Discovery of butyrylcholinesterase inhibitors among derivatives of azaphenothiazines

Figures & data

Figure 1. Structures of phenothiazine derivatives: neuroleptic drugs, MTC and other selective BuChE inhibitors.

Table 1. Details of the target preparation and docking settings for the selected complexes of inhibitors with AChE.

PDB code	Histidine tautomers	Stored water molecules (toggle enabled)	Determination of the active site	Scoring function	Parameters of the genetic algorithm (GA)
1ACJ	Nɛ	616, 634, 643	Amino acids within 12 Å from the inhibitor	Goldscore	Standard GA
2CKM	Nɛ	None	Amino acids within 10 Å from the inhibitor	Goldscore	Standard GA

Figure 2. Dose-dependent plot for active BuChE inhibitors 14–18.

Table 2. Inhibition of AChE^a and BuChE^b at 10 µmol/L screening concentration by azaphenothiazine derivatives selected by virtual in silico screening.

		Inhibition of AChE^c ± SEM	Inhibition of BuChE^c ± SEM	Inhibition of BuChE^c IC50 ± SEM	Value of evaluation function
Cmp.	Structure	[%]	(%)	(nM) 95% CI (nM)	1ACJ^e	2CKM^e
7		7.01 ± 0.59	7.29 ± 1.34	n.d.	88.15	91.65
8		5.70 ± 1.55	4.45 ± 0.24	n.d.	86.77	102.53
9		1.41 ± 0.23^d	0.88 ± 0.65^d	n.d.	83.05	85.87
10		6.47 ± 2.10	5.75 ± 2.69	n.d.	86.84	81.47
11		3.75 ± 0.20	4.51 ± 0.98	n.d.	84.47	87.73
12		2.60 ± 0.32	7.30 ± 0.24	n.d.	84.44	89.24
13		1.50 ± 1.10	5.66 ± 2.32	n.d.	84.65	79.29
14		34.77 ± 0.31	99.76 ± 0.03	11.78 ± 1.31 9.38−14.78	70.07	63.35
15		27.04 ± 2.28	99.88 ± 0.04	24.43 ± 1.93 20.75−28.75	69.06	61.60
16		24.86 ± 0.73	98.88 ± 0.00	122.2 ± 11.8 100.3−148.9	66.63	59.24
17		38.75 ± 3.20	83.20 ± 0.17	2961.0 ± 199.5 2577.0−3403.0	84.94	85.90
18		19.46 ± 1.83	79.99 ± 0.99	743.4 ± 95.2 571.2−967.6	85.25	77.73
19		4.04 ± 0.80	5.02 ± 0.44	n.d.	82.98	73.47
20		4.85 ± 0.98	5.31 ± 0.70	n.d.	84.45	82.08
21		2.88 ± 0.20	3.82 ± 0.48	n.d.	75.36	74.36
Tacrine		99.72 ± 0.03	99.92 ± 0.06	5.36 ± 1.00 4.39 ± 6.55	62.93	88.45

n.d., Not determined.

^aAChE (EC 3.1.1.7) from Electrophorus electricus.

^bBuChE (EC 3.1.1.8) from horse serum.

^cListed results are the mean of three independent experiments.

^dIn vitro screening for compound 9 was performed at 1 µmol/L concentration (due to solubility).

^eTypes of complexes: 1ACJ – AChE with tacrine, 2CKM – AChE with bis-tacrine.

Table 3. Physicochemical properties of selected, potential cholinesterases inhibitors: MW, Log P (average Log P from ALOGPS 2.1), molecular volume (Mvol from MarvinSketch).

Compound	MW	Log P	Mvol (Å^3)
7	526.672	6.63	454.07
8	512.646	6.23	437.59
9	498.619	5.92	420.16
10	496.044	6.21	412.05
11	475.626	5.40	415.20
12	510.070	5.99	429.11
13	433.975	4.28	358.41
14	426.63	6.35	391.07
15	421.68	6.26	387.67
16	410.02	6.28	365.96
17	464.03	5.26	391.53
18	447.43	5.22	366.54
19	413.920	5.50	349.92
20	488.560	6.26	410.41
21	326.414	6.04	299.18

Figure 3. Lineweaver–Burk plots resulting from substrate-velocity curves of BuChE activity of compound 14 in concentration of 10, 25 and 75 nM.

Figure 4. Binding mode of compound 14 in the active site of butyrylcholinesterase. Marked spheres (yellow) show hydrophobic interactions while the arrow (green) indicates a hydrogen bond between the nitrogen atom and the serine 198 amino acid.

Figure 5. Binding mode of compounds 15 and 16 in the active site of butyrylcholinesterase. Marked spheres (yellow) show hydrophobic interactions. (a) Compound 15 and (b) Compound 16.