Flavonoids with M1 muscarinic acetylcholine receptor binding activity

Article abstract of DOI:10.3390/molecules19078933

Muscarinic acetylcholine receptor- Active compounds have potential for the treatment of Alzheimer's disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M₁ muscarinic receptors. Several compounds were found to possess competitive binding affinity (K _i = 40-110 μM), comparable to that of acetylcholine (K_i = 59 μM). Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

Full text of DOI:10.3390/molecules19078933

Molecules 2014, 19, 8933-8948; doi:10.3390/molecules19078933
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Flavonoids with M1 Muscarinic Acetylcholine Receptor
Binding Activity
Meyyammai Swaminathan 1, Chin Fei Chee 2,3, Sek Peng Chin 1, Michael J. C. Buckle 1,*,
Noorsaadah Abd. Rahman 2, Stephen W. Doughty 4 and Lip Yong Chung 1,*
1
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,
Malaysia; E-Mails: oosh_mey@yahoo.com (M.S.); xueping.chin@gmail.com (S.P.C.)
2
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia; E-Mails: cfchee@yahoo.com (C.F.C.); noorsaadah@um.edu.my (N.A.R.)
3
School of Pharmacy and Health Sciences, International Medical University, Jalan Jalil Perkasa 19,
Bukit Jalil, 57000 Kuala Lumpur, Malaysia
4
School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih,
Selangor Darul Ehsan, Malaysia; E-Mail: stephen.doughty@nottingham.edu.my
* Authors to whom correspondence should be addressed; E-Mails: buckle@um.edu.my (M.J.C.B.);
chungly@um.edu.my (L.Y.C.); Tel.: +60-3-7967-4959 (M.J.C.B. & L.Y.C.);
Fax: +60-3-7967-4964 (M.J.C.B. & L.Y.C.).
Received: 27 February 2014; in revised form: 20 June 2014 / Accepted: 23 June 2014 /
Published: 27 June 2014
Abstract: Muscarinic acetylcholine receptor-active compounds have potential for the
treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones
and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human
cloned M1 muscarinic receptors. Several compounds were found to possess competitive
binding affinity (Ki = 40–110 µM), comparable to that of acetylcholine (Ki = 59 µM).
Despite the fact that these compounds lack a positively-charged ammonium group under
physiological conditions, molecular modelling studies suggested that they bind to the
orthosteric site of the receptor, mainly through non-polar interactions.
Keywords: Alzheimer’s disease; muscarinic acetylcholine receptor; binding activity;
flavonoids; molecular modelling

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1. Introduction
Muscarinic acetylcholine receptor (mAChR)-active compounds have promising potential for the
treatment of Alzheimer’s disease (AD) [1]. The main pathological hallmarks of AD are cholinergic
system dysfunction and the formation of extracellular neuritic plaques containing the β-amyloid peptide
and neurofibrillary tangles composed of hyperphosphorylated forms of the tau protein. M1 mAChR
agonists would therefore be expected not only to give symptomatic relief by improving cognitive
functions but also to modify the progression of AD through the promotion of the non-amyloidogenic
processing of amyloid precursor protein and inhibition of tau hyperphosphorylation [2,3].
Flavonoids are polyphenolic compounds which are commonly found in vegetables, fruits and
drinks [4] and their human dietary intake is of the order of 100 mg/day [5]. They are known to possess
a wide range of pharmacological properties, including neuroprotective effects and antiamyloidogenic
activity [6–9]. There is also evidence from epidemiological studies that a higher dietary intake of
flavonoids reduces the risk of AD and cognitive decline [10,11].
Most mAChR active ligands contain a positively-charged ammonium group under physiological
conditions, but the fact that there are examples of active compounds that lack this feature [12–15]
suggests that this may not be an absolute requirement. In our laboratory, we have previously identified
a group of six polyoxygenated flavones from the bark extract of Melicope subunifoliolata (Stapf) T.G.
Hartley which show competitive binding activity at muscarinic acetylcholine receptors [16]. In the
current study, we have assayed a series of 13 structurally-related, natural and synthetic flavones and
flavonols, with varying degrees of hydroxyl and methoxyl substitution at positions 5–7, 3'–5' and 3 in
the A, B and C rings, respectively (Figure 1), in order to investigate whether this property might be a
general characteristic of such compounds and have performed molecular modelling studies to explore
further which structural features may be beneficial.
Figure 1. General structure of the investigated flavones and flavonols.
R5'
R4'
B
R7
R6
O
C
R3'
A
R3
O
R5
2. Results and Discussion
2.1. Synthesis of Flavones and Flavonols
Flavones 1–4 were prepared from the corresponding 2'-hydroxyacetophenones and benzoyl
chlorides via the Baker-Venkataraman reaction [17,18] to produce 1,3-diketones, followed by
cyclisation under acidic conditions (Scheme 1). Flavonols 9–10 were synthesised from the
corresponding 2'-hydroxyacetophenones and benzaldehydes to produce chalcones, followed by
cyclisation under the Algar-Flynn-Oyamada [19,20] reaction conditions (Scheme 2). Apigenin (5),

Molecules 2014, 19
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luteolin (6), diosmetin (7), fisetin (8), kaempferol (11), quercetin (12) and myricetin (13) were
obtained from commercial sources.
Scheme 1. Synthesis of flavones 1–4.
R'
OH
O
OH
O
O
c
a, b
R'
R
R'
R
+
R
Cl
O
O
O
Reagents and conditions: a—pyridine, 110 °C, 2–3 h; b—KOH, pyridine, 60 °C, 4 h, 60%–80%
(over 2 steps); c—AcOH, H2SO4, reflux, 16 h, 35%–55%.
Scheme 2. Synthesis of flavonols 9–10.
R'
OH
O
OH
O
O
b
a
R
+
R'
R
R'
R
H
OH
O
O
Reagents and conditions: a—50% aq. KOH, EtOH, rt, 24 h, 65%–90%; b—30% H2O2, 20% aq.
NaOH, MeOH, 0 °C, 10 h, 50%–75%.
2.2. M1 mAChR Radioligand Binding Assay
The compounds were assayed in vitro according to the 96-well microplate method previously
described [16] for their ability to displace [3H]N-methylscopolamine (0.2 nM) using cloned human M1
mAChRs (Perkin-Elmer, Boston, MA, USA). Three of the compounds, 3',4',5',5,6,7-hexamethoxyflavone
(4), luteolin (6) and ombuin (9), were found to have comparable affinities (Ki = 40–110 µM) for
the M1 mAChR with those observed for the standard agonists, acetylcholine (ACh) and pilocarpine
(Ki = 59 and 2.7 µM, respectively) (see Table 1 and Figure 2).
Table 1. Substituent patterns and M1 mAChR binding data of compounds 1–13 and standard agonists.
Cpd
1
2
3
4
5
6
7
8
Trivial Name
R3
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
R5
OMe
OMe
OMe
OMe
OH
OH
OH
H
OH
H
OH
OH
OH
R6
H
H
OMe
OMe
H
H
H
H
H
R7
R3'
OMe
OMe
OMe
OMe
H
OH
OH
OH
OH
OMe
H
R4'
OMe
OMe
OMe
OMe
OH
OH
OMe
OH
OMe
OMe
OH
OH
OH
R5'
H
OMe
H
OMe
H
H
H
H
H
H
IC50 µM
>10000
>10000
>10000
257 ± 27
>10000
226 ± 14
>10000
>10000
101 ± 6
>10000
>10000
>10000
>10000
142 ± 12
6.5 ± 0.1
(Ki µM)
OMe
OMe
OMe
OMe
OH
OH
OH
OH
OMe
OMe
OH
OH
OH
Sinensetin
107 ± 11
94 ± 6
Apigenin
Luteolin
Diosmetin
Fisetin
9
Ombuin
42 ± 3
10
11
12
13
OMe
H
H
Kaempferol
Quercetin
Myricetin
Acetylcholine
Pilocarpine
H
H
OH
OH
OH
H
59 ± 5
2.7 ± 0.1

Molecules 2014, 19
Figure 2. Displacement curves for the binding of [3H]N-methylscopolamine to the M1
8936
mAChR in the presence of (A) 3',4',5',5,6,7-hexamethoxyflavone (4), luteolin (6) and
ombuin (9) and (B) acetylcholine (ACh) and pilocarpine. Receptor membranes (13 µg
protein/well) were incubated with N-methylscopolamine (0.2 nM) in the presence of the
compounds at 27 °C for 120 min. Each data point is expressed as a mean ± SEM (n = 2).
Compound:
100
80
60
40
20
0
4
6
9
-12
-10
-8
-6
-4
-2
0
log [compound] (M)
(A)
ACh
100
Pilocarpine
80
60
40
20
0
-12
-10
-8
-6
-4
-2
0
log [compound] (M)
(B)
2.3. Molecular Modelling
To explore the possible binding modes of the compounds and their interactions with the human M1
mAChR, molecular modelling studies were carried out. A homology model based on the crystal
structure of the rat M3 mAChR (PDB code: 4DAJ) [21] was constructed using Prime v3.0 and the
orthosteric binding site was refined by induced-fit docking of the endogenous agonist (ACh) using
Glide v5.7, as recently described by us elsewhere [22]. Upon docking the three active compounds into
the M1 mAChR model using AutoDock v4.2 [23], they were all found to bind to the orthosteric site,
mainly through non-polar (van der Waals, π-π and hydrophobic) interactions with a number of residues

Molecules 2014, 19
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which have been identified by site-directed mutagenesis experiments as being important for the
binding of orthosteric ligands [24,25], including Y106, W157, W378, Y381 and Y404.
In an effort to probe the reasons for the differences in activity between structurally-similar
compounds, e.g., ombuin (9) versus quercetin (12) (see Figures 3 and 4), induced-fit docking using
Glide and Prime was carried out. The results suggested that the active compounds may be able to make
better contacts with key residues, D105, through substituents in ring B, and T192 and N382, through
the carbonyl oxygen of the ketone moiety in ring C or the hydroxyl substituent at position 5 in ring A
(where present). Alternatively, they may form additional hydrogen bonds to other active site residues.
Figure 3. 3D representations of the docking poses of (A) ombuin (9) (brown) and
(B) quercetin (12) (blue-green) in complex with the human M1 mAChR model obtained
from induced-fit docking using Glide v5.7 and Prime v3.0 (Schrödinger LLC) with side
views and top views (from the extracellular surface) depicted in the left hand and right
hand panels, respectively. For the purpose of clarity, only D105 and W378 from among the
active site residues are depicted in stick representation and some of the transmembrane
helices are not shown.

Molecules 2014, 19
Figure 4. 2D representations of the ligand-receptor interactions of (A) ombuin (9) and
8938
(B) quercetin (12) in complex with the human M1 mAChR model obtained from
induced-fit docking using Glide v5.7 and Prime v3.0 (Schrödinger LLC), showing pi-pi
stacking interactions (green lines), hydrogen bonds involving backbone atoms (solid
purple arrows) and hydrogen bonds involving side-chain atoms (dashed purple arrows).
Negatively-charged, polar and hydrophobic residues are depicted with red, light blue and
green circles, respectively.
However, the use of the Prime MM-GBSA module to predict the free energy of binding revealed no
significant energy differences for the active compounds compared to the inactive compounds. This may
be due to limitations in the docking and scoring functions used, such as not allowing full conformational
flexibility in the receptor and excluding the involvement of water molecules in binding.
The positively-charged ammonium group that is normally found in mAChR active ligands has been
observed to form ionic interactions with the conserved aspartate residue of transmembrane 3 in the two
crystal structures of ligands bound to muscarinic receptors that have been solved to date [21,26].
However, the data obtained from this study suggests that the non-polar interactions between a ligand
and residues in the hydrophobic cavity of the receptor may be sufficient to compensate for the absence
of ionic interactions. The proposal that such non-polar interactions may be of relatively greater
importance compared to ionic interactions in the binding of ligands to muscarinic receptors has
previously been made from observed differences in affinity within a series of 1,3-oxathiolane
compounds [27]. Furthermore, good correlation was observed between the majority of the other key
ligand-receptor interactions for acetylcholine and those for the active compounds (see Figure 5). In
particular, it appears that the carbonyl oxygen of the ketone moiety in ring C in the flavonoid
structures may mimic the carbonyl oxygen in the ester moiety of acetylcholine. However, it should be
noted that the effects of the compounds studied as agonists or inverse agonists/antagonists cannot be
determined by either radioligand binding or docking studies. These effects would need to be
investigated using functional studies.

Molecules 2014, 19
Figure 5. The structural binding analysis map for acetylcholine and a composite map for
8939
the active flavonoids, 3',4',5',5,6,7-hexamethoxyflavone (4), luteolin (6) and ombuin (9),
obtained from molecular modelling studies. Negatively-charged, polar and hydrophobic
residues are depicted in red, blue and dark green, respectively.
Interestingly, luteolin (6) has previously been observed to exert procognitive effects in vitro and
in vivo. These effects include cholinesterase inhibitory activity [28], protective effects on hydrogen
peroxide-induced apoptosis in SH-SY5Y neuroblastoma cells [29], anti-amnesic effects against amyloid
β25-35 peptide-induced toxicity in mice [30] and attenuation of cognitive decline in streptozotocin-induced
diabetic rats [31]. In addition, ombuin (9) has recently been found to act as a potent inhibitor of
DYRK1A/CLK1 kinases, whose increased activity has been linked to neurodegenerative diseases [32].
Hence, the compounds identified in this study have the potential to act as multitarget-directed
therapeutic agents, capable of addressing different aspects in the treatment of AD.
To assess the therapeutic potential of the compounds in vivo, bioavailability issues should also be
considered. Although orally-administered polyoxygenated flavonoids have been shown to be able to
penetrate the blood-brain barrier in rats [33], flavonoids bearing hydroxyl substituents are also known
to undergo extensive Phase II metabolism in the course of intestinal absorption. In an effort to predict
the effect of such metabolism on the activity of the compounds identified in this study, known
metabolites of luteolin (6) [34], the 3'-, 4'- and 7-monoglucuronides and the 3'-sulfate, and metabolites
of ombuin (9) that might be predicted from those that have been observed for quercetin [35,36], the
3'- and 3-monoglucuronides and the 3'-sulfate, were docked into the M1 mAChR model using
induced-fit docking. They were all found to be able to bind to the active site, but in a different
orientation to the aglycones, with the conjugated moiety pointing towards the extracellular surface of
the receptor (see Figure 6), suggesting that the activity of the compounds studied may be modified
in vivo. However, there is also evidence that glucuronoconjugated metabolites of flavonoids are
hydrolysed by β-glucuronidase, releasing the parent aglycone [37–40]. Hence further in vivo studies
should be carried out to determine the usefulness of compounds which have been found to have good
activity in vitro.

Molecules 2014, 19
Figure 6. 3D representations of the docking poses of (A) ombuin-3'-glucuronide and
8940
(B) ombuin-3'-sulfate in complex with the human M1 mAChR model obtained from
induced-fit docking using Glide v5.7 and Prime v3.0 (Schrödinger LLC) with side views
and top views (from the extracellular surface) depicted in the left hand and right hand panels,
respectively. For the purpose of clarity, only D105 and W378 from among the active site
residues are depicted in stick representation and some of the transmembrane helices are not
shown. The carbon atoms of the glucuronide moiety in ombuin-3'-glucuronide are coloured
in light green.
Lastly, it should be noted that the human dietary intake of flavones and flavonols is only in the
range 10–20 mg/day [5], predominantly in the form of quercetin and kaempferol, with luteolin just
being found (at levels of 5–20 mg/kg) in specific vegetables, such as celery and capsicum pepper [4].
Furthermore, less commonly available flavonols, such as ombuin, may only be obtained from
medicinal plants. Coupled with the bioavailability issues described above, this means that the
compounds identified in this study would probably need to be administered in purified form in order to
exert significant therapeutic effects in vivo.

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3. Experimental Section
3.1. Chemistry
All melting points were taken on a Mel-Temp II melting point instrument. 1H and 13C-NMR spectra
were obtained using Jeol EX 270 (270 MHz) and ECA 400 (400 MHz) NMR spectrometers with TMS
as the internal standard. All chemical shifts are reported in ppm. Column chromatography and
analytical thin-layer chromatography (TLC) were performed using silica gel 60 (230–400 mesh) and
pre-coated aluminum silica gel sheets (Kieselgel 60 F-254) from Merck (Darmstadt, Germany),
respectively. The physical and spectroscopic data for flavones 1–4 and flavonols 9–10 was in
agreement with that previously reported for these compounds. All other chemicals used were of
analytical grade and were obtained from TransMIT (Marburg, Germany), Sigma-Aldrich (St. Louis,
MO, USA) or Merck.
3.1.1. General Procedure for the Synthesis of Flavones
A mixture of the corresponding 2'-hydroxyacetophenone (1 equiv) and benzoyl chloride (1.1 equiv)
in pyridine (10 mL/mmol of acetophenone) was heated at 110 °C for 2–3 h. The mixture was then
poured into ice and HCl and extracted with ethyl acetate. The extract was washed with water and
aqueous sodium carbonate and then dried over sodium sulfate. The solvent was evaporated under
reduced pressure to yield the crude phenolic ester. Powdered KOH (4–5 g) was added to a solution of
the ester in pyridine (10–15 mL) and the mixture was stirred vigorously at 60 °C for 4 h. The mixture
was then poured into ice and HCl, extracted with ethyl acetate and dried over sodium sulphate to give
the 1,3-diketone in 60%–80% yield. To a yellow suspension of the 1,3-diketone (2 mmol) in glacial
acetic acid (7 mL), concentrated sulfuric acid (a few drops) was added and refluxed overnight. The
resulting mixture was cooled and water added until no further white precipitate was formed. The crude
solid was filtered, purified using column chromatography and crystallised from methanol to give the
flavone in 35%–55% yield.
3',4',5,7-Tetramethoxyflavone (1): mp 192 °C, lit. 193 °C [41]; 1H-NMR (270 MHz, DMSO-d6): δ 7.60
(d, 8.1 Hz, 1H), 7.49 (s, 1H), 7.07 (d, 8.1 Hz, 1H), 6.86 (s, 1H), 6.70 (s, 1H), 6.46 (s, 1H), 3.86, 3.83,
13
3.79, 3.78 (s, 3H each); C-NMR (67.9 MHz, DMSO-d6): δ 176.48, 164.24, 160.58, 160.48, 159.59,
152.03, 149.35, 123.33, 119.94, 112.08, 108.41, 107.21, 96.66, 93.83, 56.50, 56.27, 56.11.
3',4',5',5,7-Pentamethoxyflavone (2): mp 195 °C, lit. 198–199 °C [42]; 1H-NMR (270 MHz, DMSO-d6):
δ 7.29 (s, 2H), 6.91 (d, 2.2 Hz, 1H), 6.50 (d, 2.2 Hz, 1H), 6.86 (s, 1H), 3.90, 3.89, 3.82, 3.72 (s, 3H
13
each); C-NMR (67.9 MHz, DMSO-d6): δ 175.84, 163.78, 160.24, 159.44, 159.23, 153.29, 140.22,
126.25, 108.15, 103.63, 96.33, 93.55, 60.24, 56.22, 56.10, 56.07.
3',4',5,6,7-Pentamethoxyflavone (sinensetin) (3): mp 176 °C, lit. 174–176 °C [43]; 1H-NMR (270 MHz,
DMSO-d6): δ 7.56 (d, 8.0 Hz, 1H), 7.43 (s, 1H), 7.26 (d, 8.0 Hz, 1H), 7.07 (s, 1H), 6.88 (s, 1H), 3.85,
3.78, 3.74, 3.69, 3.66 (s, 3H each); 13C-NMR (67.9 MHz, DMSO-d6): δ 176.42, 160.98, 158.02,
154.47, 152.17, 151.97, 149.45, 140.23, 123.51, 120.10, 112.42, 112.19, 109.51, 106.75, 97.93, 62.44,
61.57, 57.08, 56.42, 53.65.

Molecules 2014, 19
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1
3',4',5',5,6,7-Hexamethoxyflavone (4): mp 150 °C, lit. 147–149 °C [44]; H-NMR (270 MHz, DMSO-
13
d6): δ 7.32 (s, 2H), 7.13 (s, 1H), 6.94 (s, 1H), 3.91, 3.85 (s, 6H), 3.75, 3.71, 3.68 (s, 3H each); C-
NMR (67.9 MHz, DMSO-d6): δ 176.34, 160.47, 157.96, 154.34, 153.56, 151.74, 140.59, 140.13,
126.49, 112.25, 107.63, 103.97, 97.91, 62.26, 61.39, 60.58, 56.95, 56.68.
3.1.2. General Procedure for the Synthesis of Flavonols
A mixture of the corresponding 2'-hydroxyacetophenone (1 equiv) and aldehyde (1 equiv) in EtOH
(5 mL/1 mmol of acetophenone) was stirred at RT for 30 min. Then a solution of 50% w/w aqueous
KOH (1 mL/1 mmol) was added. The reaction mixture was stirred at RT and monitored by TLC until
all the aldehyde was consumed. The mixture was then poured into ice-water, acidified with 3N HCl.
The crude product was filtered, purified using column chromatography and crystallised from ethanol to
give the chalcone in 65%–90% yield. To a solution of the 2'-hydroxychalcone (1 equiv) in methanol
(10 mL), 20% w/w aqueous NaOH (2 mL) was added. The solution was cooled to 0 °C and 30%
hydrogen peroxide (1 mL) was added dropwise. After 10 h in ice, the product was liberated by ice-cold
acetic acid, washed with water, and crystallized from methanol or diluted acetic acid to give the
flavonol in 50%–75% yield.
1
3ʹ,3,5-Trihydroxy-4ʹ,7-dimethoxyflavone (ombuin) (9): mp 204 °C, lit. 201–203 °C [45]; H-NMR
(400 MHz, DMSO-d6): δ 12.45 (s, 1H), 9.34 (bs, 1H), 7.63 (s, 1H), 7.64 (d, 8.4 Hz, 1H), 7.03 (d, 8.4
13
Hz, 1H), 6.64 (d, 2.0 Hz, 1H), 6.29 (d, 2.0 Hz, 1H), 3.81, 3.80 (s, 3H each); C-NMR (100 MHz,
DMSO-d6): δ 176.45, 165.38, 161.69, 156.69, 149.84, 148.33, 146.79, 136.65, 123.90, 120.56, 116.07,
115.06, 104.49, 98.73, 93.93, 56.84, 55.43.
1
3-Hydroxy-3ʹ,4ʹ,6,7-tetramethoxyflavone (10): mp 225–226 °C, lit. 226 °C [46]; H-NMR (400 MHz,
CDCl3): δ 7.76 (d, 9.0 Hz, 1H), 7.73 (s, 1H), 7.43 (s, 1H), 6.92 (d, 9.0 Hz, 1H), 6.89 (s, 1H), 3.95,
3.93, 3.92, 3.89 (s, 3H each); 13C-NMR (100 MHz, CDCl3): δ 172.03, 154.60, 151.64, 150.37, 148.77,
147.40, 144.35, 137.30, 123.91, 120.95, 113.95, 110.84, 110.41, 103.51, 99.54, 56.46, 56.31, 56.00, 55.91.
3.2. M1 mAChR Radioligand Binding Assay
Radioligand binding assays were carried out as previously described [16], but using M1 mAChR
membranes from stably transfected CHO-K1 cell lines (Perkin Elmer, Waltham, MA, USA). Around
13 µg/well of M1 mAChR membrane suspension in 170 µL/well PBS (pH 7.4) was added to a mixture
of test compound in 0.25% v/v of DMSO (10 µL/well; 10−2 M–10−8 M, final concentration),
[3H]N-methylscopolamine (Amersham Pharmacia Biotech., Little Chalfont, Buckinghamshire, UK)
(10 µL/well; 0.2 nM, final concentration), guanosine triphosphate (10 µL/well; 0.1 mM, final
concentration). After 120 min of incubation at 27 °C, the reaction mixture was filtered with a GF/C
filter plate on a UniFilter cell harvester (Perkin-Elmer) and washed with 200 µL/well of ice cold
50 mM Tris-HCl buffer (pH 7.4) (nine times). The GF/C filter plate was then air-dried for 24 h before
the application of Bottom Seal and addition of MicroScint-O scintillation cocktail (25 µL/well)
(Perkin-Elmer). The top of the plate was then sealed with TopSeal A. After 1 h the plate was counted
for 1 min/well with a TopCount NXT microplate scintillation counter (Perkin-Elmer). Non-specific

Molecules 2014, 19
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binding was determined by adding atropine (10 µL/well; 5 µM, final concentration) to the reaction
mixture. The percentage of specific binding of radioligand was calculated using the standard data
reduction algorithm:
([B − NSB]/[TB − NSB]) × 100
where B is the binding in the presence of a test compound, NSB is the non-specific binding in the
presence of excess reference ligand and TB is the total binding.
Data analysis was performed with PRISM® v5.0 (GraphPad, San Diego, CA, USA). Values of Kd
(the equilibrium dissociation constant) for the radioligand, Bmax (maximum number of binding sites for
the radioligand) and IC50 (the half maximal inhibitory concentration) for the test compounds were
obtained as the best-fit values for the data from non-linear regression analysis using the least-squares
method. Inhibition constant (Ki) values were calculated using the Cheng-Prusoff Equation [47]:
Ki = IC50/(1 + [L]/Kd)
where [L] is the radioligand concentration that was used. Values are expressed as mean (n = 2) ± SEM.
3.3. Molecular Modelling
3.3.1. Homology Modelling of the M1 mAChR
The sequence of the human M1 mAChR (accession number: P11229) was retrieved from the
Swiss-Prot sequence database [48]. Guided alignment of the human M1 receptor sequence and that
obtained from the structure of the rat M3 mAChR complexed with an inverse agonist (PDB code:
4DAJ) [21] was carried out using Expresso [49]. A homology model of the M1 receptor was then
constructed using Prime v3.0 (Schrödinger LLC, New York, NY, USA), as previously described [22].
Briefly, ECLs and ICLs connecting the TM domains were modelled according to the template structure
except ICL3, which was excluded from the modelling. Binding site refinement was carried out by
induced-fit docking (IFD) of the endogenous muscarinic receptor agonist (ACh) using Glide v5.7
(Schrödinger LLC) and side chain refinement using Prime v3.0. The IFD protocol began with a
constrained minimization of the receptor structure, followed by initial Glide docking of ACh using a
softened potential, to allow more poses of the ligand to be generated and collected. One round of Prime
side-chain prediction was then carried out for the residues found within 5 Å of the ligand, followed by
minimization of each ligand-receptor complex. Finally, the ligand was re-docked into the induced-fit
receptor structure, without softened potential and the binding energy or IFD score was obtained.
After multiple iterations of docking and binding site refinement, an initial set of models was chosen
on the basis of showing the expected ligand-receptor interactions. The resulting ligand-receptor
conformations were inspected visually to ensure that the quaternary amine group of the docked ACh
was directed towards D105 and the side chains of the orthosteric site residues were facing inwards
towards the inner channel of the TM region. The best model was then selected on the basis of their
ability to differentiate active compounds from decoys.

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3.3.2. Docking Studies
Initial docking studies were performed with AutoDock 4.2, using a Lamarckian genetic algorithm [50]
with a flexible ligand and a rigid receptor, a population size of 300, a maximum of 250,000 generations
and 2,500,000 evaluations for 100 GA runs. AutoDockTools4 [23] was used to prepare the input files
and docking grids and the grid box was set to cover the TM domain. The root mean square deviation
(RMSD) tolerance was set to 2.0 Å for the clustering of docked results. Ligand-receptor interactions
were viewed using Discovery Studio Visualizer v3.1 (Accelrys, San Diego, CA, USA).
Induced-fit docking was performed using the same parameters and protocol as the binding site
refinement of the model (see Section 3.3.1). The best docked conformation were selected based on the
induced-fit docking score, GScore, and the observed interactions with the active site residues. Docking
poses and ligand receptor interactions were viewed using PyMOL and Maestro (Schrödinger LLC),
respectively. The obtained poses were re-scored using the Prime MM-GBSA module to predict the free
energy of binding (∆Gbind) with the following equation:
∆Gbind = Gcomplex − (Gprotein + Gligand
)
where Gcomplex, Gprotein and Gligand are the optimized free energies of binding for the protein-ligand
complex, the free protein and free ligand, respectively [51,52]. Each energy term was calculated by a
combination of molecular mechanics energy, implicit solvation energy and surface area energy.
Residues in the binding pocket of the receptor were treated as flexible.
4. Conclusions
In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their
ability to inhibit radioligand binding at human cloned M1 muscarinic acetylcholine receptors. Several
compounds were found to possess competitive binding affinity (Ki = 40–110 µM), comparable to that
of acetylcholine (Ki = 59 µM). Molecular modelling studies suggested that the compounds bind to the
orthosteric site of the receptor, mainly through non-polar interactions. Although no significant energy
differences were observed for the binding of the active compounds compared to the inactive compounds,
this may be due to limitations in the docking and scoring functions used. Further studies are required in
order to determine whether the compounds act as agonists or inverse agonists/antagonists, possess any
sub-type selectivity and have sufficient bioavailability in vivo. The results from such studies will be
able to give more of an indication of the potential of flavonoid compounds for the treatment of AD.
Acknowledgments
This work was supported by grants from the Ministry of Education, Malaysia (ER002-2012A) and
the University of Malaya (FS143/2007C and PS201/2010B). We would also like to acknowledge
David Chalmers and Elizabeth Yuriev (Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Australia) for assistance with the molecular modelling studies and
computational resources provided by the Victorian Life Sciences Computational Infrastructure through
grant VR0133.

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Author Contributions
M.S.: radioligand binding assay, data analysis and manuscript preparation. C.F.C.: chemical
synthesis. S.P.C.: molecular modelling. M.J.C.B: project design and co-ordination and manuscript
preparation. N.A.R., S.W.D. and L.Y.C.: project design and co-ordination.
Conflicts of Interest
The authors declare no conflict of interest.
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(http://creativecommons.org/licenses/by/3.0/).