Structure-Based Design and Discovery of New M2 Receptor Agonists

Article abstract of DOI:10.1021/acs.jmedchem.7b01113

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M₂ receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Full text of DOI:10.1021/acs.jmedchem.7b01113

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Article
Structure-based design and discovery of new M2 receptor agonists
Inbar Fish, Anne Stoessel, Katrin Eitel, Celine Valant, Sabine Albold, Harald Hübner, Dorothee Möller,
Mary J. Clark, Roger K. Sunahara, Arthur Christopoulos, Brian K. Shoichet, and Peter Gmeiner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01113 • Publication Date (Web): 02 Nov 2017
Downloaded from http://pubs.acs.org on November 2, 2017
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Structureꢀbased design and discovery of new M2
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Inbar Fish1,5,‡, Anne Stößel2,‡, Katrin Eitel2,‡, Celine Valant3, Sabine Albold3, Harald Huebner2,
Dorothee Möller2, Mary J. Clark4, Roger K. Sunahara4, Arthur Christopoulos3, Brian K.
Shoichet1,b,*, Peter Gmeiner2,*.
‡ Contributed equally.
* Corresponding authors
1Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158,
USA.
2Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich
Alexander University, Schuhstraße 19, 91052 Erlangen, Germany.
3Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville VIC 3052, Australia.
4Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
5Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences,
TelꢀAviv University, Ramat Aviv, Israel.
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ABSTRACT
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Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or
quaternary amine and its distance to an ester or related center. Based on the active state crystal
structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists
that lacked the highly conserved functionalities of previously known ligands. Using structureꢀ
guided pharmacophore design followed by docking, we found two agonists (compounds 3 and
17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted
to form a hydrogenꢀbond conserved among known agonists. Structural optimization led to
compound 28, which was fourꢀfold more potent than its parent 3. Fortified by the discovery of
this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments,
which revealed another three micromolar agonists unrelated either to 28 or known muscarinics.
Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal
opportunities for the structureꢀbased design and the discovery of new chemotypes.
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INTRODUCTION
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With the determination of the atomic resolution structures of ever more G proteinꢀ
coupled receptors (GPCRs), the question arises of how to exploit them for ligand discovery and
design. Though over 30 years of work against soluble proteins have taught a close integration
between medicinal chemistry, computation, and structureꢀdetermination, GPCRs present special
challenges. One often wants not only molecules that complement and inhibit a GPCR (inverse
agonists), as with enzyme inhibitors, but also agonists that activate the receptors, and the
determination of the structures of receptors in their activated states remains rare. Also, most
GPCRs have subtypes that recognize identical endogenous agonists but that signal in different
organs and that couple to different G proteins, making specificity particularly important and
problematic. Finally, structureꢀbased design against GPCRs struggles with the facile
determination of coꢀcomplex structures, especially for new ligand series for which affinity is
initially weak1, 2.
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Agonist discovery for the M2 muscarinic receptor illustrates the opportunities and
challenges facing GPCRs. On the one hand, there are compelling therapeutic and chemicalꢀprobe
arguments for new muscarinic agonists, ideally with new scaffolds. The muscarinic
(acetylcholine GPCR) receptors are ubiquitous in human organs, regulating functions ranging
from heartbeat, to smooth muscle contraction, to glandular secretion, to cognition3, 4. The
receptors are attractive targets for the treatment of conditions like chronic obstructive pulmonary
disease, Alzheimer’s disease, and overactive bladder syndrome4ꢀ7, and the use of selective
muscarinic ligands has recently been discussed for diseases including cancer, diabetes,
cardiovascular disease, pain and inflammation3, 8ꢀ10. Selectivity is challenging, however, owing to
the multiple subtypes with related orthosteric sites signaling in often opposed ways in different
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organs. Among the five major muscarinic receptor subtypes, the M1, M3, and M5 receptors
couple to the G protein Gq, activating phospholipase C, while the M2 and M4 subtypes couple to
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Gi, mediating inhibition of adenylyl cyclase without stimulating PLC, and the differences among
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the orthosteric sites can be as little as a single amino acid (e.g., the orthosteric sites of the M2 and
M3 receptors differ only by a PheꢀLeu)11ꢀ14. This makes other muscarinic subtypes the major
offꢀtarget for muscarinic drugs. Meanwhile, for agonists, which are wanted to treat diseases like
glaucoma, Alzheimer’s disease, and Sjögren's syndrome, the design criteria are very tight. Most
muscarinic agonists derive from small natural products such as the eponymous muscarine,
pilocarpine, and arecoline, and the activated state crystal structure of the M2 receptor15 confirms
that the binding site for agonists is highly constrained (Figure 1A).
Figure 1. A) The crystal structure of M2 active state in a complex with iperoxo (PDB ID 4MQS).
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Residues Asn6.52 and Asp3.32 are represented as sticks and hydrogen bonds as red broken lines. Iperoxo fits
tightly in the binding site; BꢀD) Docking poses of pilocarpine (B) muscarine (C), and arecoline (D), in the
M2 active state structure.
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The restricted agonist site in the M2 receptor, and the tight chemotypes of even the
natural product agonists (Figure 1BꢀD), suggested a focused search for new agonist scaffolds.
Accordingly, we began with complexed conformation of iperoxo bound to the M2 receptor in its
activated state. We initially sought new ligands with an aromatic moiety, substituted with a
hydrogen bond acceptor for the interaction with Asn6.52 (BallesterosꢀWeinstein numbering
system16), and a quaternary amine to ionꢀpair with Asp3.32. This simple strategy succeeded in
finding a new scaffold, but to explain the functional effects of the resulting agonists and
antagonists, we needed to dock them into the M2 receptor structures. We used the predicted
docking poses to exclude compounds unable to interact with Asn6.52, which we expected would
prioritize compounds that can activate the receptor; other contactꢀbased filters, such as
interactions with the tyrosines that are an important ligand recognition element in the site, did not
add to selectivity among the docking hits. As ever, a primary prioritization criterion was docking
scores. Almost all predicted agonists received scores in the range of ꢀ27 to ꢀ41 kcal/mol, with
compounds 25 and 28 only slightly outside that range at ꢀ23 and ꢀ23.5 kcal/mol. Conversely, all
compounds predicted not to be agonists received docking scores higher than (worse than) 0
kcal/mol, except for compound 18 that received a docking score of ꢀ15.5 kcal/mol. Whereas
docking scores are notoriously inaccurate, this range represents a substantial separation. The
resulting model allowed us to prioritize the design of still newer analogs, the most promising of
which was a dihydrobenzofuran 28 (Figure 2), which only shares a ECFP4ꢀbased Tanimoto
coefficients (Tc values) of 0.31 from previous M2 ligands, and appears to be a new scaffold .
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With this new chemotype defined, we cast a final, broader net, screening a large library for
molecules with similar physical properties but greater chemotype diversity. This led to three
more agonists in two distinct scaffolds. The hierarchy of approaches used here, beginning with a
pharmacophore from the crystallographic conformation of an agonist bound to the active state of
the receptor, followed by detailed structural placement, and ending with a large library screen,
though inverted from the more typical discoveryꢀandꢀoptimization flow, may be pragmatic for
agonist design against other receptors.
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Figure 2: Structureꢀbased discovery of new muscarinic agonists: project flow. Structureꢀguided
design from the M2R /iperoxo complex (top left) led to 19 candidate ligands chemically distinct from
previous agonists. Optimization led to the improved compound 28, a new agonist scaffold (bottom right).
A docking screen of a large fragment library (bottom middle) led to three still newer agonists (bottom
left).
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RESULTS
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Structureꢀbased design of new muscarinic agonists. The manual design of initial set of
19 compounds depended on the use of a benzene unit as a central scaffold. The aromatic ring
was functionalized by a hydrogen bond with Asn6.52 – accepting group and a quaternary
ammonium salt (to form an ionꢀpair with Asp3.32 ) that was linked to the aromatic system by
various spacers consisting of 1ꢀ4 carbons (Figure 2, Table 1). To probe distinct orientations of
the functional groups to each other, we synthesized different regioisomers. For some compounds,
conformationally restricting elements were installed bridging either the ammonium salt or the
Hydrogen bond acceptor group with the benzene scaffold. The analogs included
conformationally restricted tertiary and quaternary methoxy or hydroxy substituted
aminotetralins. Analogous benzofuran derivatives were also prepared. To fineꢀtune the distance
between the aromatic ring and the ammonium ion, we synthesized aminomethyl substituted
analogs and tetrahydroisochinolines bearing an endocyclic nitrogen atom. Furthermore, we
prepared a set of monocyclic derivatives, where the methoxyphenyl and the quaternary
ammonium head group are linked by a methylene, ethylene, propylene, cyclopropylene,
propenylene or propynylene chain. The methoxy moiety was added either in the orthoꢀ or metaꢀ
position of a benzene ring, or incorporated into a fused furan. The compounds were synthesized
using solution phase chemical reactions including amide coupling, reductive amination, Henry
reaction, nucleophilic displacement, reduction of amides and nitroolefins, methylene transfer or
palladiumꢀcatalyzed coupling reactions (Supporting Information). Overall, the 19 molecules had
Tc values to previously known muscarinic ligands, annotated in ChEMBL and DrugBank,
ranging from 0.19 to 0.47.
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Table 1: Activities and structural complementarity of compounds 1 to 19 to the M1R, M2R and
M3R receptor subtypes (Ki in µM).
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compound
X
Ki (µM)a
IP accumulation assayb
docking
Hꢀbond predicted
to Asn6.52 agonist?
EC50
Emax
[%]d
docking score
active state
n
M1R
M2R
M3R
[µM]c
2ꢀOMe
2ꢀOMe
3ꢀOMe
3ꢀOMe
1
2
2
3
26±15
4.9±1.1
>50
>50
4.7±1.2
>50
18±3.3
9.1±3.9
12±2.8
/
51±15
21±7.8
75±7.5
<10e
ꢀ8.13
ꢀ27.11
ꢀ32.89
ꢀ15.56
N
N
Y
N
ꢀ
ꢀ
1
2
3
4
5.7±2.3
14±6.1
33±6.6
+
ꢀ
0.33±0.073
0.63±0.17
0.62±0.20
ꢀ
ꢀ
16±0.88
20±4.9
22±2.6
50±25
21±4.5
20±5.9
/
/
/
/
3.86
N
N
ꢀ
ꢀ
5
6
24.43
N/N/
Y/Y
81.61/5.74/
9.66/15.82h
ꢀ
7.3±1.1
15±2.5
8.7±4.4
/
/
ꢀ
7
5ꢀOMe
8ꢀOMe
8ꢀOMe
8ꢀOH
8ꢀOMe
5ꢀOMe
5ꢀOMe
8ꢀOMe
8ꢀOH
ꢀ
ꢀ
14±3.6
17±8.5
1.0±0.19
2.9±1.1
1.8±0.35
2.5±0.35
17±8.5f
2.0±1.5f
>50f
26±14
6.3±2.3
0.93±0.15
4.2±2.5
1.3±0.18
4.4±2.2
14±8.9f
1.2 ±0.88f
>50f
24±9.4
5.8±2.2
2.0±0.53
16±3.5
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
ꢀ18.61
ꢀ10.97
N
N
ꢀ
ꢀ
8
ꢀ
9
0
0
1
0
1
0
0
1
2
ꢀ
79.83/33.04h
ꢀ4.32/7.83h
28.12/ꢀ6.16h
93.73/89.59h
14.86/11.16h
74.55/34.28h
ꢀ6.56/6.86h
ꢀ27.79
N
ꢀ
10
11
12
13
14
15
16
17
18
19
Y
ꢀ
3.9±0.26
4.0±0.42
25±2.8f
0.83±0.22f
28±13f
Y
ꢀ
Y
ꢀ
N/Y
Y/N
Y/Y
Y
ꢀ
ꢀ
ꢀ
0.023±0.0053 0.14±0.049 0.041±0.011 0.22±0.21 ꢀ14±18
+
ꢀ
ꢀ
0.063±0.012 0.23±0.028 0.14±0.053 0.027±0.022 12±13
ꢀ15.49
Y
ꢀ
0.38±0.063
350±50g
5.8±1.5
0.79±0.17 0.69±0.090
2.3±1.9
0.28±0.088g 125±2.3
ꢀ8.3±8.0 39.28/28.65h
N/N
Y
ꢀ
iperoxo
4.9± 0.60g
0.39±0.084
4.1±1.1
550±73g
4.7±1.3
51±12
NA
NA
+
+
acetylcholine
carbachol
0.056±28
0.89±0.22
92±5.5
100
ꢀ28.22
ꢀ32.23
Y
63±12
Y
aKi values ± SEM derived from 3ꢀ8 individual competition binding experiments using the radioligand [3H]Nꢀmethylꢀ
scopolamine bromide and membranes from HEK cells transiently expressing the human M1R, M2R or M3R.
bSecond, less sensitive IP accumulation assay with COS cells coexpressing M2R and Gαqi5HA. EC50 values ± SEM
from 3ꢀ8 individual experiments each done in triplicate. dEmax values relative to the full effect of carbachol. Emax at
10 µM (no complete doseꢀresponse curve was available). fKi values ± SD derived from two independent competition
c
e
binding experiments. gValues are displayed in nM ± SEM. hRacemic mixture. “/” = not determined
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Structural complementarity from docking. All 19 compounds fit the loose
pharmacophore described above, but to guide specific structural complementarity we wanted a
more quantitative metric. In parallel with the synthesis, and blind to biological testing, the 19
compounds were docked into the structures of the active and inactive states of the M2 receptor
(PDB IDs 4MQS15 and 3UON17, respectively). Docking complexes were scored for
electrostatic18, 19 and van der Waals complementarity, corrected for ligand desolvation20, 21, and
the top scoring configuration of each molecule was retained.
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Against the inactive conformation of M2R, all analogs docked favorably, with energy
scores ranging from ꢀ38.05 to ꢀ45.13 kcal/mol, and all posed to interact with Asp3.32 (Figure S1;
Table S1). Conversely, against the active state, only analogs 3 and 17 complemented the more
constrained agonist conformation of the orthosteric site, making favorable interactions with
Asp3.32 and with Asn6.52 and scoring well, with scores of ꢀ32.89 and ꢀ27.79, respectively (Figures
3A and S1; Table 1). All the other compounds in the first set either scored poorly with
unfavorable score, typically above 0 kcal/mol (see above), or did not hydrogen bond with
Asn6.52. Superposition of the pose of iperoxo in the active state structure of M2R with the docked
pose of compound 3 (Figure 3B) shows that the tertiary amine of compound 3, as well as the
oxygen forming the hydrogen bond with the Asn6.52 are in the same spatial position as the
corresponding moieties of iperoxo.
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Figure 3. A) Superposition of compound 17 (green) and compound 3 (purple). Residues Asn6.52, Asp3.32
and Ser107 are represented as sticks. Both compounds hydrogen bond (black broken lines) with Asn6.52
.
B) Superposition between the iperoxo (silver) pose in the M2R active state structure (PDB ID 4MQS15)
and the docked pose of compound 3 (purple). Both compounds appear to hydrogen bond with Asn6.52, ion
pair with Asp3.32, and are enclosed by an aromatic cage composed of Tyr104, Tyr403 and Tyr426.
Binding and functional studies at M1, M2 and M3 muscarinic receptors. Radioligand
binding studies were conducted to evaluate the 19 compounds for their M1R, M2R and M3R
affinity, using [3H]Nꢀmethylꢀscopolamine bromide and membrane preparations from transiently
transfected human embryonic kidney cells (HEK)22, 23. To detect agonists, the ability of the
compounds to activate the M2 receptor was first investigated using a sensitive IP accumulation
assay (HTRF detection, IPꢀOne®) in HEK cells transiently expressing the human M2R together
with the hybrid Gꢀprotein Gαqi5HA24. Promising compounds were tested in a second, less
sensitive but more informative IP accumulation assay in kidney cells from African green monkey
(COS) transiently expressing the human M2R and Gαqi5HA25; it is the results of this second,
higher fidelity assay on which we focus here. The affinity and efficacy profiles of the analogs
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were compared to those of the neurotransmitter acetylcholine, the approved drug carbachol, and
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the superagonist iperoxo (Tables 1 and S2).
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Compounds 2, 3 and 5ꢀ15 had M1R, M2R and M3R Ki values in the singleꢀ or doubleꢀ
digit micromolar range, and compound 3 had nascent specificity. The conformationally
restrained ligands 17ꢀ19 had nanomolar Ki values (0.023 to 0.79 µM). The IP accumulation assay
for M2R activation revealed inverse agonism to strong partial agonism, with Emax values ranging
from ꢀ14% for compound 17 (i.e., inverse agonism), to 75% for compound 3. Antagonist or very
weak agonist effects were observed for the bicyclic compounds 8ꢀ17. While the monocyclic
derivatives 5ꢀ7, bearing conformationally restrained moieties, did not substantially stimulate
M2R, the methoxyphenyl compounds 1ꢀ3 with the flexible alkylene unit had Emax values ranging
from 21 to 75% and EC50 values in the 10 ꢀM range. These observations agreed broadly with the
docking predictions: compound 3 was the most active, compound 17 was among the tightest
binding of the 19 ligands, and 16 compounds were correctly classified as antagonists or inverse
agonists (Table 1). Admittedly, the docking prioritization was imperfect: compound 1, which
turned out to be a decent agonist, was misꢀpredicted as an antagonist, and compound 17,
notwithstanding its affinity, turned out to be an antagonist.
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Structureꢀguided optimization. We sought to optimize for activity by designing a
7
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9
second set of ligands. Two approaches were used: 1) docking of a library of analogs and 2)
structureꢀbased design from the docking pose. All nine of the resulting analogs preserve the
ethylene linker between the aromatic moiety and the ammonium head group of compound 3 that
seem important to superpose with iperoxo, and wellꢀcomplement the activated conformation of
the M2R.
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In the first approach, a library of 54 analogs was generated and docked against the active
state structure. Out of these, four compounds were predicted to be agonists from their ability to
hydrogen bond with Asn6.52 and their favorable docking scores. These include the secondary
amine 20, the hydroxy analog 21, the secondary alcohol 23 and compound 22 (Tables 2 and S1,
Figure S1). On synthesis and testing, compound 22 displayed an improved Ki of 1 ꢀM while
retaining specificity over the M1 and M3 receptors and substantial agonist activity (Tables 2 and
S2). Of the other three compounds, the Ki for 20 also improved to micromolar but it lost agonism
and specificity, 21 retained activity but was less active than the lead agonist, 3, and 23 lost both
affinity and most of its activity. These results, which represent docking failures, are consistent
with the idea that while the method can select for fit, both optimization and selection for
activation remain challenging it.
In the second approach, which also began with the docking pose, we manually designed
and then synthesized analogs of compound 3 by replacing the metaꢀmethoxy substituent by an
ethoxy, chloro or trifluoromethoxy group (25ꢀ27). Additionally, the ketone 24 and the
dihydrobenzofuran analog of compound 17 (compound 28) were prepared. The conformationally
restricted ligand 28 was expected to be a closer surrogate of the lead 3 than the previous
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unsaturated analogs 17ꢀ19, because the electronic properties of the sp3 oxygen with two lone
pairs are more isosteric than the respective sp2 atom of the benzofuran system. On testing,
compound 24 had improved affinity and 25 retained substantial efficacy, but only compound 28,
the conformationally restricted analog, retained both decent affinity and agonist efficacy for the
M2 receptor in the higher fidelity IP accumulation assay (Tables 2 and S2). Overall, the docking
prioritized compounds 3 and its phenylꢀfluorinated analog 22, along with the conformationally
restricted 28, emerged as the most active of the new analogs.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
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40
41
42
43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2: Screening of the second set compounds 20 to 28 (receptor binding affinities to M1, M2
and M3 (Ki in µM), M2 receptor activation and docking data).
IP accumulation
compound
R1
Ki [µM]a
docking
Hꢀbond
assayb
docking
score
active state
EC50
Emax
[%]d
predicted
agonist?
R2
X
M1R
M2R
M3R
to
[µM]c
Asn6.52
ꢀ
ꢀ
ꢀ
0.74±0.13
20±11
1.0±0.25
14±4.7
0.78±0.10
13±4.8
8.2±5.5
10±4.1
23±10
ꢀ12±1.5
44±10
ꢀ40.99
ꢀ36.29
ꢀ29.73
Y
Y
Y
+
+
+
20
21
22
OH
OMe
H
F
H,H
H,H
7.1±2.5
0.98±0.61
14±5.9
44±4.7
ꢀ32.23/
ꢀ30.97g
Y/
N
+/
ꢀ
OMe
H
H,OH
13±5.5
29±8.7
12±4.2
ꢀ
<10e
23
OMe
OEt
Cl
H
H
H
H
ꢀ
O
H,H
H,H
H,H
ꢀ
0.17±0.014f 2.4±0.42f 0.21±0.028f
ꢀ
<10e
40e
<10e
<10e
62±6.6
ꢀ34.56
ꢀ23.00
ꢀ39.07
ꢀ28.6
Y
Y
N
Y
Y
+
+
ꢀ
24
25
26
27
28
42±14
4.5±1.1
7.8±1.6
6.4±0.96
27±9.9
4.8±1.4
9.2±2.1
13±3.3
43±10
5.8±1.8
11±6.6
6.3±1.7
ꢀ
ꢀ
ꢀ
OCF3
+
+
ꢀ
21±7.6
ꢀ23.47
aKi values ± SEM derived from 3ꢀ8 individual competition binding experiments using the radioligand [3H]Nꢀmethylꢀ
scopolamine bromide. bSecond, less sensitive IP accumulation assay with COS cells coexpressing M2R and Gαqi5HA
.
d
cEC50 values ± SEM from 3 individual experiments each done in triplicate. Emax values ± SEM relative to the full
effect of carbachol. eMaximum effect at 100 µM; no complete doseꢀresponse curve could be determined. fKi values ±
SD derived from 2 individual competition binding experiments. gRacemic mixture.
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With these results in hand, we decided to more thoroughly investigate 3 and 28 in whole
cell assays, using Chinese Hamster Ovary (CHO) cells stably expressing the muscarinic receptor
subtypes M1, M2 or M3, (Table 3, Figure 4). Unlike the assays reported in Tables 1 and 2, these
cells recapitulate the native Gi/0 coupling of the M2 receptor, and the Gq/11 coupling of the M1 and
M3 receptors. Whereas the binding affinity and selectivity for compound 3 remained little
changed, the apparent affinity of compound 28 improved in the CHO cells to 2 ꢀM, while its
selectivity over M1 and M3 receptors improved to 4ꢀ and 10ꢀfold, respectively (Table 3 and
Figures 4AꢀC). Looking at [35S]GTPγS binding assay, a classic functional assay for Gi/o
proteinꢀcoupled receptors, 28 was a full agonist with an EC50 of 3.3 µM and a 100% Emax
compared to acetylcholine (Figure 4F); compound 3 was also a full agonist in this assay with an
EC50 only slightly higher at 8 ꢀM. These values were largely confirmed by a cAMP
accumulation assay (Figure 4G). In IP accumulation assays (IPꢀOne® assay), which flows from
Gq/11 stimulation through the M1 and M3 receptors, both 3 and 28 showed only weak agonist
behavior, suggesting specificity for the M2R, with potencies consistent with their binding
affinities (Figure 4DꢀE), while no IP accumulation was measured via M2, as expected given its
native Gi/o coupling. In a more downstream functional assay, looking at the level of ligandꢀ
mediated ERK1/2 phosphorylation, both 3 and 28 behaved as full agonists (Emax = 100%) with
potencies of 0.83 and 0.23 µM for M2R (Figure 4J). Against the M1 receptor only 3 had reliable
agonist activity, displaying partial agonist profile, while against M3 neither compound showed
substantial activation, suggesting that amplification of signal corresponds with amplification of
the new agonists’ subꢀtype selectivity (Figure 4IꢀK). Finally, we determined the ability of 28 to
stimulate ßꢀarrestin recruitment via M2R activation. Compared to acetylcholine, 28 was a less
10
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17
18
19
20
21
22
23
24
25
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27
28
29
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potent but more efficacious agonist, and showed 20ꢀfold bias toward arrestin, versus
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acetylcholine as a reference in the cAMP assay (Table 3, Figure 4H).
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These results suggest that compounds 3 and 28 are micromolar to subꢀmicromolar full
agonists of the M2 receptor in native signaling, with 28 having moderate arrestin bias versus
acetylcholine. The aryl methoxy groups on the “leftꢀhand side” of each molecule represent a new
chemotype for the muscarinic receptors, while the distal quaternary nitrogen is wellꢀestablished.
To investigate the replacement of this cationic group, and find still newer scaffolds, we turned to
large library docking.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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Table 3: Signaling selectivity of new muscarinic agonists 3 and 28 versus acetylcholine.
test compounds
receptor
test system
subtype
acetylcholine
4.64 ± 0.04 / 23
6.42 ± 0.06 / 0.38
5.17 ± 0.07 / 6.8
3
28
pKi [M] / Ki [µM]
pKi [M] / Ki [µM]
pKi [M] / Ki [µM]
M1R
M2R
M3R
4.28 ± 0.09 / 52
5.04 ± 0.08 / 9.1
4.09 ± 0.15 / 81
5.17 ± 0.03 / 6.8
5.69 ± 0.07 / 2.0
5.11 ± 0.10 / 7.8
whole cell
bindinga
pEC50 [M] / EC50 [µM]
Emax [% ± SEM]c
6.21 ± 0.11 / 0.62
100
5.04 ± 0.17 / 9.1
48 ± 5
5.32 ± 0.33 / 4.8
8 ± 2
M1R
IP accumulationb pEC50 [M] / EC50 [µM] M2R
ꢀ
ꢀ
ꢀ
pEC50 [M] / EC50 [µM]
M3R
6.62 ± 0.16 / 0.24
100
4.98 ± 0.07 / 10
43 ± 2
5.43 ± 0.23 / 3.7
17 ± 2
Emax [% ± SEM]c
pEC50 [M] / EC50 [µM] M1R
pEC50 [M] / EC50 [µM]
Emax [% ± SEM]c
ꢀ
ꢀ
ꢀ
6.29 ± 0.07 / 0.51
100
5.11 ± 0.06 / 7.8
100
5.48 ± 0.07 / 3.3
100
GTPγS bindingd
M2R
pEC50 [M] / EC50 [µM] M3R
pEC50 [M] / EC50 [µM] M1R
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
inhibition of
cAMP
pEC50 [M] / EC50 [µM]
M2R
7.31 ± 0.19 / 0.049
100
5.20 ± 0.18 / 6.3
108 ± 9
4.96 ± 0.13 / 11
102 ± 16
Emax [%]c
accumulatione
pEC50 [M] / EC50 [µM] M3R
ꢀ
ꢀ
ꢀ
pEC50 [M] / EC50 [µM] M1R
pEC50 [M] / EC50 [µM] M2R
ꢀ
ꢀ
ꢀ
ßꢀarrestin
recruitmentf
5.72 ± 0.09 / 1.9
4.85 ± 0.05 / 14
4.76 ± 0.11 / 17
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Emax [%]c
100
133 ± 7
ꢀ
118 ± 9
ꢀ
pEC50 [M] / EC50 [µM] M3R
ꢀ
6
7
8
9
pEC50 [M] / EC50 [µM]
M1R
7.33 ± 0.15 / 0.047 6.27 ± 0.23 / 0.54
100 53 ± 6
7.90 ± 0.08 / 0.013 6.08 ± 0.14 / 0.83
n.a.h
Emax [%]c
pEC50 [M] / EC50 [µM]
M2R
6.62 ± 0.11 / 0.24
~100
ERK1/2
phosphorylationg
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28
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60
Emax [%]c
100
7.64 ± 0.08 / 0.023
100
92 ± 8
n.a.h
pEC50 [M] / EC50 [µM]
M3R
n.a.h
Emax [%]c
aAffinity to M1R, M2R or M3R from whole cell receptor binding performed with CHO cells stably expressing a given
receptor subtype and with the antagonist radioligand [3H]Nꢀmethyl scopolamine; pKi ± SEM values are the means of
b
4ꢀ6 individual experiments each in duplicate. Gqꢀprotein mediated functional activity was measured with the same
cells using the IPꢀOne® assay (Cisbio); pEC50 ± SEM values are the means of 3ꢀ4 individual experiments each in
c
d
duplicate. Maximum efficacy vs. acetylcholine. Gi/oꢀProtein activation was measured using [35S]GTPγS binding
with membranes from the same cells stably expressing M2R; pEC50 ± SEM values are mean values from 3 individual
experiments each in duplicate. eInhibition of cAMP accumulation was done with HEK cells stably expressing the Epac
f
cAMP sensor and M2R; pEC50 ± SEM were means 4ꢀ5 individual experiments each in duplicate. ßꢀarrestin
recruitment assay was performed with HEK cells stably expressing the βꢀarrestinꢀTEVprotease and transiently
transfected with M2ꢀTEVꢀtTA; pEC50 ± SEM values are the means of 4ꢀ5 individual experiments, each in triplicate.
gERK1/2 phosphorylation was measured by AlphScreen with CHO cells stably expressing M1R, M2R or M3R; pEC50 ±
SEM values are the means of 3ꢀ4 individual experiments, each in duplicate. hn.a.: could not be estimated.
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A
B
C
5
6
7
8
100
80
60
40
20
0
9
acetylcholine
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22
23
24
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28
29
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3
28
-11 -10 -9 -8 -7 -6 -5 -4 -3 -2
compound [log M]
D
E
100
80
60
40
20
0
100
80
60
40
20
0
acetylcholine
acetylcholine
3
3
28
28
-11 -10 -9 -8 -7 -6 -5 -4 -3
compound [log M]
-11 -10 -9 -8 -7 -6 -5 -4 -3
compound [log M]
F
G
H
100
acetylcholine
80
3
60
40
20
0
28
-11 -10 -9 -8 -7 -6 -5 -4 -3
compound [log M]
I
J
K
Figure 4. Detailed investigation of the new muscarinic agonists 3 and 28. AꢀC: Binding behavior at
whole cells expressing the muscarinic receptor subtypes M1 (A), M2 (B) and M3 (C) in comparison to the
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natural ligand acetylcholine. DꢀG: M2 selective signaling of 28 indicated by a weak activation of M1 and
M3 stimulated IP accumulation (D and E, respectively) and full agonist effect in M2 mediated GTPγS
binding (F) and inhibition of cAMP accumulation (G). H: M2 mediated ßꢀarrestin recruitment displays
full agonist effect for 28. IꢀK: Downstream signaling shows M2 selective agonist properties for 28 as
determined in a ERK1/2 phosphorylation assay for M1 (I), M2 (J) and M3 (K).
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Prospective fragments library docking screen–selection of 10 compounds. Heartened
by the discovery of the new agonists, we sought still more novel agonists from a structureꢀbased
screen of a larger chemical library. We screened the “clean fragments” subset of the open access
27
ZINC26,
(http://zinc15.docking.org), then just over 2.2 million commercially available
compounds, with xlogP ≤ 3.5, molecular weight ≤ 250 Dalton and rotatable bonds ≤ 5, with
DOCK3.628. Each library molecule was screened in an average of 337.5 orientations in the
orthosteric site, and in each orientation an average of 32.6 conformations was sampled. Overall,
over 24 billion molecular complexes were evaluated (in a leadꢀlike screen, by comparison, we
might evaluate 50ꢀfold more complexes, as the increased molecular size demands more
sampling). Configurations were ranked by their electrostatic (using a point charge model of the
30
PoissonꢀBoltzmann equation, as implemented in QNIFFT)29,
and van der Waals
complementarity (using the AMBER potential31) to the M2 active state, corrected for contextꢀ
dependent ligand desolvation28 (using GB/SA electrostatics implemented in AMSOL20, 21), and
the top scoring configuration of each molecule was retained. The screen took 37.6 total core
hours, or less than an hour of elapsed time on our lab cluster.
The result of the calculation was a ranked list of fragments, from most to least
complementarity to the M2 orthosteric active state pocket. The top ranked 1000 (best 0.05%)
fragments were inspected for those that interacted with both Asp3.32 and Asn6.52. Ten were
selected for testing by radioligand displacement and IP accumulation, again using the sensitive
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(IPꢀOne®) and the more informative IP accumulation assay ([3H]inositol based) (Table 4, Table
S3). Three of the ten fragments had micromolar EC50 values in the more stringent IP
accumulation assay of between 9.9 and 29 ꢀM, and M2 receptor Emax values ranging from 60 to
74% (Table 4, Figure 5). Most of the other fragments had midꢀmicromolar affinities for the
M2R, and several even had substantial agonism in the IP screening assay, but this activity was
not retained in the more stringent functional assay (Table S3). By design, the three new agonists
have little similarity to known muscarinic ligands, with ECFP4ꢀbased Tc values ranging from
0.20 to 0.34 to annotated ligands in ChEMBL and DrugBank (Table 4). While all three retain the
ubiquitous cation of aminergic agonists, the conserved ester/amide of muscarinic agonists has
been replaced with either a thiazole (29) or a pyridine (30 and 31), which has little precedence; in
the docked configurations, these heterocycles interact with the same Asn6.52 with which the
carbonyl system of classic agonists interact. Consistent with the degree of these changes, further
alkylation of the aminergic group, which ordinarily would increase activity, for the new agonists
diminished it substantially (Tables 4 and S3). Structurally, these new agonists represent an even
greater departure from known agonist scaffolds than even compounds 3, 22, and 28.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
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Table 4: Experimentally active molecules from docking and their synthesized analogs.
5
6
7
IP accumulation
compound
Ki [µM]a
docking
ZINC IDs of
assayb
8
9
Tc to closest
muscarinic
ligand
Ran
k
EC50
Emax
[%]d
closest
muscarinic
ligande
Structure
M1
M2
M3
•
[µM]c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
324
383
4.2±0.65 6.0±2.5 4.1±1.6 9.9±1.7
74±4.6
74±14
0.20
0.34
C13739835
C34802190
29
30
18±9.7 11±2.0 36±6.7
10±2.9 17±4.9 10±2.8
29±13
13±4.8
449
60±6.5
0.33
C27984351
33
NA
NA
6.1±1.8 10±1.1 12±1.4
10±4.3
>100
28±1.2
38±4.8f
0.28
0.33
C00000346
C34802190
29a
30a
38±16
39±4.4 75±6.4
NA
20±3.2 46±2.0 42±2.2
50±7.4
33±10
0.31
C27984351
33a
aKi values ± SEM derived from 3ꢀ8 individual competition binding experiments using the radioligand [3H]Nꢀmethylꢀ
scopolamine bromide. bSecond, less sensitive IP accumulation assay with COS cells coexpressing M2R and Gαqi5HA
cEC50 values ± SEM from 4ꢀ6 individual experiments each done in triplicate. dEmax values ± SEM relative to the full
.
e
f
effect of carbachol. 2D structures are presented in Table S4. Maximum effect at 100 µM; no complete doseꢀ
response curve could be determined.
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Figure 5. Docking poses of compounds 29 (A), 30 (B) and 33 (C) in the M2R active state structure (PDB
ID 4MQS). Residues Asn6.52, Asp3.32, Ser107 and Tyr403 are represented as sticks. Hydrogen bonds are
represented in black.
Like many primary neurotransmitters, acetylcholine activates receptors from more than
one protein family; such crossꢀfamily polypharmacology provides an uniquely chemical
organization for signaling32, 33. Besides the five muscarinic GPCR subtypes, acetylcholine also
activates ligandꢀgated ion channels as primary targets. These nicotinic acetylcholine receptors
(nAChRs) are widely expressed throughout the central and peripheral nervous system and at the
neuromuscular junction. Functional nAChRs are a heterogenic group of pentameric ion channels
composed of various subunits. Whereas the new docking compounds were chosen for their
novelty against known muscarinic ligands, we initially did not consider their similarity to
nicotinic ion channel ligands. Unexpectedly, certainly not by design but during the review of this
manuscript, we discovered that compounds 3, 28, 29, 30, 33, 29a, 30a and 33a had meaningful
similarities to known nicotinic ligands, with Tc values ranging from 0.33 to 0.62 (SI Table S5).
Whereas none were identical to known nicotinic ligands, these similarities are high enough to
suggest the new M2 muscarinic agonists might also activate the nicotinic receptor, a cross
pharmacology that remains relatively rare, though not completely unknown, outside of
acetylcholine itself, and its close cogeners.
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Accordingly, the affinity for the (ꢀ)ꢀnicotine binding site at α4β2 nAChR was determined
for representative compounds 28, 30 and 33, against the potent radioligand [3H]cytisine.
Although all three compounds display nearly identical Ki values for M2 mAChR (11ꢀ17 µM),
their affinity for the α4β2 nAChR differs in more than three orders of magnitude, resulting in
distinct selectivity profiles. While 28 shows equipotent affinities to the muscarinic and nicotinic
acetylcholine receptor (Ki = 9.7 µM for α4β2 nAChR), the alkyloxymethylpyridines 30 and 33
have pronounced selectivity towards nAChR (Ki values 200 nM for 30 an extraordinary 1.6 nM
for 33, respectively). Indeed, the affinity of 33 for nAChR resembles the 1.5 nM affinity of
nicotine itself. Notably, compounds 30 and 33 share structural similarity with selective α4β2
nAChR ligands of the 3ꢀpyridylether family such as pozanicline (ABTꢀ089, 2ꢀmethylꢀ3ꢀ(2ꢀ(S)ꢀ
pyrrolidinylmethoxy)pyridine). Pozanicline, epibatidine and other high affinity α4β2 nAChR
(partialꢀ)agonists have been under clinical investigation as cognitive enhancers, including for
attention deficit/hyperactivity disorder and Alzheimer’s disease, and as analgesics and
anxiolytics. Moreover, the α4β2 nAChR partial agonist varenicline is used to effectively
enhance smoking cessation. Whereas the functional activity of compounds 30 and 33 is out of
scope for this study, such properties may merit future study, as too their role as joint activators of
the acetyl cholinergic circuit.
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DISCUSSION
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Two key observations emerge from this study. First, whereas classical muscarinic
agonists reflect a highly constrained pharmacophore, structural complementarity to an activated
receptor reveals novel agonists topologically unrelated to those previously known. The modeling
that discovered the new agonists suggests that they are recognized via the same interactions
made by the classic agonists, but using different agonist functional groups. This suggests that
there might be many more agonist recognition motifs readily accessible, as neither our
pharmacophoreꢀlike design nor our library screen pretend to comprehensiveness. Several of these
new molecules, like the more optimized 28, have intriguing signaling properties, including a 20ꢀ
fold bias toward arrestin signaling vs the endogenous acetylcholine, and a signaling specificity
for the M2 vs the M1 or M3 receptor subtypes. Second, the new agonists flowed both from a large
library docking screen and also from byꢀhand modeling, the traditional domain of the medicinal
chemist. This study supports an alloy between the designing chemist and facile quantitative
techniques by which their inspiration can be rapidly checked.
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Certain caveats bear mentioning. None of the new agonists have strong activities against
muscarinic receptors—none are even at probe levels of activity or specificity, far less what one
would expect from a therapeutic lead. And while novel chemotypes can lead to new biological
effects34ꢀ36, the evidence for such here, even with the nascent selectivity and signaling bias of 28,
remains at an early stage. While both the byꢀhand design and the unbiased molecular docking
screen both support the possibility of discovering new agonists for the muscarinic receptors, the
docking hits stumbled into chemotypes with high activities against the nicotinic ion channel,
against which muscarinic GPCR activity would ordinarily be optimized against (the coꢀactivity
against both the ionotropic and metabotropic acetyl choline receptors may itself merit further
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study).
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Still, this study supports a structureꢀbased effort to discover new chemotypes, even in a
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field as wellꢀploughed as the muscarinic. We have only undertaken an early reconnaissance into
the design of or screens for such agonists; we suspect many more are readily accessible, and a
screen of a larger, more elaborated leadꢀlike or drugꢀlike molecules might reveal a broader array
of more potent and more selective molecules, as would optimization of the early agonists
discovered here. An advantage of these new chemotypes is that by engaging the receptor with
new functionalities they can stabilize activated ensembles in manners unexplored by the
precedented agonists, engaging effectors in new ways—biased signaling is one example of that.
More concretely, they provide templates for the optimization of pharmacokinetic properties that
have long been exploited among muscarinic ligands, such as blood brain penetration, typically
defined by tertiary vs. quaternary ammoniums. The new agonists, with their new responses to
wellꢀestablished optimization moves, provide new points of departure for medicinal chemistry
and probe development programs.
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EXPERIMENTAL METHODS
Docking against active and inactive state of M2R. We used DOCK3.628 to dock
molecules against the M2R active state crystal structure bound to the agonist iperoxo (PDB ID
4MQS15), and to the M2R inactive state bound to QNB (PDB ID 3UON17). The same program
was used in a docking screen of the “fragmentsꢀnow” subset of the ZINC database
(http://zinc15.docking.org). Partial charges from the unitedꢀatom AMBER force field were used
for all receptor atoms except for Asn6.52 for which the magnitude of the local partial atomic
charges were increased to accentuate electrostatic interactions with this particular residue (the net
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charge of the residue remained neutral)—a technique we have widely used previously36, 37
.
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Fortyꢀfive matching spheres were used. The number of ligand orientations sampled is determined
by the values of the bin size, bin size overlap and distance tolerance, set at 0.3 Å, 0.1 Å and 1.2
Å, respectively, for both the matching spheres and the docked molecules. The ligand
conformations sampled were preꢀcalculated using Openeye’s Omega program38 (Openeye
Software, Santa Fe NM). Ligand charges and initial solvation energies were calculated using
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AMSOL (http://comp.chem.umn.edu/amsol/)20, 21
.
BallesterosꢀWeinstein (BW) numbering. Receptor residues are referred to by their
threeꢀletter code, followed by their BallesterosꢀWeinstein (BW) number. In this method, TM
residues are identified by a superscript numbering system, in which the residue corresponding to
the Family A GPCRs most conserved residue in a given TM is assigned the index X.50, where X
is the TM number, and the remaining residues are numbered relative to this position16.
Tanimoto coefficient (Tc) calculation. We extracted a dataset of 2422 ligands from
CHEMBL2039ꢀ41 and DrugBank42. Using the GenerateMD program (version 5.10.3) in the
Chemaxon package we calculated the EFCP4 fingerprints which were used to calculate the Tc43
between our hits and all of the 2422 ligands in Table 4 .
Membrane based radioligand binding experiments. Affinities of the test compounds
towards the human M1, M2, and M3 receptor were determined using homogenates of membranes
as described previously22, 23, 44. In brief, HEK293T cells were transiently transfected with the
cDNA of the appropriate receptor (purchased from cDNA Resource Center, Bloomsberg, PA)
using a solution of linear polyethyleneimine in PBS45. Receptor binding experiments were
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performed in 96ꢀwell plates using homogenates of the corresponding receptor together with the
radioligand [3H]Nꢀmethylꢀscopolamine bromide (specific activity of 70 Ci/mmol, PerkinElmer,
Rodgau, Germany) at a final concentration of 0.20 – 0.30 nM for M1R and M2R, and 0.10 – 0.20
nM for M3R at a receptor density (Bmax) of 1500 ± 260 fmol/mg, 1400 ± 140 fmol/mg, and 2200
± 530 fmol/mg, a protein concentration of 3ꢀ6 µg/test tube, 5ꢀ10 µg/test tube, and 2 – 10 µg/test
tube, and a Kd value of 0.18 ± 0.052 nM, 0.20 ± 0.018 nM, and 0.086 ± 0.005 nM, for M1R,
M2R, and M3R, respectively. Unspecific binding was determined in the presence of 10 µM
atropine. Protein concentration was established by the method of Lowry using bovine serum
albumin as standard46. Resulting competition curves were analyzed by nonlinear regression using
the algorithms for oneꢀsite competition of PRISM 6.0 (GraphPad, San Diego, CA).
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Whole cell radioligand binding assays. Radioligand binding experiments were
performed on CHOꢀFlpIn whole cells stably expressing the human M1, M2, and M3. receptor
constructs of choice. After plating 20,000 cells in complete DMEM into 96ꢀwell ISOPLATE TC
plates (all amounts are per well), cells were allowed to grow overnight at 37 oC. The next day,
cells were washed with phosphateꢀbuffered saline (100 mL) and reꢀsuspended in binding buffer
(10 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4). Assay mixtures, in a total volume of
100 ꢁL with a 1/10 dilution of drug, were incubated at room temperature (22 oC) for 6 h. Assays
were terminated by buffer removal followed by rapid washing, twice, with iceꢀcold 0.9% NaCl
(100 ꢁL). Plates were allowed to dry inverted for 30 min; OptiPhase Supermix scintillation
cocktail (100 ꢁL) was added, plates were sealed (TopSealTM) and radioactivity was measured in
a MicroBeta2 LumiJET microplate counter. Saturation binding experiments were performed in
the absence or presence of atropine (10 ꢁM) with 0.003 – 3 nM [3H]NMS. Inhibition binding
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experiments were performed with ~0.2 nM [3H]NMS (the approximate K) in the presence of
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various concentrations of analogues.
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IP Accumulation assay: For validation of the screening data derived with the IPꢀOne®
assay (see supporting information) the most promising compounds were tested on M2R
activation with an IP accumulation assay as described previously15. In brief, COSꢀ7 cells were
transiently cotransfected with M2R and Gαqi5ꢀHA (Gαq protein with the last five amino acids at the
Cꢀterminus replaced by the corresponding sequence of Gαi; grateful gift from The J. David
Gladstone Institutes, San Francisco, CA) applying the TransITꢀ2020 Mirus transfection reagent
(MoBiTec, Goettingen, Germany). 18 h before the test cells were incubated with myoꢀ
[3H]inositol (specific activity = 20.1 Ci/mmol, PerkinElmer, Rodgau, Germany). Test
compounds (six different concentrations for each compound, total range from 0.1 pM up to 300
µM) were incubated for 2 h at 37 °C in triplicate and resulting radioactivity was measured by
scintillation counting. Activation curves were normalized to the maximum effect of carbachol
(100%) and buffer (0%) and analyzed using the algorithms for nonlinear regression in PRISM
6.0. For all compounds 3ꢀ8 individual doseꢀresponse curves were measured, the corresponding
EC50 and Emax values of each mean curve were calculated and summarized to get the average
EC50 and Emax values ± SEM.
IPꢀOne Accumulation Assays. The IPꢀOne® assay kit (Cisbio, France) was used for the
direct quantitative measurement of myoinositol 1ꢀphosphate (IP1) in FlpIn CHO cells stably
expressing hM1 and hM3 mAChRs. This is a competitive immunoassay that measures the
homogeneous timeꢀresolved fluorescence signal transferred between a cryptateꢀlabeled IP1ꢀ
specific monoclonal antibody and d2ꢀlabeled IP1. The fluorescence signal measured is inversely
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proportional to the concentration of native IP1. Briefly, cells were seeded into 96ꢀwell plates at
20,000 cells per well and allowed to grow overnight at 37 °C, 5% CO2. The following day, cells
were washed once with PBS then incubated with stimulation buffer (HEPES 10 mM, CaCl2
1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, pH 7.4) for
60 min at 37 °C, 5% CO2. Following this incubation, ligands were added at 10x their final
concentrations (ACh or test compounds) and incubated for a further 40 min prior terminating the
ligandꢀmediated stimulation by removing the buffer and adding 25 ꢁL of lysis buffer. Finally,
14 ꢁL of lysate was transferred into 384ꢀwell Optiplate, followed by the addition of 3 ꢁL of IP1ꢀ
d2, then 3 ꢁL of AbꢀCryp, and incubated for 60 min at room temperature. Time resolved
fluorescence resonance energy transfer (HTRF) was determined using the Envision plate reader
(Perkin Elmer).
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[35S]GTPγS Binding Assay. Membrane homogenates (15 ꢁg) were equilibrated in a
500 ꢁL total volume of assay buffer containing 10 mM guanosine 5’ꢀdiphosphate and a range of
concentrations of ACh or test compounds (1 nM–100 mM) at 30 °C for 60 min. After this time,
50 ꢁL of [35S]GTPγS (1 nM) was added, and incubation continued for 30 min at 30 °C.
Termination of the reaction and determination of radioactivity were performed by rapid filtration
through Whatman GF/B filters using a Brandell cell harvester (Gaithersburg, MD). Filters were
washed three times with 3 mL aliquots of iceꢀcold 0.9% NaCl buffer and dried before the
addition of 4 mL of scintillation mixture (Ultima Gold, PerkinElmer Life Sciences). Vials were
then left to stand until the filters became uniformly translucent before radioactivity was
determined in dpm using scintillation counting.
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cAMP Accumulation assay: HEK293 cells stably expressing the Epac cAMP sensor47,
obtained as a gift from Jesper Mathiesen, were stably transfected with M2AChRꢀtetO (a gift from
Brian Kobilka). HEKꢀEpacꢀM2tetO cells were grown to confluency and then treated with 2
µg/mL doxycycline and 1 mM sodium butyrate for 40 h to induce expression of the M2AChRꢀ
tetO. Cells were harvested with lifting buffer (0.68 mM EDTA, 150 mM NaCl, 20 mM HEPES,
pH 7.4), centrifuged, resuspended in HBSSꢀHEPES (Hank’s Balanced Salt Solution plus 20 mM
HEPES, pH 7.4) and pipetted into a 96ꢀwell plate (black with clear bottom). After 20 min in the
dark at 37 oC, the basal CFP/YFP ratio of the EpacꢀcAMP FRET sensor was measured at 436 exc
and 480/535 ems for 2 min using a SpectraMax M5. Then forskolin (2 µM final), IBMX (1 mM
final), and test compound (0 – 100 µM final) in HBSSꢀHEPES, pH 7.4 were added and the
CFP/YFP ratio area under the curve was measured for 10 min at 37 oC. Basal values were
subtracted and data was analyzed using GraphPad Prism 6.
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βꢀArrestin recruitment assay: HEK293 cells stably expressing tTA dependent
luciferase and βꢀarrestinꢀTEVprotease were transiently transfected with M2ꢀTEVꢀtTA (cells and
DNA construct were a gift from Bryan Roth) for measurement of M2AChR stimulated of βꢀ
arrestin
recruitment,
basically
as
described
at
https://pdspdb.unc.edu/pdspWeb/content/PDSP%20Protocols%20II%202013ꢀ03ꢀ28.pdf. The day
after transfection, cells were lifted, resuspended in DMEM with 1% FBS, and plated into a Polyꢀ
D
ꢀLysine coated 384ꢀwell clearꢀbottom plate at 15,000 cells/well. After at least 6 h, 0 – 100 µM
test compounds in HBSSꢀHEPES (Hank’s Balanced Salt Solution plus 20 mM HEPES, pH 7.4)
were added to the cells. The following day, media was replaced with diluted BrightꢀGlo Reagent
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