Discovery of butyrylcholinesterase inhibitors among derivatives of azaphenothiazines

Abstract

The study presents the discovery of novel butyrylcholinesterase (BuChE) inhibitors among derivatives of azaphenothiazines by application of in silico and in vitro screening methods. From an in-house library of compounds, 143 heterocyclic molecules derived from the azaphenothiazine scaffold were chosen for virtual screening. Based on results of the docking procedure, 15 compounds were identified as exhibiting the best fit for the two screening complexes (ligand – AChE and ligand – BuChE). Five compounds displayed moderate AChE and good BuChE inhibitory activity at screening concentrations of 10 µM. The IC₅₀ values for active BuChE inhibitors were in the 11.8–122.2 nM range. Three of the most active inhibitors are tetra- or pentacyclic derivatives of azaphenothiazines with the same N-methyl-2-piperidinethyl substituent.

• Azaphenothiazines
• butyrylcholinesterase inhibitors
• cholinesterases
• molecular docking
• virtual screening

Introduction

Chemical modifications of structures of known pharmaceutical agents are a well-established approach to drug discovery. The tricyclic nitrogen- and sulfur-containing scaffold known as phenothiazine is a potent pharmacophoric moiety; thus phenothiazines are found among several important classes of drugs which possess neuroleptic, antiemetic and anti-histaminic properties. Phenothiazine derivatives exhibit wide-ranging biological activity – they are variously described as antipyretic, analgesic, anticonvulsant, anti-cancer, anti-bacterial, anti-fungal, anti-inflammatory, anti-malarial and immunosuppressive agents. Progress in researching the properties of phenothiazine derivatives has recently been reviewed1^,2. Moreover, phenothiazine-like compounds possess potential anti-Alzheimer’s properties, due to their inhibitory potency against acetyl- and butyrylcholinesterase3^,4, neuroprotective activity5^,6 as well as the ability to inhibit formation of amyloid beta (Aβ) fibrils and to inhibit the aggregation of tau protein7. Such properties of bioactive molecules are important from the point of view of neurodegeneration processes and dementia.

Various phenothiazine derivatives have been reported to inhibit cholinesterases8^,9 – among them neuroleptics such as chlorpromazine, thioridazine, derivatives of N-acetylaminophenothiazines, alkylamide and arylamide derivatives of phenothiazine10^,11. Generally, most of these compounds are selective inhibitors of butyrylcholinesterase12. Structures of selected derivatives of phenothiazine which inhibit cholinesterases are presented in Figure 1. It is interesting to note that the methylthioninium chloride (MTC) compound also known as methylene blue – an old antiseptic dye and a representative of phenothiazines – is currently in phase II clinical trials as potential drug candidate in the treatment of Alzheimer’s disease (AD). This compound has been shown to display multifunctional properties, including influence on the formation of β-amyloid plaques and neurofibrillary tangles, on mitochondrial function and on cholinergic, serotoninergic and glutamatergic neurotransmitter systems13^,14.

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

AD is a multifactorial neurodegenerative disorder which is classically characterized by extracellular accumulation of senile plaques composed of Aβ along with intracellular accumulation of the tau protein. AD is complex and still not completely understood as many different processes are involved in neuropathological changes leading to neuronal death. In addition, there is a body of evidence establishing the role of neurotransmitter systems, oxidative stress, metal ion dysregulation and inflammation in AD pathogenesis15. In particular, loss of cholinergic neurotransmission in the brain contributes to the substantial cognitive and behavioral symptoms of AD16. Most drugs currently available on the market for symptomatic treatment of AD are cholinesterase inhibitors such as donepezil, rivastigmine and galantamine17. In healthy brain tissue, cholinesterases are involved in cholinergic neurotransmission by hydrolysis of acetylcholine (ACh), although at different rates: AChE is the main enzyme responsible for ACh hydrolysis, while BuChE plays a secondary (supportive) role18. As AD progresses, the balance of AChE and BuChE changes. In the AD brain, BuChE level in regions of the brain involved in cognition and behavior is significantly elevated while AChE level in these areas is decreased. Several in vivo experiments indicated that BuChE is able to compensate for the lack of AChE, enabling continued regulation of cholinergic neurotransmission19^,20. It has also been shown that cholinesterases display several non-classical properties associated with Aβ and neurofibrillary tangles, and are therefore important for AD pathogenesis21–25. Recent studies have shown that BuChE exerts an influence on the modulation of motor control, awareness, cognition and behavior by regulation of ACh levels in the central nervous system (CNS)26.

Both enzymes remain valuable targets in anti-AD drug discovery studies27–30. Many selective or dual acting inhibitors of both enzymes were developed31^,32, and among them compounds which display activity also on Aβ aggregation and neurofibrillary tangles33–37.

Our research focuses on novel cholinesterase inhibitors with additional properties as potential multifunctional anti-Alzheimer’s drugs38–40. The aim of the presented study has been the discovery of novel cholinesterase inhibitors among derivatives of azaphenothiazines by application of in silico and in vitro screening methods.

Methods

Chemistry

Synthesis of tested azaphenothiazines (7–21) was described earlier41–44. Their structure and purity has been confirmed by the NMR spectra which were recorded on Bruker Fourier 300 spectrometers (¹H at 500 MHz, ¹³C at 125 and 75 MHz) in CDCl₃ or DMSO-d₆ and fast atom bombardment mass spectra which were run on a Finnigan MAT 95 spectrometer at 70 eV. These data were presented for azaphenothiazines (7–9, 14, 17, 20)41 for 2142 and for remaining compounds were described elsewhere43^,44.

Molecular modeling

Three-dimensional structures of potential cholinesterases inhibitors (143 ligands) were properly prepared and optimized for processing. In order to prepare compounds for docking with an appropriate geometry the CORINA (Molecular Networks) online application was used to create three-dimensional structures in the pdb format45. The resulting compounds were processed using the SYBYL 8.0 (St. Louis, MO) tool46. Types of atoms were checked and corrected with each molecule assigned adequate formal and partial charges. Prepared compounds were stored in the mol2 format. Optimization of protein models used in the screening and docking was performed using GOLD 5.147. Results of docking were visualized using PyMOL48. Detailed procedure was described and validated49.

Biological tests

The cholinesterase inhibition assay is based on the use of acetylthiocholine iodide (ATCh), and butyrylthiocholine iodide (BTCh) and 5,5′-dithio-bis (2-nitrobenzoic) acid (DTNB) to generate a yellow chromophore (5-mercapto-2-nitrobenzoic acid)50. Reagents and chemicals – DTNB, ATCh, BTCh, AChE from Electrophorus electricus (425.96 U/mg solid), and BuChE from horse serum (2.5 units/1 mL), were purchased from Sigma-Aldrich (Poznań, Poland). All assays were performed using the Perkin Elmer Lambda 12 device. Detection was performed at 412 nm. The assay was carried out as per a previously described protocol39^,40 using tacrine hydrochloride control. The compounds tested were dissolved in DMSO giving stock solution (100 µM). Each compound was tested at concentration 10 µM which was obtained by dissolving appropriate amount of stock solution in water. Two kinds of assays were conducted: the peak activity of the enzyme was determined by using a blank sample, followed by reference measurements (for tacrine) and measurements for the synthesized compounds. Data from concentration-inhibition experiments were integrated through non-linear regression analysis using the GraphPad Prism program (GraphPad Prism Software Inc. 2005, La Jolla, CA), producing estimates of IC₅₀.

Kinetic studies were performed using Ellman’s method for compound 14. The test was carried out in 0, 10, 25, and 75 nM concentration of 14 for BuChE. Substrate of the reaction, butyrylthiocholine, was used in the following concentrations: (0.05, 0.1, 0.2, 0.3, 0.5 mM). Stock solution of ATC (0.5 mM in a well) was prepared in water and diluted before use. For each concentration of tested compound ATC was used in concentration of 0.05, 0.1, 0.2, 0.3, 0.5 mM in a well. The obtained data were used to create substrate-velocity curves, which were transformed in GraphPad Prism program to Lineweaver–Burk plots (Figure 3).

Results and discussion

From an in-house library of compounds, 143 heterocyclic molecules derived from the azaphenothiazine scaffold were chosen. All these compounds had previously been synthesized (by Pluta and co-workers41–44^,51–53) and their structures are listed in the supplement. Most of these compounds represent 6-substituted derivatives of pentacyclic diquino-1,4-thiazines and substituted derivatives of tetracyclic benzoazaphenothiazines. Their preliminary biological evaluation focused on anti-cancer, anti-proliferative and immunosuppressive properties. Some of the investigated derivatives of azaphenothiazines exhibited significant anti-cancer activity against several types of human cells as well as immunosuppressive activity in human and mouse models42–44^,52^,53. Knowing that various phenothiazines possess inhibitory activity against cholinesterases, derivatives of azaphenothiazines were selected for our study.

In the first step, virtual screening was performed for all molecules (143 azaphenothiazines). For this molecular modelling study, we applied our custom method which has recently been described and validated49. In order to accurately check for possible interactions between enzymes with different conformations and test substances, two types of complexes with acetylcholinesterase inhibitors were used: 1ACJ – enzyme with tacrine and 2CKM – enzyme with bis-tacrine54. These complexes were chosen due to possible similarity of interaction between tricyclic tacrine or bis-tacrine and our tetra- or pentacyclic azaphenothiazines. In the search for potential inhibitors of both enzymes (AChE and/or BuChE), our in silico experiments focused on complexes with AChE since it is known that inhibitors of AChE have to meet stricter structural requirements then those of BuChE. Details of the target preparation and docking settings are presented in Table 1. Based on results from the docking procedure, 15 compounds were chosen for the in vitro screening. Among 32 pentacyclic azaphenothiazines 5 compounds (7–9, 17, 20), and from the remaining tetracyclic azaphenothiazines 7 compounds (10–13, 16, 18, 19, 21) were identified as exhibiting the best fit for the two complexes used in screening and containing different substituents. Values of the evaluation function for these selected set of compounds were in the 83.05–88.15 and 82.98–86.84 range for the 1ACJ-AChE complex with tacrine, while 82.08–102.53 and 73.47–89.24 for the 2CKM-AChE complex with bis-tacrine, respectively. Moreover, 3 compounds (14–16) with lower values of the scoring function (70.07, 69.06, 66.63 for the 1ACJ-AChE complex with tacrine and 63.35, 61.60, 59.24 for the 2CKM-AChE complex with bis-tacrine, respectively) but with N-methylpiperidine substituent as important pharmacophoric moiety were selected. Values of the scoring function for reference compounds (complex with 2CKM) were 88.45 and 99.67 for tacrine and bis-tacrine, respectively.

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

In the next step, the inhibitory potency of the selected compounds against AChE (from electric eel) and BuChE (from horse serum) was evaluated via the spectrophotometric Ellman’s method40. Experiments were carried out at 10 µM concentration of potential inhibitors. The measured activity is given as a percentage of enzyme inhibition and as IC₅₀ values which were only determined for compounds with better than 50% inhibitory activity at 10 µM concentration. Structures of tested compounds and results of in vitro screening against cholinesterases are collected in Table 2. Ten compounds displayed weak AChE (1.41–7.01%) and weak BuChE (0.88–7.30%) inhibitory activity while five compounds showed moderate AChE (19.46–38.75%) and good BuChE (79.99–99.88%) inhibitory activity at 10 µM screening concentration. The IC₅₀ values for active BuChE inhibitors were in the 11.8–122.2 nM range (Figure 2 and Table 3). Three of the most active inhibitors (14–16) are derivatives of tetra- or pentacyclic azaphenothiazines with the same N-methyl-2-piperidinethyl substituent. The inhibitory activity of their analogs with the 2-chloroethyl-propylurea substituent was significantly lower (17–18).

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

The mechanism of BuChE inhibition was investigated in enzyme kinetic studies using Ellman’s test and compound 14, the most potent inhibitor. Enzyme kinetic was analyzed by recording substrate-velocity curve in the absence and presence of compound 14. Graphical analysis of Lineweaver–Burk plots of BuChE activity (Figure 3) showed different K_m and constant V_max value in different inhibitor concentrations. This suggest a competitive inhibition, revealing that this compound compete for the same binding site (CAS) as the substrate ACh.

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

Molecular docking studies showed that ligands, despite structural similarity, were characterized by large variations in their binding mode with BuChE. For compounds with the N-methyl-piperidine substituent (14–16), the penta- or tetracyclic fragment occupies a large cavity in the active site of the enzyme, stacking along the gorge, while the piperidine substituent is oriented toward Trp82 or outside, in the direction of the peripheral binding site. Compounds 17 and 18 in which the 2-chloroethyl-propylurea substituent is much longer enable these azaphenothiazines to reach the bottom of the active site; however, their hydrophobic interactions are weaker. The most active compound 14 represents a large and strongly hydrophobic pentacyclic ring system, which is placed along the gorge of the enzyme between Trp82 and Ile442 at the bottom of the gorge, and Tyr332 near the entrance to the active site. The parallel arrangement of the aromatic rings of the ligand with Trp82 can create hydrophobic interactions and π–π stacking. The more hydrophilic N-methyl piperidine substituent of the ligand is oriented toward the catalytic triad and its arrangement enables interaction with the Ser198 residue. The protonated piperidine nitrogen atom creates cation-π interactions with Trp231 (Figure 4).

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.

Derivatives of diazatetracene (15 and 16) which were less active than the pentacyclic ligand 14 generally display similar interactions within the active site of the enzyme; however, some differences have been observed (Figure 5). Their arrangement also differs from that of compound 14. The smaller aromatic fragment creates weaker hydrophobic interactions with Trp82, which explains the lower activity of these compounds. In 15 the thiomethyl substituent, which is larger than a chlorine atom, may enter into hydrophobic interactions with the methyl group of Thr120 close to the entrance to the gorge.

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.

For all studied compounds physicochemical properties such as molecular weight (MW), lipophilicity (logarithm of partition coefficient, log P) and molecular volume (Mvol) were calculated. Data are collected in the Supplement and for tested inhibitors 7–21 in Table 3. MW for active inhibitors (14–18) was lower than that given by the rule of five (<500; 410.02–463.03), however log P values were in the 5.22–6.35 range, which indicated high lipophilicity55. Such properties of compounds probably enable them to penetrate the blood–brain barrier and reach the CNS56, however from the point of view of pharmacokinetics they should still be optimized. The calculated molecular volumes for active inhibitors (14–18) were in the 365.99–391.53 ų range. These values are in accordance with the results of biological tests and may explain selectivity toward BuChE. Both enzymes differ in terms of the calculated volume of the active site gorge; for AChE the corresponding volume is rather small (320 ų), while for BuChE it is substantially larger (502 ų)9.

Conclusions

In conclusion, the role of BuChE as a therapeutic target in the treatment of AD remains a focus of pharmacological and clinical studies57. This paper reports the discovery of novel selective BuChE inhibitors among azaphenothiazine derivatives by application of virtual screening and in vitro assays. This led to identification of three BuChE inhibitors, derivatives of tetra- or pentacyclic azaphenotiazines. Molecular modeling studies proved the essential role of the N-methyl-2-piperidinethyl substituent in these azaphenothiazines regarding interactions at the active site of BuChE. These studies indicated that results of virtual screening (value of scoring function) were useful for identification of inhibitors while with a wide range of activity (from weak to strong). However, important were also results of analysis of binding mode of ligands in the active site of enzyme. The inhibitory potency of the most active compound 14 (IC₅₀ = 12 nM) was comparable to that of reference tacrine (IC₅₀ = 5 nM). Such activity against BuChE has previously been described for derivatives of phenothiazines. Our results present previously unacknowledged inhibitory activity against BuChE by tetra- or pentacyclic azaphenothiazies.

Supplementary material available online

Supplementary Table.

Declaration of interest

This work was supported by the Jagiellonian University Collegium Medicum Student’s Grant no. gs 1/7/2011. The authors have declared no conflict of interest.