoidance test showed that 1 or 5 mg/kg TFK restored memory impairment in scopolamine-induced Alzheimer's disease-like dementia in rats. Pretreatment of mice with 5 mg/kg TFK, 2-3 hrs before challenge by 2xLD(50) POX provided a modest and short protection against POX toxicity. Futhermore, analysis of POX-induced neuronal degeneration by using fluoro-jade B staining showed that TFK pretreatment, at the dose 5mg/kg before POX challenge, significantly reduced the density of apoptotic cells in hippocampus and entorhinal cortex of mice. Thus, TFK is capable of reducing POX-induced neurotoxicity.

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with K(i) = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with K(i)* = 0.53 nM. Reversible binding of TFK provides a long residence time, = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

Association of cholinesterase with beta-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine (18F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.

The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.

Ten meta-substituted aryl trifluoromethyl ketones (m-XC6H4COCF3; X = H, CH3, CF3, C2H5, isopropyl, t-butyl, NH2, NMe2, N+Me3, NO2) have been evaluated as inhibitors of acetylcholinesterases from Electrophorus electricus and Torpedo californica. Trifluoro ketones that have small meta substituents (X = H, CH3, CF3, C2H5, NH2, NO2) are rapid reversible inhibitors, whereas the remaining compounds in this study show time-dependent inhibition. Dissociation constants (Ki values) for these compounds span a range of approximately 10(7)-fold, with trifluoroacetophenone (X = H) being the least potent and m-(N,N,N-trimethylammonio)trifluoroacetophenone (X = Me3N+) being the most potent inhibitor. For the latter compound Ki values are 1.5 and 15 fM for inhibitions of the respective acetylcholinesterases (Nair, H. K., Lee, K., & Quinn, D. M. (1993) J. Am. Chem. Soc. 115, 9939-9941). Linear correlations of log(kcat/Km) for substrate turnover versus pKi of inhibitors have slopes of approximately 0.6, which suggest that aryl trifluoro ketones bind to AChE in a manner that structurally resembles transition states in the acylation stage of catalysis. Substituent variation in the inhibitors allows one to gauge the importance for AChE function of molecular recognition in the quaternary ammonium binding locus of the active site. This locus is frequently termed the "anionic site" and consists of E199, W84, and perhaps Y130 and F330. Correlations of pKi versus hydrophobicity constant are linear for alkyl and trifluoromethyl substituents but fail for nitrogen-containing substituents. However, three-dimensional correlations of pKi versus sigma m and molar refractivity of substituents indicate that dispersion interactions in the anionic locus contribute approximately 10(5)-fold (delta delta G = 7 kcal mol-1) to the above-mentioned 10(7)-fold range of inhibitor potencies. The remaining approximately 100-fold arises from the inductive electronic effects of substituents on the stability of the tetrahedral adduct that forms between the ketone carbonyl of inhibitors and S200 in the esteratic locus of the active site. Values of k(on), the second-order rate constant for binding of time-dependent inhibitors, monitor a diffusion-controlled process. Moreover, k(on) for the quaternary ammonio inhibitor is 20-70-fold higher than for inhibitors that have uncharged meta substituents, which likely reflects the effect of the electrical field of AChE on ligand and substrate binding.