Development of a photoactivatable allosteric ligand for the M1 muscarinic acetylcholine receptor

Article abstract of DOI:10.1021/cn500173x

The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M₁ muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor.

Full text of DOI:10.1021/cn500173x

Letter
pubs.acs.org/chemneuro
Development of a Photoactivatable Allosteric Ligand for the M1
Muscarinic Acetylcholine Receptor
Briana J. Davie,†,‡ Patrick M. Sexton,‡ Ben Capuano,† Arthur Christopoulos,*,‡ and Peter J. Scammells*,†
‡
†Medicinal Chemistry and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville,
Victoria 3052, Australia
S
* Supporting Information
ABSTRACT: The field of G protein-coupled receptor drug discovery has
benefited greatly from the structural and functional insights afforded by
photoactivatable ligands. One G protein-coupled receptor subfamily for which
photoactivatable ligands have been developed is the muscarinic acetylcholine
receptor family, though, to date, all such ligands have been designed to target the
orthosteric (endogenous ligand) binding site of these receptors. Herein we report
the synthesis and pharmacological investigation of a novel photoaffinity label,
MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M1
muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment
of cognitive deficits. MIPS1455 may be a valuable molecular tool for further
investigating allosteric interactions at this receptor.
KEYWORDS: Muscarinic, allosteric, photoactivatable, photoaffinity, BQCA, cognition
hotoaffinity labeling is a robust technique that is highly
amenable to the study of ligand interactions with G
P
protein-coupled receptors (GPCRs).1 The utility of photo-
affinity labeling arises from the range of photoactivatable
functionalities available for incorporation into both ligands and
receptors,2 the potential for applying this technique to study
any ligand−receptor1 or, indeed, receptor−receptor complex,3
and the ability to use the technique in tandem with more
recently developed computational, genetic, and crystallographic
technologies.
Figure 1. Three commonly used photoactivatable functionalities.
efficiency compared to diazirines and aryl azides. However,
their bulk means that they may result in a less accurate
representation of the parent ligand−receptor interaction.
Diazirines and aryl azides are hence more prudent choices if
incorporation of a benzophenone is likely to result in steric
clashes with the binding pocket.
Diazirines, which photoactivate at 350−380 nm, may also
prove to be an advantageous choice if considerable nonspecific
cross-linking is anticipated; they display low cross-linking
efficiency such that a covalent complex will only form if the
ligand has very high affinity for the receptor.5 A considerable
drawback is that their synthesis can be complicated and time-
consuming. Aryl azides, while more synthetically accessible that
diazirines, require photoirradiation at <300 nm; wavelengths
likely to cause nonspecific damage to the biological system
under investigation.6
Photoaffinity labeling involves taking a known ligand or
receptor and incorporating a photoactivatable moiety; a
functional group capable of forming a reactive species (typically
a carbene, nitrene, or diradical) upon electronic excitation at a
specific wavelength of ultraviolet (UV) light. This species is
then able to rapidly react or “cross-link” with a chemical bond
of a neighboring entity in close proximity. Photoactivatable
ligands offer the significant advantage of temporal control of
reactivity, compared to affinity-labeled ligands containing, for
example, inherently reactive electrophilic functionalities. The
receptor may be pretreated with the “ground state” ligand prior
to photoactivation, increasing the likelihood of “capturing” the
ligand−receptor complex of interest. However, as with affinity-
labeled ligands, reasonable affinity for the receptor and a similar
pharmacological profile to the parent molecule are important to
ensure selective labeling of the binding site of interest.4
Photoactivatable functionalities that are routinely incorpo-
rated into known ligands include benzophenones, diazirines
and aryl azides (Figure 1).5 Benzophenone-containing probes
undergo photoactivation at 350−360 nm and have the notable
advantages of higher chemical stability and higher cross-linking
Photoaffinity labeling has considerably advanced our under-
standing of ligand−receptor and receptor−receptor interactions
at Family A GPCRs. The technique has enabled the
identification and structural characterization of ligand binding
sites,1,7 provided early evidence for receptor dimerization,3 and
© XXXX American Chemical Society
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facilitated receptor purification that has subsequently aided
GPCR crystallization.8 Muscarinic acetylcholine receptors
(mAChRs) are Family A GPCRs that, due to their widespread
expression in the central nervous system,9 have been extensively
investigated as therapeutic targets for enhancing cognition and
alleviating psychotic symptoms in conditions such as
schizophrenia and Alzheimer’s disease.10 Examples of photo-
activatable ligands for mAChRs include aryl azide derivatives of
the potent orthosteric antagonists tropine,11 3-quinuclidinyl
benzilate (3QNB),12 and N-methyl-4-piperidyl benzilate
(4NMPB).12,13 In addition to the highly structurally conserved
orthosteric ligand binding site, the existence of multiple,
topographically distinct allosteric ligand binding sites at
mAChRs has been extensively experimentally validated.14
While an array of mAChR allosteric ligands have subsequently
been developed,15 our knowledge about the precise location
and structure of their binding sites and the interactions of
allosteric ligands within them is in its infancy. Hence,
photoactivatable ligands for allosteric sites may be of significant
worth to this field of research.
incorporate the benzophenone group into our ligand design
without concern about steric disruption of the ligand−receptor
binding interaction or functional activation.
Using [3H]N-methylscopolamine (NMS) competition radio-
ligand binding and ERK1/2 phosphorylaton assays performed
at human M1 mAChR-expressing Chinese hamster ovary
(CHO) cells, we confirm that the allosteric binding and
functional profile of BQCA, as well as the parent molecule’s
high mAChR subtype selectivity, is preserved in MIPS1455.
Importantly, saturation radioligand binding assays using hM1
mAChR-expressing CHO cell membranes reveal the ability of
MIPS1455 to irreversibly bind to the receptor following
photoactivation.
The quinolone carboxylic acid 5 was prepared in three steps
as previously described.24 Radical bromination of 4-methyl-
benzophenone gave a mixture of the desired monobrominated
6 and a small amount of dibrominated product. These
compounds display a similar retention time and were unable
to be separated by chromatographic methods. Fortunately, the
crude mixture was successfully employed in the N-benzylation
step to form the target molecule 4 in an acceptable yield (54%).
The allosteric binding and functional properties of BQCA
(1) and MIPS1455 (4) were assessed, utilizing methods
previously described,24 by interacting the ligands with ACh in
[3H]NMS competition binding assays and ERK1/2 phosphor-
ylation functional assays conducted in CHO cells stably
expressing the hM1 mAChR (Figure 3). MIPS1455 exhibited
a similar affinity to BQCA (MIPS1455 KB = 15 μM compared
to BQCA KB = 36 μM) and displayed positive binding (α = 61)
and functional (β = 1.1) cooperativity with the agonist ACh
and negative binding cooperativity with the antagonist
[3H]NMS (log α[NMS] = −3.00) consistent with the two-state
model of receptor activity reported for BQCA.25 MIPS1455
also behaved as an allosteric agonist in its own right
(MIPS1455 τB = 11 compared to BQCA τB = 28). [3H]NMS
competition binding curves and ERK1/2 phosphorylation
concentration−response curves for BQCA and MIPS1455 in
the absence of orthosteric agonist are available in Supporting
Information Figure 1.
Herein we describe the development of a photoactivatable
irreversible allosteric ligand, MIPS1455 (4, Figure 2), for the
M1 mAChR that may be useful in further probing the structural
and functional mechanisms governing allosteric ligand-mAChR
interactions.
Next, we repeated the [3H]NMS competition binding assays
at the M2-M5 mAChR subtypes to evaluate the selectivity
profiles of BQCA and MIPS1455. Neither significant positive
cooperativity with ACh nor negative cooperativity with
[3H]NMS was observed with either ligand, suggesting that
BQCA and MIPS1455 are unable to bind to, or else display
neutral cooperativity with, the M2-M5 mAChRs (Supporting
Information Figure 2).
Finally, we performed [3H]NMS saturation membrane
binding assays to ascertain whether MIPS1455 is capable of
binding irreversibly to the M1 mAChR. A protocol previously
reported by our laboratory24 was modified to incorporate a
photoactivation step. Specifically, following a 1 h preincubation
of M1 mAChR-expressing CHO cell membranes with
MIPS1455, the sample was exposed to 350 nm UV light for
30 min at 4 °C;2 optimal conditions to affect photolysis,
diradical formation, and covalent cross-linking by the
benzophenone functionality (Figure 4).
Figure 2. Structures of BQCA (1), two BQCA analogues (2 and 3),
and the photoactivatable irreversible allosteric ligand MIPS1455 (4).
The extensive structure−activity relationship studies16
performed on the parent molecule, the highly selective M1
mAChR positive allosteric modulator benzyl quinolone
carboxylic acid (BQCA, 1, Figure 2),17 guided our selection
of the benzophenone group as our photoactivatable function-
ality. Many high potency BQCA analogues contain biaryl and
fused aryl systems.18−21 Specifically, replacement of the 4-
methoxy group with a phenyl ring (2 and 3, Figure 2) resulted
in improved affinity (pKB), positive cooperativity (αβ), and
allosteric agonism (τB).22,23 This enabled us to confidently
[3H]NMS saturation membrane binding assays revealed that
MIPS1455 is binding irreversibly to the M1 mAChR; a
significant reduction in Bmax (total receptor density) was
observed following MIPS1455 preincubation and photo-
activation compared to vehicle (Bmax (% vehicle) = 50.2%;
Figure 5A,D). This is indicative of reduced [3H]NMS binding
B
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Figure 3. Pharmacological characterization of BQCA (1) and MIPS1455 (4) in a [3H]NMS-ACh competition binding assay (left) and an ERK1/2
phosphorylation functional assay (right). Experiments performed in hM1 mAChR-expressing CHO cells in the presence of increasing concentrations
of ACh with or without increasing concentrations of BQCA (1) or MIPS1455 (4) (and, in the case of the binding assay, in the presence of a KD
concentration of radiolabeled orthosteric antagonist [3H]NMS (0.1 nM)). The binding cooperativity factor between [3H]NMS and each allosteric
ligand was constrained to an arbitrary low value (log α[NMS] = −3.00) consistent with very high negative cooperativity. Values represent the mean
SEM from at least three experiments performed in duplicate.
capable of binding irreversibly to the M1 mAChR following
photoactivation with 350 nm UV light (Figure 5).
We postulate that this ligand may possess advantageous
properties compared to our previously reported irreversible
allosteric ligand MIPS1262 (see the Supporting Information
Figure 3 for structure),2 which contains an electrophilic
isothiocyanate moiety.24 MIPS1262 is limited to reacting with
nucleophilic amino acid residues such as cysteine, serine and
lysine. Such a requirement may result in the ligand adopting a
distinct binding pose from that of BQCA in the allosteric
binding site in order to orient itself around such residues
(which may in turn lead to stabilization of a different receptor
conformation), making it difficult to extrapolate any structural
or functional findings to BQCA. Furthermore, the isothiocya-
nate moiety of MIPS1262 is inherently reactive, increasing the
likelihood that the ligand binds nonselectively to multiple sites
on the M1 mAChR, in addition to the BQCA allosteric binding
site. In a more complex in vitro setting, this reactivity is also
likely to result in considerable off-target binding. In contrast,
the benzophenone MIPS1455 affords the advantages of
temporal control of reactivity, the ability to rapidly cross-link
with any neighboring methylene unit (within 3.1 Å4) once
activated, and more closely mimicking the structure of higher
potency BQCA analogues than does MIPS1262. Furthermore,
we demonstrate that the high mAChR subtype selectivity of
BQCA is preserved with MIPS1455 (Supporting Information
Figure 2). While these experiments were performed with the
“ground state”, nonphotoactivated MIPS1455, the results
suggest that this ligand has little to no affinity for the other
mAChRs and the likelihood of it cross-linking with these
receptors following photoactivation is greatly reduced. Taken
together, while MIPS1262 may serve as a useful ligand for
investigating allosteric interactions in a recombinant in vitro
setting, MIPS1455 may likely be a more favorable pharmaco-
logical tool for selectively probing structural and functional
allosteric interactions at the M1 mAChR in both a recombinant
and native in vitro setting.
Figure 4. Scheme depicting UV photolysis, generation of a diradical
intermediate, and covalent bond formation by a benzophenone
functionality.
as a result of irreversible allosteric site occupation by
MIPS1455, with which the radioligand has high negative
cooperativity. No significant difference in Bmax was observed
following UV exposure of membranes preincubated with
BQCA (Bmax (% vehicle) = 88.6%, Figure 5B,D). Importantly,
no change in Bmax was observed following preincubation with
MIPS1455 if the UV exposure step was omitted (Bmax (%
vehicle) = 90.3%, Figure 5C,D).
Herein we report the development of MIPS1455, a
photoactivatable allosteric ligand for the M1 mAChR. The
synthesis of the ligand was completed following previously
established methods (Scheme 1). In vitro [3H]NMS competi-
tion binding assays and ERK1/2 phosphorylation functional
assays confirmed that MIPS1455, like the parent molecule
BQCA, acts as an M1 mAChR positive allosteric modulator
(Figure 3). Importantly, we demonstrate that MIPS1455 is
C
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Figure 5. Pharmacological characterization of MIPS1455 (4) and BQCA (1) in [3H]NMS saturation binding assays performed in hM1 mAChR-
expressing CHO cell membranes in the presence of increasing concentrations of radiolabeled orthosteric antagonist [3H]NMS. (A) 1 h
preincubation with vehicle or MIPS1455, followed by 30 min exposure to 350 nm UV light. (B) 1 h preincubation with vehicle or BQCA, followed
by 30 min exposure to 350 nm UV light. (C) 1 h preincubation with vehicle or MIPS1455, without 30 min exposure to 350 nm UV light. (D) Bmax
(% vehicle) bar graphs where values significantly different from vehicle are indicated by asterisk(s) (*) (where * = p < 0.05, ** = p < 0.01, *** = p <
0.001, **** = p < 0.0001, and ns = not significant). Values represent the mean SEM from three experiments performed in duplicate.
Graphics workstation. Chemical shifts (δH) for all 1H NMR spectra are
a
Scheme 1. Synthesis of MIPS1455 (4)
reported in parts per million (ppm) using the center peak of the
deuterated solvent chemical shift as the reference: CDCl3 (7.26) and
d6-DMSO (2.50).26 Each resonance was assigned according to the
following convention: chemical shift (δ) (multiplicity, coupling
constant(s) in Hz, and number of protons). Coupling constants (J)
are reported to the nearest 0.5 Hz. In reporting spectral data, the
following abbreviations have been used: s, singlet; d, doublet; t, triplet;
q, quartet; p, pentet; m, multiplet; br, broad; app, apparent; as well as
combinations of these where appropriate.
13C NMR spectra were routinely recorded at 100 MHz using a
Bruker Avance 400 Ultra Shield Plus spectrometer equipped with a
̈
Silicon Graphics workstation. Chemical shifts (δC) for all 13C NMR
spectra are reported in parts per million (ppm), using the center peak
of the deuterated solvent chemical shift as the reference: CDCl3
(77.16) and d6-DMSO (39.52).26
HSQC, HMBC, and COSY spectra were obtained using the
standard Bruker pulse sequence to assist with structural assignment of
̈
the compounds.
a
Reagents and conditions: (a) 6, DIPEA, ACN, 80 °C, 54%; (b) NBS,
Liquid chromatography−mass spectrometry (LCMS) was per-
formed on an Agilent 1200 Series coupled to the 6120 quadrupole
mass spectrometer. Elution was also monitored at 254 nm. High
resolution mass spectrometry (HRMS) analyses were recorded in the
specified ion mode using a LCT Premier XE TOF mass spectrometer
coupled to a 2795 Alliance Separations Module at cone voltages of 45
V (ESI+) and 60 V (ESI−).
Analytical reverse-phase high performance liquid chromatography
(HPLC) was performed on a Waters HPLC system using a
Phenomenex Luna C8 (2) 100 Å column (150 × 4.6 mm, 5 μm)
and a binary solvent system; solvent A, 0.1% TFA/H2O; solvent B,
0.1% TFA/80% MeOH/H2O. Isocratic elution was carried out using
the following protocol (time, % solvent A, % solvent B): 0 min, 100, 0;
10 min, 20, 80; 11 min, 20, 80; 12 min, 100, 0; 20 min, 100, 0; at a
flow rate of 1.0 mL/min monitored at 254 nm using a Waters 996
Photodiode Array detector.
benzoyl peroxide, EtOAc, microwave 120 °C.
METHODS
■
Chemistry. General. All materials were reagent grade and
purchased commercially from Sigma-Aldrich or Matrix Scientific.
Anhydrous solvents were obtained from a MBraun MB SPS-800
Solvent Purification System. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 precoated plates (0.25 mm,
Merck ART 5554) and visualized by ultraviolet light, iodine, or
ninhydrin as necessary. Silica gel 60 (Fluka) was used for silica gel flash
chromatography. Microwave reactions were performed in a CEM
Discover microwave reactor. Melting points (mp) were determined on
a Mettler Toledo MP50 Melting Point System.
1H NMR spectra were routinely recorded at 400 MHz using a
̈
Bruker Avancell Ultrashield Plus spectrometer equipped with a Silicon
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Characterization requirements for intermediate compounds were
set as mp, 1H NMR, 13C NMR, LCMS and HPLC (254 nm) or LCMS
purity. Characterization requirements for final compounds were set as
made up in HEPES-buffered saline at 10× the required concentration.
A stock solution of atropine (10−4 M) was made up at 10× the
required concentration. Membranes (100 μL per tube) were
equilibrated at 37 °C for 1 h with 100 μL of HEPES-buffered saline
(specific wells) or 100 μL of atropine (nonspecific wells) and 100 μL
per well of each concentration of [3H]NMS, in the required amount of
HEPES-buffered saline to make a total volume of 1 mL per tube.
Assay Termination and Data Collection. Assays were terminated
by harvesting the tubes in the Brandel harvester and rinsing tubes
twice with 2 mL of ice-cold 0.9% NaCl solution. Membranes were
dried under the heat lamp, picked, and placed into 6 mL scintillation
tubes. Then 4 mL per tube of Ultima Gold scintillation liquid was
added, and the tubes capped, vortexed, and left on the bench for 1 h at
RT. The levels of bound radioligand, and therefore the degree of
radioligand blockade by any irreversibly bound test ligands, was
measured in disintegrations per minute (dpm) on the TriCarb 2910
TR liquid scintillation analyzer (PerkinElmer).
1
mp, H NMR, 13C NMR, LRMS, HRMS, and HPLC (254 and 214
nm) purity >95%.
1-(4-Benzoylbenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
Acid (4). 4-Oxo-1,4-dihydroquinoline-3-carboxylic acid (180 mg, 952
μmol), crude 4-(bromomethyl)benzophenone (6) (393 mg, 1.43
mmol), and acetonitrile (8 mL) were placed in an RBF and cooled to
0 °C in an ice bath. N,N-Diisopropylethylamine (DIPEA) (4 equiv)
was added, and the reaction stirred at 80 °C for 22 h. The reaction
mixture was cooled to room temperature, and the precipitated product
isolated as a white solid by vacuum filtration and washed with diethyl
ether (197 mg, 54%); mp 235−236 °C. δH (d6-DMSO) 15.15 (s, 1H),
9.36 (s, 1H), 8.43 (dd, J = 8.0, 1.0 Hz, 1H), 7.93−7.82 (m, 2H), 7.75−
7.62 (m, 6H), 7.58−7.51 (m, 2H), 7.45 (d, J= 8.5 Hz, 2H), 6.02 (s,
2H). δC (d6-DMSO) 195.1, 178.1, 165.9, 150.5, 140.2, 139.5, 136.8,
136.6, 134.3, 132.8, 130.2, 129.6, 128.6, 126.6, 126.5, 126.0, 125.7,
118.5, 108.1, 56.1. LCMS m/z [M + H]+: 384.1. m/z HRMS (TOF
ES+) C24H17NO4 [M + H]+ calcd 384.1230, found 384.1226. HPLC:
tR = 9.88 min, purity (254) = 97.0%, purity (214) = 96.3%
4-(Bromomethyl)benzophenone (6). 4-Methylbenzophenone (200
mg, 1.02 mmol), N-bromosuccinimide (NBS) (218 mg, 1.22 mmol),
and benzoyl peroxide (12.3 mg, 51 μmol) were placed in a microwave
vial with ethyl acetate (4 mL), and the reaction mixture was stirred
briefly to dissolve the reagents. The vessel was sealed and reacted in
the microwave for 15 min at 120 °C. The reaction mixture was filtered
to remove any precipitated NBS, and then the solvent evaporated in
vacuo. The target molecule was reacted on without any further
purification (crude mass: 577 mg); δH (CDCl3) 7.76−7.69 (m, 4H),
7.46−7.39 (m, 5H), 4.47 (s, 2H). δC (CDCl3) 178.4, 142.2, 137.4,
137.3, 132.7, 130.6, 130.4, 130.1, 129.0, 128.4, 126.6, 32.4. LCMS m/z
[M(81Br) + H]+: 277.0. LCMS: tR = 6.78 min, purity = 85.0%
monobrominated product, 15.0% dibrominated byproduct.
ASSOCIATED CONTENT
* Supporting Information
■
S
(1) [3H]NMS competition binding curves and ERK1/2
phosphorylation concentration−response curves for BQCA
(1) and MIPS1455 (4) alone, (2) [3H]NMS competition
binding selectivity study of BQCA (1) and MIPS1455 (4) in
hM2−hM5 mAChR-expressing CHO cells, "(3) structure of
MIPS1262, and (4) HPLC chromatograms (254 and 214 nm)
for compound 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*For P.J.S.: Phone: +61 (0)3 9903 9542. E-mail: Peter.
Scammells@monash.edu.
■
Pharmacology. [3H]NMS competition whole cell binding assays,
ERK1/2 phosphorylation assays, and data analysis were performed as
previously described.24 [3H]NMS saturation membrane binding assays
were performed as follows.
Membrane Preparation. Preprepared FlpIn-CHO cell membranes
stably expressing human muscarinic M1 receptors (hM1 mAChR
FlpIn-CHO) were thawed, diluted to a concentration of 1250 μg/mL,
and resuspended using a hand-held homogenizer (at slow speed to
minimize frothing). Ultracentrifuge tubes (one per buffer/ligand
treatment) were labeled, 500 μg of membrane pipetted into each, the
volume made up to 9 mL with cold HEPES-buffered saline, and the
tubes kept on ice.
*For A.C.: Phone: +61 (0)3 9903 9067. E-mail: Arthur.
Christopoulos@monash.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Author Contributions
B.J.D. conducted all of the experimentation, all authors
contributed to experimental design and the production of the
manuscript.
Preincubation with Controls and Test Ligands. Stock solutions of
MIPS1455 and BQCA (10−2 M) were made up in DMSO. Ligands
were diluted to 10−3 M in HEPES-buffered saline. A buffer control
(10% DMSO/HEPES-buffered saline) was also made up. One
milliliter buffer or ligand additions, for a final volume of 10 mL,
were made, and the membrane tubes incubated in a water bath at 37
°C for 1 h. Membrane tubes were placed immediately on ice for 3 min
and then exposed to 350 nm UV light at 4 °C for 30 min. Next, 10 mL
of ice cold HEPES-buffered saline was added, and then additional ice
cold buffer was added to bring all tubes to equal mass. Tubes were
centrifuged on the Sorval ultracentrifuge at 4 °C for 20 min at 40 000
rpm. The supernatant was discarded, the membranes were washed and
resuspended thoroughly in 1 mL of ice cold HEPES-buffered saline (to
ensure the removal of any noncovalently bound ligand), an additional
19 mL of ice-cold buffer added, and the tubes brought to equal mass
again. Tubes were centrifuged again on the Sorval ultracentrifuge at 4
°C for 20 min at 40 000 rpm. The supernatant was discarded, and the
membranes resuspended in 400 μL of cold HEPES-buffered saline. A
Bradford assay was performed to ascertain the concentration of
membrane in each treatment tube, and then the required amount of
cold HEPES-buffered saline was added to bring the concentration of
each to 30 μg/mL.
Funding
This research was supported by Discovery Grant DP110100687
of the Australian Research Council and Program Grant
APP1055134 of the National Health and Medical Research
Council (NHMRC) of Australia. A.C. and P.M.S. are Principal
Research Fellows of the NHMRC.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to kindly thank Drs. Sebastian Furness
and Karen Gregory for discussions that contributed to the
experimental design.
ABBREVIATIONS
■
ACN, acetonitrile; BQCA, benzyl quinolone carboxylic acid (1-
(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid); CHO, Chinese hamster ovary; COSY, correlation
spectroscopy; DIPEA, N,N-diisopropylethylamine; DMSO,
dimethyl sulfoxide; ERK, extracellular-regulated kinase; ESI,
Assay Protocol. Stock solutions of [3H]NMS (10−7.5 and 10−8 M)
were made up in HEPES-buffered saline. Dilutions of [3H]NMS were
E
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electrospray ionization; GPCR, G protein-coupled receptor;
HMBC, heteronuclear multiple-bond correlation; HPLC, high
performance liquid chromatography; HRMS, high resolution
mass spectrometry; HSQC, heteronuclear single quantum
coherence; LCMS, liquid chromatography−mass spectrometry;
LRMS, low resolution mass spectrometry; mAChR, muscarinic
acetylcholine receptor; MP, melting point; NBS, N-bromosuc-
cinimide; NMPB, N-methyl-4-piperidyl p-azidobenzilate; NMR,
nuclear magnetic resonance; NMS, N-methylscopolamine;
QNB, quinuclidinyl benzilate; RBF, round-bottomed flask;
SEM, standard error of the mean; TFA, trifluoroacetic acid;
TLC, thin layer chromatography; TOF, time-of-flight; UV,
ultraviolet
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