1-(3- Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies

Abstract

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, τ = 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.

Keywords: acetylcholinesterase; organophosphorus; slow-binding inhibition; transition state analog.

Figure 1

Chemical structure of related trifluoromethylketone molecules: (A): 1-[3-(trimethylamino)phenyl]-2,2,2-trifluoro-1-ethanone (TMTFA); (B): 2,2,2-trifluoro-1-[m-(trimethylsilyl)phenyl]-1-ethanone (Zifrosilone); (C): 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK).

Figure 2

Typical progress curves (without treatment for smoothing noise) for slow-binding inhibition of AChE by TFK. [E] = 0.08 nM, [ATC] = 0.1 mM and [TFK] ranged from 0.1 to 50 nM. ΔOD₄₁₂ is the concentration of ATC hydrolysis product (in Equation (1), [P] = thiocholine = thionitrobenzoate), expressed as the absorbance increase at 412 nm. The reciprocal of k_obs is the lag time (vertical dotted line at t = 1.75 min for 50 nM TFK) before steady state. Progress curves fit to Equation (1).

Figure 3

Dixon plot (A) and Cornish-Bowden plot (B) for determination of fast reversible inhibition constant.

Figure 4

Typical dependance of k_obs as a function of TFK concentration for inhibition in the presence of 0.1 mM ATC. The k_obs values were determined from nonlinear fitting of progress curves in Figure 2 at three substrate concentrations. Data were fitted to Equation (3): ordinate is k₋₄ and asymptote is k₋₄ + k₊₄ in Scheme of Figure 5.

Figure 5

Slow-binding inhibition model of type B.

Figure 6

SBI and subsequent acylation of 2 × 10⁻⁹ M AChE by 2 × 10⁻⁸ M TFK, followed by slow deacylation. Activity was assayed with 1 mM ATC with DTNB as the thiol probe (black, blue, red curves) and with Probe IV as the thiol probe (green curve). The black curve is enzyme activity (control) in the presence of 2% acetonitrile; the red curve is enzyme activity monitored up to 180 min in the presence of 2 × 10⁻⁸ M TFK. Blue curve: after steady-state SBI, the system was diluted 10 times, so that TFK dropped to 2 × 10⁻⁹ M after t = 20 min. Green curve: after steady-state SBI, the system was diluted 1000 times, then TFK final concentration dropped to 2 × 10⁻¹¹ M after t = 20 min. The first-order rate constant of deacylation (k_reac) was estimated from the slope of Ln increase in activity vs. time.

Figure 7

Binding of TFK to AChE: (A) main docked poses in the gorge: in the PAS (most populated cluster, carbon atoms colored red), at the gorge rim partly blocking the bottleneck (carbon atoms orange), and in the active site ready to react with the catalytic serine (least populated cluster, carbon atoms are colored white) compared to the main binding poses of TMTFA (B). Docking results with X-ray structure PDB ID: 4EY7 as a target are shown. (C) Free energy (PMF) profile of the binding process calculated with REMD-US method. Process coordinate ξ is the distance between the TFK/TMTFA trifluoroketone group and the active site, oxyanion hole and catalytic serine hydroxyl-group.

Figure 8

(A) Tetrahedral adducts for reaction between TFK (carbon atoms shown in yellow) and TMTFA (carbon atoms shown in green) and hAChE obtained by QM/MM calculations overlaid with X-ray structures of conjugates of TMTFA with Mus musculus (PDB ID: 2H9Y [53], carbon atoms shown in blue) and Torpedo californica (PDB ID: 1AMN [6], carbon atoms shown in pink). Double hAChE/TcAChE numbering is provided; (B) Non-covalent complex between TFK and hAChE, stable product resulting from the tetrahedral adduct reactivation process.

Figure 9

Slow-binding inhibition of type C with a subsequent covalent step, enzyme phosphorylation (k_p) in the present case.

Figure 10

Modulation of progressive biphasic inhibition of hAChE by CBDP CSP (0.21 µM) after pre-incubation of the enzyme for 120 min in the presence of TFK (0.1–10 nM).

Figure 11

Dependence of k_obs,max (fast process, see Equations (7) and (8)) of hAChE phosphorylation by CSP as a function of TFK concentration (after 120 min pre-incubation).

Figure 12

Observed first process (fast process) phosphorylation rate constant of hAChE by 0.21 μM CSP as a function of enzyme pre-incubation time in the presence of 10 mM TFK.

Figure 13

First-order inhibition of AChE by 50 nM paraoxon after pre-incubation of the enzyme in the presence of 10 nM TFK up to 90 min, pH 8.0, 25 °C.

Figure 14

Observed phosphorylation rate constant of hAChE by 50 nM paraoxon as a function of enzyme pre-incubation time (from 0 to 90 min) in the presence of 10 nM TFK.