Novel M2-selective, Gi-biased agonists of muscarinic acetylcholine receptors

Article abstract of DOI:10.1111/bph.14970

Background and Purpose: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. Experimental Approach: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M₂ receptors was modelled in silico. Key Results: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M₂ muscarinic receptors and displaying bias towards the G_i signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. Conclusions and Implications: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M₂ and M₄ muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

Full text of DOI:10.1111/bph.14970

Su Qiwen (Orcid ID: 0000-0003-0965-9947)
Boulos John (Orcid ID: 0000-0001-8561-6418)
Jakubík Jan (Orcid ID: 0000-0002-1737-1487)
Novel M2-selective, Gi-biased agonists of muscarinic acetylcholine
receptors
Running title: selective agonists of muscarinic ACh receptors
Alena Randáková, Dominik Nelic, Dana Ungerová, Peter Nwokoye, Qiwen Su, Vladimír
Doležal, Esam E. El-Fakahany, John Boulos, Jan Jakubík
Department of Neurochemistry, Institute of Physiology CAS, Prague, Czech Republic (AR,
DN, DU, VD, JJ), Department of Physical Sciences, Barry University, Miami Shores, FL, USA
(PN, QS, JB) and Department of Experimental and Clinical Pharmacology, University of
Minnesota College of Pharmacy, Minneapolis, MN, USA (EEE).
Correspondence to: Jan Jakubik, Department of Neurochemistry, Institute of Physiology CAS,
Videnska 1083, CZ142 20, Prague, Czech Republic, jan.jakubik@fgu.cas.cz, Phone: +420-2-
4106-2620
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/bph.14970
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Abstract
Background & Purpose
Above 30 % of currently marketed medications act via G-protein coupled receptors (GPCRs).
Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to
traditional agonists activating multiple signalling pathways, agonists activating a single
signalling pathway represent a new generation of drugs with increased specificity and fewer
adverse effects.
Experimental Approach
We have synthesized novel agonists of muscarinic acetylcholine receptors and tested their
binding and function in CHO cells expressing individual subtypes of muscarinic receptors,
primary cultures and dissipated native tissues. Binding of novel compounds was modelled in
silico.
Key Results
Two of the tested new compounds (1‐(thiophen‐2‐ylmethyl)‐3,6‐dihydro‐2H‐pyridinium and
1‐methyl‐1‐(thiophen‐2‐ylmethyl)‐ 3,6‐ dihydro‐2H‐pyridinium) only inhibited cAMP
synthesis in CHO cells, primary cultures and native tissues, with selectivity for M2 muscarinic
receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic
receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way
specific for a given agonist followed by agonist-specific changes in the conformation of the
receptor.
Conclusions & Implications
The identified compounds may serve as lead-structures in the search for novel non-steroidal
and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects
associated with activation of phospholipase C signalling pathway.
Summary
What is already known
 Selective muscarinic agonists are needed to prevent undesired side-effects.
 Only few functionally selective muscarinic agonists are known.
What study adds
 We have developed M2 selective, Gi-biased agonists.
Clinical significance
 These compounds may serve as lead-structures in the search for novel non-steroidal and
non-opioid analgesics.
Keywords
muscarinic acetylcholine receptors; functionally selective agonists; signalling bias
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Abbreviations
CHO – Chinese hamster ovary, IPX – inositol phosphates, KHB – Krebs HEPES buffer, NMS
– N-methylscopolamine, VTA – ventral tegmental area
Although individual G-protein coupled receptors (GPCRs) preferentially couple to the
particular class of G-proteins, their specificity is not absolute. They usually activate multiple
G-proteins with various potency, efficacy and kinetics (Hermans, 2003; Jakubík et al., 2006).
Besides artificial systems with over-expressed GPCRs, this coupling promiscuity was also
observed in native cells (Jakubík et al., 1996; Laugwitz et al., 1996). It is generally accepted
that structurally diverse agonists induce different changes in conformation of GPCRs that can
lead to non-uniform modulation of signalling pathways. This preferential orientation of
signalling of a given GPCR towards a subset of its signal transducers is termed signalling bias
(Kenakin and Christopoulos, 2013). The property may be employed to develop drugs that
selectively produce desired effects while avoiding side effects associated with activation of
unwanted signalling pathways (Manglik et al., 2016). Enormous research has been done on
agonists biased either towards G-protein or arrestin mediated pathway. Here, as a proof of
concept, we demonstrate signalling bias among individual classes of G-proteins.
Five subtypes of muscarinic acetylcholine receptors are expressed in the central and peripheral
nervous system as well as innervated tissues. Muscarinic receptors mediate a wide variety of
physiological functions via Gq/11 G-protein mediated stimulation of phospholipase C (M1, M3
and M5 receptors) or via Gi/o G-protein mediated inhibition of adenylyl cyclase (M2 and M4
receptors) (Eglen, 2012). Disruption of muscarinic signalling frequently contributes to many
pathophysiological conditions, including a variety of neurological and psychiatric disorders,
e.g. Alzheimer's disease and schizophrenia, and also to some internal diseases, e.g. asthma or
overactive bladder (Eglen, 2012). To target the above-mentioned disease states, selective
modulation of individual muscarinic subtypes is necessary to avoid undesired side effects.
However, high homology of the orthosteric binding site among all five muscarinic receptor
subtypes (Haga et al., 2012; Kruse et al., 2012; Thal et al., 2016) makes it very difficult to find
agonists that could bind and selectively activate individual subtypes. No affinity-based
selective agonists of muscarinic receptors have been discovered so far. The development of
biased agonists can be the right approach to overcome the conserved nature of the orthosteric
binding site and to achieve selective modulation of individual subtypes of muscarinic receptors.
However, signalling bias of muscarinic receptors particularly with respect to individual G-
proteins is still poorly explored.
Residues in the binding site that mediate activation of a specific signalling pathway are termed
functional hot-spots (Nivedha et al., 2018). Binding of an agonist to one or a subset of
functional hot-spots within the binding site results in activation of a subset of signalling
pathways and thus in ligand-mediated signalling bias. An agonist relatively small in size has a
better chance to bind to a smaller number of functional hot-spots than a larger agonist. It has
been proposed that some muscarinic agonists (e.g. analogues of arecoline) exert bias towards
Gi/o signalling pathway. These agonists do not interact with Asn6.52 (Ballesteros-Weinstein
numbering (Ballesteros and Weinstein, 1995)) in contrast to conventional agonists (Fish et al.,
2017). The scaffold containing the NCCSC backbone where the nitrogen is electron positive
and sulphur is electron negative part of a ligand has been shown to exert some M2 binding
selectivity (Boulos et al., 2013). Taking above-mentioned into account, we have designed and
synthesized small muscarinic agonists (Figure 1). Two of these compounds exclusively
inhibited synthesis of cAMP (through activation of the Gi/o signalling pathway) and did not
stimulate accumulation of inositol phosphates (Gq pathway), either in a heterologous
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expression system or in dissipated tissues and primary cultures. Although Gi versus β-arrestin-
biased agonists were reported (e.g. at opioid receptors (DeWire et al., 2013)), Gi/o bias at the
G-protein level has not been reported so far (Bologna et al., 2017). Molecular modelling
confirmed the absence of hydrogen bonding of the novel agonists with Asn6.52 in the sixth
transmembrane helix and the existence agonist-specific conformations induced by the novel
agonists.
Muscarinic agonists have strong analgesic effects. Several lines of evidence indicate that the
analgesic effects of muscarinic agonists are mediated by Gi/o G-proteins activated by M2 and
M4 receptors (Duttaroy et al., 2002; Chen and Pan, 2004; Wess et al., 2007; De Angelis and
Maria Tata, 2016). Currently, muscarinic agonists represent one of the most promising drug
development targets to find novel agents to replace addictive opioid analgesics (Tata, 2008).
Therefore, agonists, solely inhibiting cAMP synthesis, discovered in our laboratory, may serve
as a novel pharmacophore for development of new painkillers with reduced side-effects
mediated by phospholipase C pathway. However, their potential analgesic effects need to be
verified in vivo.
Materials and Methods
Materials
Reagents were purchased from Aldrich Chemical Company (St. Louis, MO) unless otherwise
noted, and all starting liquid materials were distilled before use. NMR spectra were recorded
on a Varian 300 MHz spectrometer housed at Barry University. Mass spectra were recorded
on a Perkin Elmer Clarus 560 S GC/MS system. Elemental analyses were carried out by
Galbraith Laboratories (Knoxville, TN) and biological assays were conducted at the Institute
of Physiology of the Czech Academy of Sciences in Prague. Melting points were recorded on
a MEL-TEMP II purchased from Laboratory Devices and are uncorrected. All radiolabelled
compounds (N-[3H]methyl scopolamine, myo-[2-3H(N)]inositol and [2,8-3H]adenine) were
purchased from American Radiolabelled Chemicals, Inc. (Saint Louis, MO). Common
chemicals were purchased from (Sigma, Prague, CZ) in the highest available purity.
Cell culture and membrane preparation
Chinese hamster ovary (CHO) cells stably transfected with the genes of individual human
variants of muscarinic receptors were purchased from Missouri S&T cDNA Resource Center
(Rolla, MO, USA). Fresh primary cell culture of smooth muscle cells (SMC) from rat aorta
prepared as previously described (Bacáková et al., 1997) was kindly provided by Dr. Lucie
Bačáková. Primary culture of SMCs was grown to confluency in 75 cm2 flasks in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum. Up to 6-times, 2 million
of cells were sub-cultured. Cell cultures and crude membranes from CHO cells were prepared
as described previously (Boulos et al., 2017). Cells were grown to confluency in 75 cm2 flasks
in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 2
million of cells were subcultured in 100 mm Petri dishes. Medium was supplemented with 5
mM butyrate for the last 24 hours of cultivation to increase receptor expression. When needed,
cells were cultivated with pertussis toxin at a final concentration of 25 ng/ml for the 24 hours .
Cells were detached by mild trypsinization on day 5 after subculture. Detached cells were
washed twice in 50 ml of phosphate-buffered saline and 3 min centrifugation at 250 x g.
Washed cells were suspended in 20 ml of ice-cold incubation medium (100 mM NaCl, 20 mM
Na-HEPES, 10 mM MgCl2, pH = 7.4) supplemented with 10 mM EDTA and homogenized on
ice by two 30 sec strokes using Polytron homogenizer (Ultra-Turrax; Janke & Kunkel GmbH
& Co. KG, IKA-Labortechnik, Staufen, Germany) with a 30-sec pause between strokes. Cell
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homogenates were centrifuged for 30 min at 30,000 x g. Supernatants were discarded, pellets
suspended in fresh incubation medium, incubated on ice for 30 minutes and centrifuged again.
Resulting membrane pellets were kept at -80 oC until assayed within 10 weeks at a maximum.
Rat dissipated tissues
Suspension of dissipated tissues from rat brain cortex, cerebellum, striatum, ventral tegmental
area (VTA) and submaxillary glands was prepared as described previously in supplementary
methods (Randáková et al., 2018). Adult 8-weeks old male Wistar rats were scarified by
cervical dislocation in accordance with current legislative and guidelines under permission of
Czech Ministry of Agriculture 16OZ23113/2017-17214. Tissues were dissected immediately.
Submaxillary glands were minced by scissors. Tissues were dispersed by incubation for 15 min
at 37ºC in KHB supplemented with trypsin and trituration every 5 min. Large undissipated
parts were removed by cell mesh. Then dissipated tissues were centrifuged 3 min at 250 x g,
re-suspended in KHB and centrifuged again to remove trypsin. Suspension of dissipated tissues
was used immediately in experiments. Two submaxillary glands or about 400 mg of brain tissue
was used a 96-sample assay.
Equilibrium radioligand binding experiments
All radioligand binding experiments were optimized and carried out according to general
guidelines (El-Fakahany and Jakubik, 2016). Briefly, membranes were incubated in 96-well
plates at 30 oC in the incubation medium described above. Incubation volume was 400 μl or
800 μl for competition and saturation experiments with [3H]NMS, respectively. Approximately
30 μg of membrane proteins per sample were used. N-methylscopolamine binding was
measured directly in saturation experiments using six concentrations (30 pM to 1000 pM) of
[3H]NMS for 1 hour. For calculations of equilibrium dissociation constant (KD), concentrations
of free [3H]NMS were calculated by subtraction of bound radioactivity from total radioactivity
in the sample and fitting Eq. 1 (data analysis section). Binding of tested ligands was determined
in competition experiments with 1 nM [3H]NMS. Membranes were incubated for 1 (M2), 3
(M1, M3, M4) or 5 hours (M5) at 30 °C. Inhibition constant KI was calculated according Eq. 3.
Non-specific binding was determined in the presence of 1 μM unlabelled atropine. Incubations
were terminated by filtration through Whatman GF/C glass fibre filters (Whatman) using a
Brandel cell harvester (Brandel, Geithesburg, MD, USA). Filters were dried in microwave oven
and then solid scintillator Meltilex A was melted on filters (105 °C, 90 s) using a hot plate. The
filters were cooled and counted in Wallac Microbeta scintillation counter.
Measurements of the production of cAMP and inositol phosphates
Levels of second messengers cAMP and inositol phosphates were determined in radiochemical
chromatography assays as described previously (Jakubík et al., 1996). To determine level of
cAMP, cells in suspension were pre-incubated for 1 h with 0.4 µM [3H]adenine, washed, and
incubated for 10 min in the presence of isobutylmethylxanthine and 10 µM forskolin. Then
about 200 thousand cells per 0.8 ml sample were incubated for 1 h with carbachol or tested
compounds. Incubation was ended by addition of 0.2 ml of 2.5 M HCl to the samples. Samples
were applied to alumina columns (1.5 g of alunima per column), washed with 2 ml ammonium
acetate (pH = 7.0) and eluted from columns with 4 ml ammonium acetate and measured by
liquid scintillation spectrometry. M2 and M4 receptors that preferentially inhibit cAMP
synthesis via Gi/o G-proteins were coupled to the Gq – PLC pathway. For this purpose, new
CHO cell lines stably expressing promiscuous G15 G-protein (Offermanns and Simon, 1995)
and M2 or M4 receptor were generated by transfection with pCMV/hygro vector and
hygromycin selection. To determine the rate of formation of inositol phosphates IPX, suspended
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cells were pre-incubated with 0.1 µM [3H]myo-inositol for 60 min, washed, and incubated with
10 mM LiCl for 15 min. Then about 200 thousand cells per 0.8 ml sample were incubated for
1 h with carbachol or tested compounds for 60 min. The incubation was stopped by addition of
0.5 ml of chloroform methanol HCl mixture (2:1:0.1). After separation of water and organic
phases, 0.6 ml of water phase was applied to Dowex columns (1.5 g of Dowex per column),
washed by 20 ml of 60mM ammonium formate / 5mM sodium borate buffer and eluted from
columns with 4 ml of 1M ammonium formate / 0.1 formic acid buffer and measured by liquid
scintillation spectrometry. All pre-incubations and incubations were carried in Krebs-HEPES
buffer (KHB; final concentrations in mM: NaCl 138; KCl 4; CaCl2 1.3; MgCl2 1; NaH2PO4
1.2; HEPES 20; glucose 10; pH adjusted to 7.4) supplemented with 10 mM glucose at 37°C.
Data and analysis
The manuscript complies with BJP’s recommendations and requirements on experimental
design and analysis (Curtis et al., 2018). Experiments were independent, using different
seedings of CHO cells or explanted from different animals. Binding experiments were carried
out in 6 experiments with samples in quadruplicates and functional assays were carried out in
5 experiments with samples in triplicates. Experimenters were blind to chemical structures of
tested compounds. After subtraction of non-specific binding (binding experiments) or
background/blank values (functional experiments) data were normalized to control values
determined in each experiment. IC50 and EC50 values and parameters derived from them (Ki
and KA) were treated as logarithms. All data were included in analysis, no outliers were
excluded, and normality of distribution was checked. In statistical analysis value of P<0.01 was
taken as significant for all data. In multiple comparison tests ANOVA with P<0.01 was
followed by Tukey post-test (P<0.01). Statistics was calculated using R (www.r-project.org).
[3H]NMS saturation binding
∗푥
y= 퐵
Eq. 1
MAX
x+K
퐷
where y is specific binding at free concentration x, BMAX is maximum binding capacity, and
KD is equilibrium dissociation constant.
Competition binding
100∗푥
y=100 − x+IC
Eq. 2
50
where y is specific radioligand biding at concentration x of competitor expressed as percent of
binding in the absence of competitor, IC50 is concentration causing 50 % inhibition of
radioligand binding. Inhibition constant KI was calculated as:
IC
50
퐾퐼 =
Eq. 3
[
]
퐷
1+
퐾
퐷
where IC50 is concentration causing 50 % inhibition of [3H]NMS binding calculated according
Eq. 2 from competition binding data, [D] is concentration of [3H]NMS used, and KD is its
equilibrium dissociation constant calculated according Eq. 1 from saturation binding data.
Functional response
nH
)
−1 ∗푥
y=1 + (
Eq. 4
E'
MAX
nH
nH
EC +x
50
where y is functional response at concentration of tested compound x, E'MAX is apparent
maximal response to the tested compound, EC50 is concentration causing half-efficient
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concentration and nH is slope factor (Hill coefficient). For bi-phasic response, curves were
inhibitory and stimulatory phase were fitted separately.
Operational model of functional agonism
Operational efficacy coefficient τ (Black and Leff, 1983) was determined by fitting Eq. 5 to
data from functional assay.
nH nH
퐸
∗휏 ∗푥
MAX
y= (
Eq. 5
nH
nH nH
)
x+K
+τ ∗푥
퐴
where y is functional response at concentration of tested compound x, EMAX is maximal
response of the system, KA is equilibrium dissociation constant and nH is slope factor. Eq. 5
was fitted to data from functional experiments. Eq. 5 was fitted to data by the two-step
procedure described earlier (Jakubík et al., 2019). In the first step, system EMAX was determined
using carbachol, oxotremorine and pilocarpine as internal standards by global fit to all data for
a given receptor subtype and signalling pathway. In the second step, Eq. 5 with EMAX fixed to
the value determined in the first step was fitted to individual experimental data sets.
Relative intrinsic activity
For comparison of effects of agonists at different receptors and signalling pathways, relative
intrinsic activity (RAi) was calculated according to Griffin et al. (Griffin et al., 2007):
휏
휏
∗ 퐾
퐴 푎
푐푎푟푏푎푐ℎ표푙
∗ 퐾
푅퐴푖 =
Eq. 6
푎
퐴 푐푎푟푏푎푐ℎ표푙
Where τa and KAa are half-effective concentration and apparent maximal response to the tested
compound, respectively. As Hill coefficients were equal to one, RAi values were calculated
according Eq. 7.
퐸′
퐸′
∗ 퐸퐶
50푎
푀퐴푋푐푎푟푏푎푐ℎ표푙
푅퐴푖 =
Eq. 7
∗ 퐸퐶
푀퐴푋푎
50푐푎푟푏푎푐ℎ표푙
Where EC50a and E’MAXa are half-effective concentration and apparent maximal response to the
tested compound, respectively.
Signalling bias
A putative signalling bias between pathway j1 and j2 was estimated by ΔΔlog(τ/KA) method
according to Eq. 8 (Kenakin and Christopoulos, 2013) based on Eq. 6 (Griffin et al., 2007):
휏
)
푏푖푎푠 = 10훥훥 log(
Eq. 8
Eq. 9
푗1−푗2
퐾
퐴
Where
휏
휏
휏
훥훥 log( 퐾
)
= 훥 log(
)푗1 − 훥 log( 퐾 )
푗2
퐾
퐴 퐴
푗1−푗2
퐴
Molecular modelling
Preparation of receptor structure
Model of the M2 receptor in an active state was based on the crystal structure 4MQS (Kruse et
al., 2013). Model of the M2 receptor in an inactive state was based on the crystal structure
3UON (Haga et al., 2012). Non-receptor molecules except ligand were deleted, and the
resulting receptor protein was processed in Maestro using Protein Preparation Wizard
according to Madhavi Sastry et al. guidelines (Sastry et al., 2013).
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Ligand docking
Docking of carbachol, compounds 6A and 7A was done using Schrodinger Software Suit
(Schrödinger LLC, New York, NY) with procedure described earlier (Jakubík et al., 2015).
The ligands were constructed in ChemAxon MarvinSketch (RRID:SCR_004111) and prepared
for docking in Maestro (RRID:SCR_016748) using LigPrep (RRID:SCR_016746). The
ligands were docked to the receptor using Induced Fit Docking procedure. Iperoxo was set to
define the binding site. Initial Glide docking step was set to “Standard Precision” without
explicit constraints. In Prime refinement step, amino acid residues within 5 Å form ligand were
refined in single pass. Glide re-docking step was set to “Extra Precision”. Top 20 poses were
then re-evaluated using Prime MM-GB/SA. The highest pose possessing hydrogen bond to
Asp103 was selected for further work.
Simulation of molecular dynamics
To evaluate ligand binding to the receptor and to evaluate its interactions with the receptor,
conventional molecular dynamics (MD) was simulated using Desmond/GPU ver. 5.3
(RRID:SCR_014575) . The simulated system consisted of receptor-ligand complex in 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane set to receptor helices in
water and 0.15 NaCl. The system was first relaxed by the standard Desmond protocol for
membrane proteins and then 1.2 ns NPT (Noose-Hover chain thermostat at 300 K, Martyna-
Tobias-Klein barostat at 1.01325 bar, isotropic coupling, Coulombic cutoff at 0.9 nm) followed
by 120 ns NVE molecular dynamics without restrains was simulated. MD was run 3-times with
various initial velocities. The quality of molecular dynamics simulation was assessed by
Simulation Quality Analysis tools of Maestro and analysed by Simulation Event Analysis tool.
Ligand-receptor interactions were identified using Simulation Interaction Diagram tool.
Further MD trajectories were analyzed in VMD (RRID:SCR_004905). Using tcl scripts
(Supplementary Information, Molecular Modelling section) distances and RMSDs of various
parts of the receptor were measured. Namely, the distance between gravity centres of Cα of
residues 94 to 124 (TM3), 385 to 411 (TM6), 204 to 212 (TM5) and 435 to 443 (TM7), all
atoms of Arg121 at the edge of TM3 and Glu382 at the edge of TM6, all atoms of Tyr206 at
the edge of TM5 and Tyr440 at the edge of TM7 and RMSD of Arg436, Pro437 and Tyr440
were measured. Using Dynamical Network Analysis tool box (Sethi et al., 2009) relay of
conformational changes from the ligand to Arg121 and Glu382 via allosteric interaction
networks was analyzed. In this analysis ligand and each amino acid were treated as separate
nodes.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).
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Results
New compounds were tested in CHO cell lines stably expressing the human variants of
individual subtypes of muscarinic receptors. Expression level of individual subtypes of
muscarinic receptors was determined in N-methylscopolamine ([3H]NMS) saturation binding
studies and are listed alongside with [3H]NMS equilibrium dissociation constants in
Supplementary information, Table S1. Expression level of individual classes of G-proteins was
determined by [35S]GTPγS scintillation proximity assay (Jakubík et al., 2006) and is
summarized in Supplementary information, Figure S1.
Affinity of new compounds (Supplementary information, Figure S2) was assessed in
competition with 1 nM [3H]NMS (Supplementary information, Table S2). All compounds
completely inhibited the binding of [3H]NMS, suggesting competitive mutually exclusive
interaction. All compounds bound to all subtypes of muscarinic receptors with affinity in the
micromolar range. Inhibition constants Ki ranged from 0.7 µM for compound 7D at M2
receptors to 54 µM for compound 6A at M4 receptors. Compounds with methylated nitrogen
(7A, 7B and 7C) had higher affinity than non-methylated counterparts (6A, 6B and 6C) at all
receptor subtypes. Affinities of compounds with halogenated thiophene at the 5-position (7C
and 7D) ring were higher than those of non-halogenated congeners, especially at M2 receptors.
About 20 % binding to M2 and 10 % of binding to M4 of compounds 6B and 7B occurred with
high affinity (about 30 nM). Reference agonists displayed high and low affinity binding
(Supplementary information, Table S3).
M1, M3 and M5 receptors preferentially activate phospholipase C and increase the level of
inositol phosphates (IPX) via coupling with Gq/11 G-proteins. M2 and M4 receptors preferentially
inhibit production of cAMP via coupling with Gi/o G-proteins. The ability of tested compounds
to activate preferential signalling pathways of muscarinic receptors was determined in
functional experiments by measuring accumulation of IPX at M1, M3 and M5 receptors or
inhibition of forskolin-stimulated production of cAMP at M2 and M4 receptors. These results
were then compared to responses evoked by the classical non-selective orthosteric agonists,
carbachol, oxotremorine and pilocarpine (Supplementary information, Table S4). Except for
compound 6E (methyl group at the 4-position of the thiophene ring), all compounds acted as
partial to full agonists at all receptor subtypes, although in many cases, especially at M1, M3
and M5 receptors, their effects were negligible (Supplementary information Table S4, Figure
S3). In particular, compounds 6A and 7A displayed at M2 and M4 response (EMAX) comparable
to that of carbachol. Their potency (pEC50) at M2 and M4 receptors was higher than at other
subtypes (Figure 2). Overall, all compounds were more potent and more efficacious at M2 and
M4 receptors than at other receptors.
Muscarinic receptors are also able to stimulate non-preferential signalling pathways. The
ability of the two most efficacious compounds 6A and 7A to activate non-preferential responses
via muscarinic receptors was determined by measuring of forskolin-stimulated production of
cAMP at M1, M3 and M5 receptors or accumulation of IPX at M2 and M4 receptors co-
transfected with promiscuous G15 G-protein (Figure 3). Carbachol activated non-preferential
signalling pathways at all receptor subtypes. It increased cAMP level at M1 and M3 receptors
(Gs) and stimulated accumulation of IPX (G15) at M2 and M4 receptors. At M5 receptors,
carbachol inhibited forskolin-stimulated production of cAMP at nanomolar concentrations
while at micromolar concentrations it stimulated cAMP production. In contrast to carbachol,
compounds 6A and 7A inhibited cAMP production at M1, M3 and M5 receptors but did not
stimulate IPX accumulation at M2 and M4 receptors. Taking together all functional responses,
both preferential and non-preferential, compounds 6A and 7A inhibited forskolin-stimulated
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production of cAMP at all receptor subtypes and did not stimulate the production of cAMP at
any subtype. In contrast, carbachol-stimulated production of IPx and cAMP at all subtypes.
To calculate the operational efficacy coefficient τ of preferential (Figure 2) as well as non-
preferential (Figure 3) functional response to tested agonists, the system EMAX was determined
from functional responses to the reference agonists carbachol, oxotremorine and pilocarpine
according to the procedure described recently (Jakubík et al., 2019). As carbachol and
oxotremorine did not inhibit forskolin-stimulated production of cAMP at M1 and M3 receptors,
system EMAX for these systems was determined from functional responses to pilocarpine and
compounds 6A and 7A. Then Eq. 5 was fitted to the experimental data. Values of the
operational efficacy coefficient τ of carbachol and compounds 6A and 7A (Supplementary
Information, Table S4 and S5) are plotted in Figure 4. Intrinsic activities relative to carbachol
RAi were calculated to analyze potential selectivity of 6A and 7A (Table 1, Supplementary
information Table S4 and S5). Compounds 6A and 7A had the highest RAi at M2 (6A,
M2>M3≈M4≈M5≈M1) or M2 and M4 receptors (7A: M2>M4>M1=M3≈M5) (Table 1). The
reference agonist carbachol was efficacious at stimulation of IPX (Gq) and cAMP (Gs)
production at all subtypes and in lowering cAMP level at M2, M4 and M5 receptors. Compounds
6A and 7A had no Gs efficacy at any receptor subtype. Their Gq efficacy was always lower
than efficacy of carbachol. In contrast, their Gi efficacy was (except M4) always higher than
efficacy of carbachol. Being Gi efficacious at M1 and M3 receptors compounds 6A and 7A
display an absolute Gi bias, while carbachol has no Gi efficacy at these receptors. Being solely
efficacious at Gi pathway at M2 and M4 receptors compounds 6A and 7A are also Gi biased at
these receptors (Table 1, Supplementary Information, Table S4 and S5). At M5 receptors
compounds 6A and 7A exerted about 10-fold and 30-fold Gi over Gq + G15 bias, respectively.
To exclude the possibility that activation of the non-preferential Gs pathway was obscured by
activation of the preferential Gi/o pathway, accumulation of cAMP was measured in CHO cells
expressing M2 or M4 receptors where Gi/o G-proteins were inactivated by pertussis toxin (PTX)
treatment (Figure 5). PTX treated cells were cultivated alongside with untreated cells. PTX
treatment was successful as it abolished carbachol-mediated decrease in cAMP. In fact,
carbachol caused about two-fold increase in forskolin-stimulated (Figure 5, full circles) as well
as basal (Figure 4, open circles) accumulation of cAMP in PTX-treated cells. Carbachol EC50
values in PTX treated cells were the same (M2) or 3-times lower (M4) than in non-treated cells.
Unlike carbachol, compounds 6A and 7A did not change cAMP levels even after inactivation
of Gi/o G-proteins by PTX. Thus potential stimulatory (e.g. Gs-mediated) response to
compounds 6A and 7A was not obscured by activation of Gi G-proteins.
To exclude the possibility that the observed effects of compounds 6A and 7A are the result of
overexpression in the heterologous system, functional response to the compounds was
measured ex vivo in dissipated native rat tissues. Native tissues express about 10-times less
muscarinic receptors than CHO cell lines (Supplementary information, Table S6 vs. Table S1).
Accumulation of IPX was measured in brain cortex, submaxillary gland, and ventral tegmental
area (VTA) as brain cortex and submaxillary glands are rich in M1 and in M3 receptors,
respectively, and VTA is the only region expressing significant amount of M5 receptors.
Accumulation of cAMP was measured in M2-rich cerebellum and M4-rich striatum. Carbachol
produced profound accumulation of IPX in the cortex, submaxillary gland and VTA. In contrast
to carbachol, compounds 6A and 7A produced no increase in IPX levels in submaxillary gland
and led to negligible increase in IPX in the cortex and VTA (Figure 6, left). In the cerebellum
and striatum, carbachol caused transient decrease in forskolin-stimulated cAMP level at sub-
micromolar concentrations followed by an increase at micromolar concentrations. In contrast
to carbachol, compounds 6A and 7A caused only a decrease in cAMP level. EMAX of inhibition
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of cAMP synthesis in the cerebellum and striatum by compounds 6A and 7A was comparable
to inhibition by carbachol. Taken together, compounds 6A and 7A only inhibited cAMP
synthesis in dissipated tissues, being sometimes as efficacious as the full agonist carbachol.
Effects of compounds 6A and 7A on both cAMP and IPX levels were also measured in primary
cultures of rat aorta smooth muscle cells that naturally express mainly M2 and to a lesser extent
M3 receptors (Eglen, 2012). In this primary cell culture, carbachol caused a transient decrease
in cAMP level (Figure 7, left) as well as an increase in IPX level (Figure 7, right). Similar to
dissipated native tissues, compounds 6A and 7A only inhibited cAMP synthesis. Maximal
inhibition of cAMP synthesis by compounds 6A and 7A was the same as inhibition by
carbachol. Taken together, ex vivo results are in agreement with results obtained at CHO cells.
Thus, pharmacological profile of 6A and 7A is not an artefact of heterologous high-expression
system.
Binding of compounds 6A and 7A was modelled in silico. Binding of compounds 6A, 7A,
classical agonist carbachol, and the super-agonist iperoxo to the M2 receptor in an active
conformation was modelled using the crystal structure of the M2 receptor (PDB ID: 4MQS)
and analysed by simulation of molecular dynamics (MD) of receptor-ligand complex. MD
analysis showed that all agonists interact with key Asp103 (Asp3.32 according to Ballosteros-
Weinstein numbering (Ballesteros and Weinstein, 1995)) in TM3 (Figure 8). While compound
6A interacts with Asp103 mainly via polar interactions and water bridges rest of modelled
agonists interact with Asp103 by strong ionic interactions. Compounds 6A and 7A may interact
with key Asn404 in TM6 via water bridges but the super-agonist iperoxo and the full agonist
carbachol of interact with Asn404 mainly via polar interactions. Additionally, interaction of
compounds 6A and 7A with Trp400 (Trp6.48) was much more frequent than the interaction of
iperoxo or carbachol with this residue.
Using “Dynamical Network Analysis” of MD trajectory, we analysed the network of allosteric
interactions from the ligand to the receptor G-protein interface (e.g. Arg121 and Glu382)
(Supplementary Information, Figure S8). Optimal path of allosteric interactions from ligand to
Arg121 in the edge of the TM3 helix led through Asp103 - Val106 - Ala109 – Met112 – Leu115
– Ser118 in case of iperoxo; Asn108 – Met112 – Leu115 – Ser118 in case of carbachol; Ser107
– Val111 – Leu114 – Ser118 in case of compound 6A and 7A (Figure S8, blue path). Thus,
new compounds relay conformational change to Arg121 via allosteric network different from
classical agonist that converges at Ser118. Allosteric networks of carbachol and iperoxo
converge at Met112 but are initiated by different residues within the orthosteric binding site.
Optimal path of allosteric interactions from ligand to Glu382 in the edge of TM6 helix led
through Asn404 – Thr399 – Ala 395 – Ala391 – Arg387 – Leu384 in case of iperoxo; Cys429
– Thr399 – Phe396 – Ile392 – Ile389 – Thr386 in case of carbachol; Phe195 – Trp400 – Ile397
– Leu393 – Ile389 – Val385 in case of compound 6A and 7A (Figure S8, red path). The first
suboptimal path of carbachol started at Asn404. The first suboptimal path of new compounds
started at Trp400. Thus, new compounds relay conformational change to Glu382 via allosteric
network all way different from classical agonists. Allosteric networks of carbachol and iperoxo
are also substantially different.
Measurements of intramolecular distances over MD trajectory were done to analyse
conformational changes induced by agonists. Individual agonists did not change the size of
binding site (distance between Asp103 and Asn404) significantly. In addition, the RMSD of
NPxxY switch (Arg436, Pro437, Tyr440) were similar for all agonists. Agonist induced
different degree of opening of the receptor G-protein interface (distance of TM3 to TM6 and
distance of TM5 to TM7). Respective distances in the receptor complex with modelled agonists
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varied from the ligand free receptor (apo structure, Figure 9 and S5, black). The TM3 to TM6
distance was greater (about 16 Å) in case of the receptor complex with carbachol (Figure 9 and
S5, red) than in the apo structure. At the other complexes, the TM3 to TM6 distance was similar
to that in the apo structure. Compound 7A (cyan) induced almost no tilt of TM6 away from
inactive conformation (blue) (Supplementary information, Figure S6). Carbachol (red) induced
greater tilt of TM6 than compound 7A but smaller than iperoxo (yellow). Carbachol induced
slightly small tilt and shift of TM6 in M2 receptor (yellow) than in M1 receptor (green)
(Supplementary information, Figure S6 and S7). The TM5 to TM7 distance was much smaller
(about 18 Å) in the complexes of compounds 6A and 7A (Figure 9 and S5, magenta and cyan,
respectively) than in the apo structure and iperoxo or carbachol complexes (about 21 Å).
Monitoring distances between residues at the edge of TMs, Arg121 in TM3 to Glu382 in TM6
and Tyr206 in TM5 to Tyr440 in TM7 gave similar results with greater variation among
agonists but also greater variation within individual MD trajectory. Overall opening of the
receptor G-protein interface induced by compounds 6A and 7A was smaller than opening by
carbachol or iperoxo. Taken together, molecular modelling data suggests agonist-specific
binding of compounds 6A and 7A followed by agonist-specific conformation.
Comparison of cryo-EM structures 6OIJ and 6KOI of G-protein complexes of iperoxo bound
receptors in an active conformation (Maeda et al., 2019) shows that outward tilt of TM6 and
intracellular shift of TM5 is greater in M1-G11 complex than M2-GoA complex (Supplementary
Information, Figure S7). The distance between TM3 and TM6 greater at M1 (16.9 Å) than at
M2 receptor (14.6 Å). Also the distance between TM5 and TM7 is slightly greater at M1 (20.8
Å) than at M2 receptor (19.9 Å). Insertion of C-terminus of GoA α-subunit to M2 receptor is
shallower and at different angle than insertion of C-terminus of G11 α-subunit to M1 receptor.
Overall, greater conformational change of the receptor is needed to accommodate G11 C-
terminus than GoA C-terminus.
Discussion
The major achievement of this study is the development of novel muscarinic agonists that are
selective for M2 receptors and exhibit bias for Gi proteins, resulting in selective inhibition of
production of cAMP (Figure 10).
The phenomenon of stabilization of a particular active conformation of a receptor and
consequent stimulation of some responses but not others is termed signalling bias (Kenakin
and Christopoulos, 2013). Recent reports on muscarinic agonists have shown compounds with
only modest signalling bias (Masuho et al., 2015; Fish et al., 2017). A specific signalling
pathway may be activated by the interaction of an agonist with a specific amino acid within the
orthosteric binding site. Such residues are termed functional hot-spots (Nivedha et al., 2018).
Relatively small agonists may bind only to some, not all, functional hot-spots in the binding
site, therefore activating only a subset of signalling pathways that result in ligand-mediated
signalling bias.
M2 and M4 receptors preferentially activate the Gi/o signalling pathway and inhibit cAMP
synthesis, M1, M3 and M5 receptors preferentially activate the Gq/11 signalling pathway and
stimulate phospholipase C, increasing the levels of 1,4,5-inositol phosphate and intracellular
calcium (Eglen, 2012). Muscarinic receptors also couple to non-preferential G-proteins
(Jakubík et al., 2006; Michal et al., 2007, 2014). It has been proposed that some muscarinic
agonists (e.g. analogues of arecoline) do not interact with the asparagine residue in the sixth
transmembrane α-helix (Asn6.52 Ballesteros-Weinstein numbering (Ballesteros and Weinstein,
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1995) of the orthosteric binding site and exert bias towards Gi/o signalling pathway (Fish et al.,
2017). Considering this, we synthesized small muscarinic agonists. New compounds are 1‐
substituted‐1‐[(5‐substituted
thiophen‐2‐yl)methyl]‐3,6‐dihydro‐2H‐pyridin‐1‐ium
salts
(Supplementary information, Figure S1, Table S1). Compounds displayed various potency and
efficacy to stimulate muscarinic receptors, mainly being partial agonists.
In measurements of functional response to agonists, the two most efficacious compounds (6A
and 7A) only inhibited the forskolin-stimulated synthesis of cAMP at M2 and M4 receptors
(Figure 2). In contrast to the classical non-selective agonist carbachol, compounds 6A and 7A
did not activate Gs pathway (Figure 2 and 3). To compare agonistic effects, the operational
efficacies τ (Black and Leff, 1983) and agonist equilibrium dissociation constants KA were
calculated according to a recent-developed procedure (Jakubík et al., 2019) (Figure 4,
Supplementary Information, Table S4 and S5). The classical definition of bias states that “A
biased agonist is a ligand which stabilizes a particular active conformation of a receptor thus
stimulating some responses but not others” (Lefkowitz, 2013). In line with this definition,
compounds 6A and 7A, in contrast to carbachol, only inhibited production of cAMP at M2 and
M4 receptors displaying Gi bias (Figure 2 and 3). Similarly, at M1 and M3 receptors compounds
6A and 7A inhibited cAMP production while carbachol did not. Values of τ and KA were used
to further quantify bias using the ΔΔlog(τ/KA) method (Kenakin and Christopoulos, 2013) that
is reformulation of Eq. 6 for relative intrinsic activities (RAi) (Griffin et al., 2007) (Table 1,
Supplementary Information, Table S4 and S5). Besides its “absolute” Gi bias at M1 through M4
receptors, compounds 6A and 7A exerted Gi bias at M5 receptors. RAi relative to the reference
agonist carbachol Eq. 7 (Griffin et al., 2007) of preferential functional response of compounds
6A and 7A were highest at M2 receptors, suggesting their M2 selectivity (Table 1,
Supplementary Information, Table S4 and S5). Good correlation between RAi values obtained
according to Eq. 6 and Eq. 7 suggests that τ and KA values derived from EC50 and E’MAX
according to Jakubik et al. (Jakubík et al., 2019) are correct.
Gi/o G-proteins were inactivated by pertussis toxin (PTX) treatment to exclude the possibility
that preferential coupling of M2 and M4 receptors to Gi/o G-proteins (Figure 5) obscured
signalling via Gs G-proteins. Unlike carbachol, compounds 6A and 7A did not activate Gs G-
proteins in PTX-treated cells. This activation of Gi G-proteins did not obscure potential
activation of Gs pathway by compounds 6A and 7A. It is expected that inactivation of Gi G-
proteins by PTX removes Gi G-proteins from the competition of various G-proteins for receptor
and EC50 of carbachol stimulatory response should decrease. However, carbachol EC50
remained unchanged. This may suggest that PTX only inactivates Gi G-proteins and Gi G-
proteins remain pre-coupled with M2 and M4 receptors (Jakubík et al., 2011) after PTX
treatment and still compete with Gs G-proteins.
The heterologous expression and expression level of receptors and members of the signalling
pathways, including G-proteins and effectors, may influence coupling selectivity. To exclude
contribution of this system bias, the functional response to compounds 6A and 7A was also
measured ex vivo in several dissipated rat tissues (Figure 6) and primary culture of rat smooth
muscle cells (Figure 7). Similar to CHO cells, 6A and 7A only inhibited cAMP production
(activated the Gi/o signalling pathway. The cerebellum predominantly expresses M2 and the
striatum expresses M4 and M2 receptors (Eglen, 2012). The expression level of muscarinic
receptors in these brain tissues is more than 10-times lower than in CHO cells (Supplementary
information, Table S6). Lower expression levels should lead to lower apparent E’MAX
,
especially for partial agonists (Black and Leff, 1983). However, compounds 6A and 7A
inhibited cAMP production to the same extent as carbachol (Figure 6) suggesting that they are
full agonists at these receptors. In contrast, efficacy to stimulate IPX production of compounds
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6A and 7A in brain cortex that predominantly express M1 receptors and the submaxillary gland
that predominantly express M3 receptors was negligible, suggesting that compounds 6A and
7A lack significant efficacy at these receptors. About 80% of muscarinic receptors expressed
by smooth muscle cells are M2 and the rest are mainly M3 receptors (Eglen, 2012). Compounds
6A and 7A inhibited cAMP production via the M2 receptor to the same extent as carbachol but
did not stimulate IPX production via M3 receptors, confirming that these compounds are full
agonists at M2 and partial agonists at M3 receptors.
Molecular dynamics of muscarinic agonists docked to the model of the M2 receptor in an active
conformation (PDB ID: 4MQS) confirmed the absence of interaction of compounds 6A and
7A with Asn404 in the sixth transmembrane α-helix (Asn6.52) that is essential for binding of
the super agonist iperoxo and the full agonist carbachol (Figure 8, Supplementary Information
Figure S4). Allosteric interaction networks leading from the agonist to the receptor G-protein
interface of compounds 6A and 7A were different from the allosteric network of iperoxo and
carbachol (Supplementary Information, Figure S8). Receptor conformations specific to
compounds 6A and 7A were further confirmed by the smaller distance between TM5 and TM7
upon binding of these compounds in comparison to iperoxo or carbachol (Figure 9). Taken
together, the absence of a hydrogen bond of compounds 6A and 7A with Asn404, their
differential allosteric interaction network and finding of conformations specific for compounds
6A and 7A support the theory of functional hot-spots (Nivedha et al., 2018) as well as variation
among allosteric interaction networks in dependence on receptor conformation (Miao et al.,
2013; Bhattacharya and Vaidehi, 2014). A common mechanism of GPCR activation is
“opening” of the receptor G-protein interface by moving TM6 away from TM3 and TM7 away
from TM5 (Deupi and Kobilka, 2007; Weis and Kobilka, 2018). The position of the TM6 was
especially implicated in coupling selectivity of rhodopsin and β2-adrenergic receptor (Rose et
al., 2014; Semack et al., 2016). It has been shown that coupling of various GPCRs to Gs G-
proteins requires a greater degree of TM6 movement than coupling to Gi G-proteins (Glukhova
et al., 2018). Cryo–electron microscopy (cryo-EM) structures 6OIJ and 6KOI of complexes of
muscarinic receptors with G-proteins (Maeda et al., 2019) further show that greater
conformational change of the receptor is needed to accommodate C-terminus of G11 G-protein
than GoA G-protein (Supplementary Information Figure S7). Presented molecular modelling
data showed that agonist specific conformations of compounds 6A or 7A differed from agonist
specific conformations of iperoxo or carbachol by having less effects in increasing the distance
between TM5 and TM7 than that between TM3 and TM6 (Figure 9, Supplementary
Information Figure S5 through S7). Molecular modelling data thus suggest that coupling
selectivity can be reached in various ways. In this case, data suggest that, unlike iperoxo or
carbachol interacting with Asn404, compounds 6A and 7A interact with Phe195 and/or Trp400
having a shorter lever to push TM7 away from TM5. Our findings are in accordance with the
recently published structures 6OIJ and 6OIK (Maeda et al., 2019) showing also different
orientation of TM5 and TM7 during interaction of the GoA α-subunit with the M2 receptor when
compared to interaction of the G11 α-subunit with the M1 receptor (Supplementary Information
Figure S7). Overall, presented molecular modelling data are in a good agreement with a general
rule observed in cryo-EM structures of GPCRs (Glukhova et al., 2018). Gs and G11 coupling
receptors show a greater degree of G-protein interface opening than Gi/o coupling receptors. In
turn, molecular modelling data are in a good agreement with experimental data. Compounds
6A and 7A that cause a small opening of G-protein interface in silico activate only Gi/o
signalling pathway in vitro. Carbachol that causes a great opening of G-protein interface in
silico activates all three studied signalling pathways in vitro.
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The tradition of chewing betel for its stimulating effects and pain relief dates back thousands
of years. These effects of betel have been ascribed to the muscarinic agonists arecoline and
arecaidine present in areca (betel) nut. Muscarinic agonists can produce strong analgesic
effects, both at the peripheral, spinal, and brain level. Analgesic effects of the muscarinic
agonist oxotremorine in the tail flick and hot plate tests were largely reduced in M2 knock-out
mice and absent in M2/M4 double knock-out mice (Duttaroy et al., 2002). Several lines of
evidence suggest that M2 and M4 receptors inhibit pain transmission at the spinal level (Chen
and Pan, 2004) and propose the potential of M2 agonists as analgesics (Wess et al., 2007). The
proposed mechanism of peripheral analgesia mediated by M2 receptors is in principle similar
to the analgesia mediated by opioid receptors. Activation of M2 or µ opioid receptors leads to
activation of Gi/o G-proteins. The active α subunits released from activated Gi/o G-proteins
inhibit adenylyl cyclase. The consequent decrease in cAMP causes attenuation in the activity
of tetrodotoxin-resistant voltage-gated sodium channels. The βγ dimers released from activated
Gi/o G-proteins activate inwardly-rectifying potassium channels. A decrease in the activity of
sodium channels and activation of potassium channels results in a decrease in excitability of
nociceptive sensory neurons (De Angelis and Maria Tata, 2016). Moreover, in a model of
chronic neuropathic pain, reduction of the expression of M2 receptors in the dorsal root
ganglion was accompanied by a diminished analgesic response to muscarinic agonists (Zhang
et al., 2018), confirming the role of M2 receptor in spinal nociceptive transmission. In contrast
to pain, secretion of exocrine and endocrine glands and smooth muscle contraction is mediated
primarily by Gq G-proteins activated by M3 receptors (Stengel et al., 2002; Ehlert, 2003). Sole
activation of M2 and M4 receptors may prevent some undesired side-effects of common non-
selective muscarinic agonists, including excessive sweating and salivation and, importantly,
incontinence mediated by peripheral M3 receptors. Restricting undesired side-effects of M2
activation, e.g. bradycardia or smooth muscle contraction in chronic pulmonary obstructive
disorder and other smooth muscle related diseases, will require either topical administration
(Wess et al., 2007) or advanced approaches like targeted drug delivery or tissue-selective action
(Seemann et al., 2017). If analgesic effects are confirmed in vivo, compounds 6A and 7A may
serve as leads in the search for novel non-steroidal, non-opioid analgesics.
Acknowledgements
This work was supported by the Czech Academy of Sciences support [RVO:67985823], the
Grant Agency of the Czech Republic grant [17-16182S] and the Physical Science Department
at Barry University. We thank Dr. Bačáková (Department of Biomaterials and Tissue
Engineering, Institute of Physiology CAS) for providing us with primary cell culture of smooth
muscle cells. Access to computing and storage facilities owned by parties and projects
contributing to the Czech National Grid Infrastructure MetaCentrum provided under the
programme "Projects of Large Research, Development, and Innovations Infrastructures"
(CESNET LM2015042), is greatly appreciated.
Author Contributions
AR conducted experiments, data analysis, contributed to experimental design and manuscript,
DN and DU conducted experiments and managed cell lines, PN and QS synthesised
compounds, VD and EEE contributed to experimental design and manuscript, JB managed
design and synthesis of compounds and contributed to manuscript, JJ conducted molecular
modelling, managed experimental design, experiments, data analysis and wrote manuscript.
All co-authors approved final version of manuscript.
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Conflict of Interest
The authors declare no conflicts of interest.
Declaration of transparency and scientific rigour
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design
& Analysis, and Animal Experimentation, and as recommended by funding agencies,
publishers and other organisations engaged with supporting research.
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Table 1. Efficacies of compounds 6A and 7A to elicit preferential response
Efficacies of compounds 6A and 7A to elicit preferential response (stimulation of IPX
production at M1, M3 and M5 receptors and inhibition of cAMP production at M2 and M4
receptors are expressed as intrinsic activities relative to carbachol (RAi) and as Δlog(τ/KA) to
carbachol.
6A
7A
RAi
Δlog(τ/KA)
2.98±0.26
1.22±0.08*
2.23±0.23
2.15±0.16
2.60±0.82
RAi
Δlog(τ/KA)
1.96±0.16
0.93±0.09*
1.94±0.20
1.58±0.22
3.17±0.88
M1
M2
M3
M4
M5
0.0010±0.0001
0.067±0.007*
0.0058±0.0005
0.0046±0.0004
0.0025±0.0008
0.0111±0.0002
0.140±0.008*
0.011±0.001
0.0209±0.0011
0.0007±0.0002
*, significantly different (P<0.01) from other subtypes according ANOVA and Tukey post-test.
Values are means ± SD from 5 independent experiments performed in triplicates.
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Figure 1 Comparison of binding of novel agonist and agonist iperoxo to the
orthosteric binding site of the M2 muscarinic receptor
Top, chemical structures of compound 7A (left) and iperoxo (right). Bottom, compound 7A
(left) and iperoxo (right) in the orthosteric binding site of M2 receptor in an active conformation
(PDB ID: 4MQS). View from extracellular site. Secondary structure of the receptor is coloured
according residue number in red-white-blue gradient TM1 being coloured red and TM7 blue.
Key residues of the orthosteric binding site Asp103 (Asp3.32) in the TM 3 and Asn404 (Asn6.52
in TM 6 are displayed. Green dashed line is hydrogen bond of iperoxo to Asn404.
)
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Figure 2 Preferential functional responses to agonists in CHO cells that express
individual subtypes of muscarinic receptors
Accumulation of inositol phosphates (IPX) (M1, M3 and M5 CHO cells) or forskolin-stimulated
cAMP (M2 and M4 CHO cells) at non-transfected (open symbols) or G15 G-protein transfected
cells (closed symbols) were measured after stimulation by increasing concentrations of
carbachol or tested compounds (see legend). Data are expressed as folds of matched controls
and are means ± SD from 5 independent experiments performed in triplicates.
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Figure 3 Non-preferential functional responses to agonists in CHO cells that
express individual subtypes of muscarinic receptors
Non-preferential signaling was measured, namely accumulation of forskolin-stimulated cAMP
(M1, M3 and M5) or IPX (M2 and M4 CHO cells) at non-transfected (open symbols) or G15 G-
protein transfected cells (closed symbols) after stimulation by increasing concentrations of
carbachol (circles) or compounds 6A (squares) and 7A (diamonds). Levels of IPX and cAMP
are expressed as of matched controls. Data are means ± SD from 5 independent experiments
performed in triplicates.
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Figure 4 Polar plot of operational efficacy coefficient τ
Operational efficacy coefficient τ was determined from functional response curves in Figure 2
and 3 by fitting Eq. 5. Logarithm of τ values of inhibition of forskolin-stimulated cAMP level
(Gi), cAMP stimulation (Gs) and IPX stimulation at non-transfected (Gq) or G15 G-protein
transfected cells (Gq + G15) of reference compound carbachol and compounds 6A and 7A are
plotted.
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Figure 5 Accumulation of cAMP at PTX-treated CHO cells
Accumulation of cAMP was measured in CHO cells expressing M2 (left) or M4 receptors
(right) that had been treated with pertussis toxin (PTX). Cells were stimulated by increasing
concentrations of carbachol (circles), compound 6A (squares) or compound 7A (diamonds) in
the presence (full symbols) or absence (open symbols) of 10 µM forskolin. Level of cAMP is
expressed as folds of matched controls. Data are means ± SD from 5 independent experiments
performed in triplicates.
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Figure 6 Functional responses to agonists in dissipated rat tissues
Accumulation of IPX in brain cortex (upper left), submaxillary gland (middle left) and ventral
tegmental area (VTA, lower left) or forskolin-stimulated cAMP in the cerebellum (upper right)
and striatum (middle right) after stimulation by increasing concentrations of carbachol (circles)
or compounds 6A (squares) and 7A (diamonds) was measured. Levels of IPX and cAMP are
expressed as folds of matched controls. Data are means ± SD from 5 independent experiments
performed in triplicates.
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Figure 7 Functional responses to agonists in primary culture of smooth muscle
cells
Accumulation of cAMP (left) or IPX (right) in primary culture of smooth muscle cells were
measured after stimulation by increasing concentrations of carbachol (circles) or compounds
6A (squares) and 7A (diamonds). Levels of IPX and cAMP are expressed as folds of matched
controls. Data are means ± SD from 5 independent experiments performed in triplicates.
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Figure 8 Interaction of agonists with the receptor during simulation of molecular
dynamics
Scheme of major interactions and histograms of iperoxo (upper left), carbachol (upper right)
and compounds 6A (lower left) and 7A (lower right) interactions with the receptor were
calculated from MD trajectories using Ligand Interaction Diagram in Maestro. Ligand-receptor
interactions are categorized into four types: Hydrogen bonds (green), hydrophobic (cyan), ionic
(red) and water bridges (blue). The stacked bar charts are normalized over the course of the
trajectory. Values over 1.0 are possible as some protein residues may make multiple contacts
of the same type with the ligand within one time-frame.
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Figure 9 Simulation of MD
The distance between TM5 and TM7 (abscissa) and between TM3 and TM6 (ordinate) in
individual time-frames of MD of the M2 receptor in an active conformation (4MQS) with
bound agonist iperoxo (red), carbachol (green), compounds 6A (blue) and 7A (yellow) or with
an empty binding site (black).
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Figure 10 Scheme of signalling bias of novel compounds
Comparison of activation of Gi, Gq and Gs signalling pathways by carbachol (CBC, left) and
compounds 6A and 7A (A, right) leading to inhibition of cAMP production (Gi) or stimulation
of production of IPX (Gq) or of cAMP (Gs). Thickness of arrows denotes efficacy: Thick arrow
– high efficacy, thin arrow – low efficacy, dotted arrow – no activity. Carbachol activates Gq
and Gs pathways via all receptor subtypes and Gi pathway via M2 and M4. It has no Gi efficacy
at M1 and M3. Compounds 6A and 7A activate Gi pathway with high efficacy via all subtypes
and Gq pathway with low efficacy via M1, M3 and M5. They have no Gs efficacy at any subtype.
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