Hyperbolic mixed-type inhibition of acetylcholinesterase by tetracyclic thienopyrimidines

Figures & data

Figure 1.  Inhibitors of acetylcholinesterase.

Table 1  Inhibition of acetylcholinesterase from Electrophorus electricus (EeAChE), human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) by compounds 7–14.

Compound		EeAChE				hAChE	hBChE
		IC50^a ± SEM (µM)	Ki ± SEM (µM)	αKi ± SEM (µM)	β ± SEM	IC50^a ± SEM (µM)	IC50^b ± SEM (µM)
7		> 75	nd^c	nd	nd	> 200	> 200
8		> 75	nd	nd	nd	> 200	> 200
9		1.35 ± 0.06	1.55 ± 0.09	1.08 ± 0.03	0.084 ± 0.001	14.2 ± 0.3	> 200
10		1.51 ± 0.07	2.07 ± 0.19	0.98 ± 0.13	0.24 ± 0.02	5.69 ± 0.55	51.5 ± 10.2
11		1.75 ± 0.10	1.32 ± 0.08	2.70 ± 0.59	0.14 ± 0.01	56.0 ± 4.5	88.0 ± 1.0
12		2.26 ± 0.15	1.92 ± 0.12	2.87 ± 0.20	0.17 ± 0.01	20.5 ± 1.9	131 ± 7
13		3.09 ± 0.25	2.36 ± 0.22	3.88 ± 0.22	0.16 ± 0.01	21.8 ± 1.6	89.9 ± 3.7
14		1.73 ± 0.10	1.50 ± 0.07	2.34 ± 0.17	0.12 ± 0.004	62.5 ± 6.0	119 ± 14

^aIC₅₀ values with standard error were calculated from duplicate experiments at five inhibitor concentrations at a substrate concentration [acetylthiocholine] = 500 µM. Data were fitted to Equation 1. Limits were calculated from duplicate inhibition experiments at a single inhibitor concentration.

^bIC₅₀ values with standard error were calculated from duplicate experiments at five inhibitor concentrations at a substrate concentration [butyrylthiocholine] = 500 µM. Data were fitted to the equation of a competitive inhibition (Equation 5). Limits were calculated from duplicate inhibition experiments at a single inhibitor concentration.

^cnd, not determined.

Figure 2.  Relevant nuclear overhauser effect enhancement of compound 12. The spectrum was recorded in CDCl₃.

Scheme 1.  Synthesis of compounds 7–10. Reagents and conditions: (a) PhNCS, ethanol 80°C, 7h; (a) 1. 1N NaOH, EtOH, 2. MeI, room temperature; (c) ethylenediamine or 1,3-diaminopropane, 2-methoxyethanol, 150°C, 24 h.

Figure 3.  Kinetic model for the interaction of hyperbolic mixed-type inhibitors with Electrophorus electricus acetylcholinesterase. ES, enzyme-substrate complex; ESI, enzyme-substrate-inhibitor complex; EI, enzyme-inhibitor complex.

Figure 4.  Inhibition of Electrophorus electricus acetylcholinesterase (EeAChE) by compound 10. Plot of the rates versus [I] for the inhibition of EeAChE in 100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350 µM 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), 6% acetonitrile and ∼ 0.03 U/mL EeAChE. Rates were obtained by linear regression of the progress curves and are mean values of duplicate experiments. A rate at a substrate concentration of 500 µM in the absence of inhibitor was set 100%. (A) Initial substrate concentration was 250 µM acetylthiocholine (ATCh). The solid line was drawn using the best-fit parameters from a fit according to Equation 1, which gave IC₅₀ = 1.61 ± 0.04 µM and v_[I]→∞ = 5.0 ± 0.5%. (B) Initial substrate concentration was 500 µM ATCh. Non-linear regression according to Equation 1 gave IC₅₀ = 1.51 ± 0.07 µM (see Table 1) and v_[I]→∞ = 11 ± 1%. (C) Initial substrate concentration was 750 µM ATCh. Non-linear regression according to Equation 1 gave IC₅₀ = 1.39 ± 0.09 µM and v_[I]→∞ = 17 ± 2%. (D) Initial substrate concentration was 1000 µM ATCh. Non-linera regression according to Equation 1 gave IC₅₀ = 1.38 ± 0.07 µM and v_[I]→∞ = 20 ± 2%.

Figure 5.  Specific velocity plot for the inhibition of Electrophorus electricus acetylcholinesterase (EeAChE) by compound 10. Concentrations of 10 were as follows: open circles, [I] = 1 μM; full circles, [I] = 2 μM; open squares, [I] = 3 μM; full squares, [I] = 4 μM; open triangles, [I] = 5 μM. Data were from duplicate measurements at four different substrate concentrations (250 µM, 500 µM, 750 µM, and 1000 µM). Linear regression according to Equation 2 gave values for vertical intercepts at ([S]/K_m)/(1 + [S]/K_m) = 0 (a values) and for vertival intercepts at ([S]/K_m)/(1 + [S]/K_m) = 1 (b values).

Figure 6.  Replots of the specific velocity plot shown in Figure 5. Values a/(a-1) (full circles) and b(b-1) (open circles) were plotted versus the reciprocal concentrations of inhibitor 10. Linear regression according to Equations 3 and 4 gave values K_i = 2.07 ± 0.19 µM, αK_i = 0.98 ± 0.13 µM, β = 0.24.