FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M1 Receptor

Article abstract of DOI:10.1021/acschembio.6b00828

Aiming to design partial agonists as well as allosteric modulators for the M₁ muscarinic acetylcholine (M₁AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M₁AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

Full text of DOI:10.1021/acschembio.6b00828

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Article
FRET Studies of Quinolone-Based Bitopic Ligands and
their Structural Analogues at the Muscarinic M1 Receptor
Regina Messerer, Michael Kauk, Daniela Volpato, Maria Consuelo Alonso Cañizal,
Jessica Kloeckner, Ulrike Zabel, Susanne Nuber, Carsten Hoffmann, and Ulrike Holzgrabe
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00828 • Publication Date (Web): 24 Jan 2017
Downloaded from http://pubs.acs.org on January 25, 2017
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FRET Studies of Quinolone-Based Bitopic Ligands and their Structural Analogues at the
Muscarinic M1 Receptor
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Regina Messerera, ⱡ, Michael Kaukb, c, ⱡ, Daniela Volpatoa, Maria Consuelo Alonso Canizalb,c,
Jessika Klöcknera, Ulrike Zabelb,c, Susanne Nuberb,c, Carsten Hoffmannb, c, * and Ulrike
Holzgrabea, *
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a Department of Pharmaceutical and Medical Chemistry, Institute of Pharmacy, University
of Würzburg, Am Hubland, 97074 Würzburg, Germany
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b Department of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9,
97078 Würzburg, Germany
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c Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef
Schneider Straße 2, 97080 Würzburg, Germany
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Author Information
ⱡ R.M. and M.K. contributed equally.
* Corresponding author: e-mail address:
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ulrike.holzgrabe@uni-wuerzburg.de (Prof. Dr. Ulrike Holzgrabe); tel.: +49 931 31-85460;
fax: +49 931 31-85494.
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c.hoffmann@toxi.uni-wuerzburg.de (Prof. Dr. Carsten Hoffmann); tel.: +49 931 31-48304;
fax: +49 931 31-48539.
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Keywords:
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Quinolones, Bitopic ligands, Partial Agonism, Allosteric Modulation, M1 muscarinic
receptor, FRET.
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Abbreviations:
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ACh, acetylcholine; BQCA, benzyl quinolone carboxylic acid; CFP, cyan fluorescent protein;
DAG, diacylglycerol; FlAsH, fluoresceine arsenical hairpin binder; FRET, fluorescence
resonance energy transfer; GPCR, G protein-coupled receptor; IL, intracellular loop; M1,
muscarinic acetylcholine subtype 1; mAChR, muscarinic acetylcholine receptor; PAM,
positive allosteric modulator; YFP, yellow fluorescent protein.
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Abstract
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Aiming to design partial agonists as well as allosteric modulators for the M1 muscarinic
acetylcholine (M1AChR) receptor, two different series of bipharmacophoric ligands and
their structural analogues were designed and synthesized. The hybrids were composed of
the benzyl quinolone carboxylic acid (BQCA) -derived subtype selective allosteric
modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-
dimethylbut-2-yn-1-amine (base of iperoxo)
1 or the endogenous ligand 2-
(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two
pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and
C10). Furthermore, the corresponding structural analogues of 1, 2 and of modified BQCA
3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence
resonance energy transfer (FRET) measurements in a living single cell system were
investigated in order to understand how these compounds interact with a G protein-
coupled receptor (GPCR) on a molecular level and how the single moieties contribute to
ligand receptor interaction. The characterization of the modified orthosteric ligands
indicated that a linker attached to an orthoster rapidly attenuates the receptor response.
Linker length elongation increases the receptor response of bitopic ligands, until reaching
a maximum, followed by a gradual decrease. The optimal linker length was found to be six
methylene groups at the M1AChR. A new conformational change is described that is not
of inverse agonistic origin for long linker bitopic ligands and was further investigated by
exceptional fragment based screening approaches.
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1) Introduction
Muscarinic acetylcholine receptors (mAChRs) belong to class A of G protein-coupled
receptors (GPCRs) and are divided into five M receptor subtypes (M1-M5). These subtypes
regulate the activity of many important functions of the peripheral and central nervous
system. They differ in their appearance and physiological function; e.g. the M1 muscarinic
acetylcholine receptor is mostly expressed in the central nervous system (cortex,
hippocampus and striatum) and is therefore an interesting therapeutic target for the
treatment of Alzheimer`s disease and schizophrenia.1, 2
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The orthosteric binding pocket appears to be homologues among the five receptor
subtypes.3 This issue is challenging for developing subtype selective therapeutics. To our
knowledge Spalding et al.4 described for the first time an alternative - allosteric binding
region at M1 receptors. This region does not show a high sequence identity and is thus, a
promising target for developing subtype selective allosteric modulators. Allosteric
modulators can influence the affinity of ligands bound to the topographically distinct
orthosteric site, either in a positive, neutral or negative manner.5 The benzyl quinolone
carboxylic acid (BQCA) and their analogues were among others reported by Kuduk et al.
to be positive allosteric modulators (PAMs) with respect to orthosteric agonist binding,
including the endogenous neurotransmitter acetylcholine, and function in M1 receptors.6,
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To combine the advantages of the two binding sites, the concept of bitopic ligands
was developed.10 These ligands engage the allosteric and orthosteric site simultaneously.
Recently, bipharmacophoric ligands consisting of the base of the superagonist iperoxo 111,
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and a benzyl quinolone carboxylic acid moiety 3 were designed, and found to act as
partial hM1 receptor agonists.13
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For the last two decades a series of different receptor sensors, based on fluorescence
resonance energy transfer (FRET), were generated for different GPCRs.14, 15, 16 Usually,
such sensors are tagged C-terminally with cyan fluorescent protein (CFP) and in the third
intracellular loop (IL) region with a yellow fluorescent protein (YFP) or a tetracysteine
motif capable of binding a small soluble fluorophore called fluoresceine arsenical hairpin
binder (FlAsH). These receptor sensors proved to be valuable tools for pharmacological
characterizations in intact cells, especially for monitoring receptor activation in real time
and to investigate receptor ligand interaction on a molecular level.17, 18 According to Chen
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et al., structure-activity relationships (SARs) showed that the BQCA analogue containing a
benzyl moiety at the nitrogen atom as well as a 6-fluoro substituted aromatic ring
resulted in a partial hM1 receptor agonist.13 Due to these findings, the same structural
BQCA modification was used for the herein newly designed compounds. Thus, analogues
of the superagonist 1, orthosteric agonist 2, and of the BQCA-derived subtype selective
allosteric modulator 3 were designed, synthesized and characterized, resulting in
modified analogues with varying alkyl chain length between C2 and C10 (1-Cn, 2-Cn, and
4-Cn, where n gives the length of the linker, i.e. the number of C-atoms, Scheme 1 and
Scheme 2). The attachment point was chosen on the tetramethyl moiety as this group
points out of the orthosteric binding pocket as seen for iperoxo.19 Furthermore, the
fluoro-substituted allosteric BQCA moieties were linked to the base of orthosteric
agonists iperoxo and to the base of acetylcholine, aiming to design bipharmacophoric M1
receptor agonists (5-C and 6-C, Scheme 2). The linker elongation on the BQCA modified
pharmacophore was attached on the carboxylic acid residue due to molecular modeling
studies suggesting that the carboxylic acid position points out toward the extracellular
site of the receptor.20 Furthermore, the carboxylic moiety is a suitable attachment point
for improving allosteric modulation. For the pharmacological characterization of these
ligands a novel M1 FRET sensor was used. This study provides an insight into M1 receptor
activation, receptor conformational changes monitored by the movement of fluorescent
labeled domains, and signaling behavior.
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2) RESULTS AND DISCUSSION
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2.1) Chemistry. The reaction sequences of the iperoxo analogues 1-C2 to 1-C10 and of the
acetylcholine analogues 2-C4, 2-C6, 2-C8, and 2-C10 are illustrated in Scheme 1. 4-((4,5-
dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine11 (base of iperoxo) 1 and 2-
(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively, were reacted with
the corresponding bromoalkane in acetonitrile, affording the final monoquaternary
ammonium salts 1-C2 to 1-C10 and 2-C4, 2-C6, 2-C8, and 2-C10 in 48-91% yield. The
synthesis of the acetylcholine analogues 2-C4, 2-C6, and 2-C8 were described previously
using a different synthetic pathway.21
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Insertion of Scheme 1 about here
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As already reported, the fluoro-4-oxo-quinolone skeleton of 8 was synthesized using the
Gould-Jacobs procedure, starting off with the condensation of 4-fluoroaniline with diethyl
2-(ethoxymethylene)-malonate and subsequent cyclization in boiling diphenyl
ether.22, 23, 24 Ester hydrolysis led to compound 3.25 Conversion of the ester function (9)
with the aminoalcohols of the corresponding spacer length (C4, C6, C8, C10), substitution
of the alcohol function with a bromine atom using HBr/H2SO4 and subsequent reaction
with trimethylamine in acetonitrile at 40 °C led to the monoquaternary ammonium salts
4-C4, 4-C6, 4-C8, and 4-C10 in 53-96% yield. For the preparation of the final
BQCA/iperoxo hybrids 5-C4, 5-C613, 5-C8, and 5-C10 as well as the BQCA/ACh hybrids 6-
C4, 6-C6, 6-C8, and 6-C10, the intermediate bromides were connected to the base of
iperoxo and to the base of acetylcholine, respectively, in the presence of KI/K2CO3 in
acetonitrile (Scheme 2).
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Insertion of Scheme 2 about here
2.2) Pharmacology/Fret measurements
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2.2.1) Receptor sensor and ligand characterization. In comparison to the previously
reported M1-, M3-, M5-26 and M2-ACh receptor FRET-sensors 27, we created a novel full
length FRET sensor of the human M1-ACh receptor which was not truncated in the 3rd
intracellular loop (IL3). This novel sensor consists of the native amino acid sequence fused
at the receptor C-terminus to CFP by adding the amino acids Ser/Arg encoding for an XbaI
site. Additionally, a FlAsH binding motif CCPGCC was inserted between Gly227 and Ser228
(Fig. 1a) at the N-terminal part of IL3 shortly underneath transmembrane domain 5
outside of the G protein-coupling region.28 The receptor construct M1-I3N-CFP expressed
well at the cell surface as analyzed by confocal scanning laser microscopy (Supplementary
Figure 1). Since the previously published truncated sensors were not different from wild-
type receptors in radioligand binding,17 we characterized only functional response of the
novel sensor. To evaluate the effect of the six amino acid insertion into the M1-CFP
receptor we used a dual fluorescence probe, which responds with an increase in red
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fluorescence intensity upon increase in Ca2+ and a decrease in green fluorescence upon
binding to diacylglycerol (DAG). Therefore, the probe can specifically report on the
activation of Gq-signalling.29 The dual fluorescent probe was transfected in HEK293 cells
either alone or co-expressed with the M1-CFP or the novel M1-I3N-CFP sensor and
analyzed by confocal microscopy (see supplementary information for details). The dual
probe did not respond to carbachol if no receptor was co-transfected. As shown in
Supplementary Figure 2 the carbachol stimulated response was indistinguishable for the
M1-CFP receptor or the M1-I3N-CFP sensor. In combination with previous binding
experiments on a truncated sensor version17 it was concluded that this sensor is not
disturbed in its signaling properties by insertion of the CCPGCC sequence. For further
FRET experiments the M1-I3N-CFP was stably expressed in HEK293 cells and single cells
were used for further analysis. To study dynamic conformational changes in real time the
M1-I3N-CFP sensor was exposed to the endogenous agonist ACh and the synthetic full
agonist iperoxo. To eliminate potential artefacts from changes in flow rates, the cells
were constantly superfused with buffer. Under these conditions the receptor sensor
shows a constant baseline. Upon ligand addition a sharp antiparallel movement of the
CFP and FlAsH signal was observed (Supplementary Figure 3), resulting in a concentration
dependent change in the FRET signal of 8-12% for iperoxo (Fig. 1b). When the superfusion
solution was switched back from agonist to buffer, the signal returned to the baseline. A
slight reduction of the FRET signal over time was detectable and could be due to
photobleaching. To prevent artificial underestimation of ligand efficacy reference and
ligand were measured in an alternating exposure regime. Thus, we were able to generate
concentration dependent response (Fig. 1c) curves for the endogenous agonist ACh
(EC50 = 2.91 µM) and the synthetic full agonist iperoxo (EC50 = 0.57 µM). The observed
EC50 value for ACh is in very good agreements with previously obtained value using the
truncated receptor sensor.17 Due to a five-fold higher potency of iperoxo compared to
ACh, the base of iperoxo 1 was chosen as the orthosteric building block for the studied
bitopic ligands to ensure high receptor activation via the orthosteric binding site.
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As allosteric building block a structural analogue of benzyl quinolone carboxylic acid
(BQCA) origin was chosen. In 2009, BQCA was reported to be a M1 selective positive
allosteric modulator (PAM)9 whose binding region was studied in detail.20 However, an
essential property of allosteric modulators is probe dependency. Thus, an experimental
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approach to show this cooperative effect of BQCA analogue (3) combined with iperoxo
(Fig. 1d) was designed. To study positive modulation, a concentration of iperoxo which
results in approximately 20% of the maximal observed signal (EC20) was chosen. This way
a clear signal was observed and still a large detection range to observe positive allosteric
modulation was available. As seen in Figure 1d by applying the allosteric modulator 3
alone, a small conformational change was found. This small response was not clearly
detectable for all cells measured. This might be explained by the very small signal that
could not always be distinguished from noise. When applying saturating concentrations of
iperoxo, a significant signal was monitored. Next iperoxo at 0.1 µM concentration was
applied which results in 25% signal compared to saturating ligand concentrations. Now
the superfusion was changed to a mix of iperoxo and 3 and again back to iperoxo alone. A
clear enhanced receptor response was observed by applying 3 and iperoxo at the same
time, compared with the appropriate iperoxo response. This enhanced response is higher
than a theoretical additive effect of the conformational changes induced by 3 and iperoxo
alone and can be described as positive allosteric modulation.
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2.2.2) Linker elongation attenuates orthosteric properties. Besides their orthosteric and
allosteric moieties, bitopic ligands consist of a linker region that is often not investigated
in a systematic way and hence, the linker is often inappropriately treated. To date the
molecular effects of additional carbon atoms at an orthosteric ligand for muscarinic
receptors are still unknown. Therefore, nine different structural analogues of 1 with a
linker attached to the amine group were synthesized (Scheme 1, 1-C2 to 1-C10). These
compounds were investigated via FRET for their ability to induce a conformational change
at the M1 receptor. The results are summarized in Figure 2. Figure 2a shows a single FRET
experiment comparing the effect of iperoxo and different concentrations of 1-C2 and 1-
C3. Even saturating concentrations of 1-C2 exhibit a much reduced FRET signal compared
to the reference compound iperoxo. From these data it can be concluded, that additional
methylene units significantly reduce the efficacy to 65% compared to iperoxo.
Nonetheless it was possible to generate a concentration response curve for 1-C2. Besides
the reduced efficacy a 3-fold lower affinity (EC50 (1-C2) = 1.65 µM) to the receptor sensor
became evident (Fig. 2b). For 1-C3 a more than 80% reduced conformational change was
found. These signals were too small to establish a reliable concentration response curve.
The iperoxo analogues (1-C4 to 1-C10) did not induce any conformational change at the
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receptor sensor (Fig. 2c). In order to test, whether the linker extended ligands were able
to bind the receptor sensor at the orthosteric binding site, competition experiment were
performed shown in Figure 2d. To test for binding of 1-C6 first 10 µM of iperoxo as a
concentration which induces 80% of the maximal signal response (EC80) was applied. In
the first case a single ligand to the receptor sensor was superfused (Fig 2d). Next, a 10 µM
solution of iperoxo with 100 µM of the linker analogue 1-C6 was added. As can be seen in
Figure 2d, the receptor response induced by the ligand mix was significantly reduced
compared to the signal induced by iperoxo alone, most likely indicating, that the two
ligands compete for the orthosteric binding site and that the 1-C6 analogue shows affinity
to the orthosteric binding site but does not exhibit efficacy as shown by the lack of
conformational changes in Figure 2c. In Figure 2e the results for a corresponding set of
ligands of ACh origin (Scheme 1) is displayed. The given FRET trace proves, that a distinct
linker elongation at an orthosteric ligand attenuates orthosteric efficacy. This
phenomenon could be due to a steric hindrance within the binding pocket or due to
preventing an essential receptor movement like closing the aromatic lid, which was
proposed before to be essential for receptor activation.19
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2.2.3) Evaluation of Bitopic ligands. Bitopic ligands are thought to interact with both
orthosteric and the allosteric binding site at the same time.10 They are also thought to
interact with the receptor in a dynamic binding mode, which consist at least of two
different states.30, 31 Abdul-Ridha et al.20 investigated both binding sites in great detail by
mutational analysis. Using this approach, it was possible to restrict the relative position of
both moieties to a certain area of the receptor.20 In combination with the crystal
structure of iperoxo bound to the M2-receptor subtype19 and the recently published
crystal structure of the M1-receptor subtype3, we focused on bitopic ligands with
different linker length consisting of either four, six, eight or ten carbon atoms. Thus, these
ligands cover a relative distance of 6 Å to 15 Å between both pharmacophore moieties
and hence representing the seven transmembrane helical core. A series of bitopic ligands
consisting of the base of iperoxo (1) and the BQCA analogue (3) were synthesized
(Scheme 2). Correspondingly these ligands are called 5-Cn with n reflecting the linker
length (4, 6, 8, 10) (Scheme 2). As can be seen in Figure 3a, showing a representative FRET
trace of 5-C6, clear concentration dependent increase in conformational change has
taken place. This is the largest conformational change at this FRET sensor compared to all
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other structural analogues in this series. All other ligands were also able to induce a
conformational change at the M1 receptor sensor. Figure 3b shows the maximal ligand
induced changes in comparison to iperoxo. The dashed line indicates the maximal
observed signal for the allosteric moiety 3 when tested alone. Since all corresponding
iperoxo linker analogues (1-C4 to 1-C10) alone did not induce a conformational change
(cf. Fig. 2c), the difference in conformational changes of the hybrids above the dashed
line should either result from the positive cooperativity between the orthosteric and
allosteric moieties, or an alternative binding pose. Moreover, for the M1 receptor the
optimal linker length for the combination of 1 and 3 is in the range of six methylene
groups (see Fig. 3b), whereas for the M2-receptor subtype longer linker lengths are
necessary to improve bitopic ligand efficacy.30 Thus, the optimal linker length as well as
the nature of the linker chain are crucial for receptor activation and are different for each
receptor subtype, depending on their tertiary structure. A first indication for receptor
subtype selectivity among acetylcholine receptors is shown in supplementary figure 4.
Compound 5-C6 was tested at a previously published M3-ACh receptor FRET sensor26 and
did not evoke a conformational change as observed for iperoxo. Furthermore, after
reaching an optimal linker length, the efficacy of bitopic ligands decline by further linker
elongation (Fig. 3b). Surprisingly, when investigating compound 5-C10 a concentration
dependent signal with opposite signal direction compared to iperoxo was found (Fig. 3C).
Since changes in FRET signals in general represent a relative distance change this property
was used as a readout for conformational changes at GPCRs. The opposite FRET signal
induced by 5-C10 was not observed before for any bitopic ligand at M-receptors.
Comparable inverse FRET signals were reported for inverse agonistic responses at
constitutively active mutants of the α2a adrenergic receptor32 and the M3 mutant33. At
constitutively active M3 receptors, atropine behaves as an inverse agonist. Hence, to
compare the effect found for 5-C10 at the M1-I3N-CFP with eventual constitutive activity,
the effect of atropine at this construct was studied. However, neither atropine nor
tiotropium did induce any conformational change (see Fig. 3d or Supplementary Figure 5),
arguing against constitutive activity as an explanation for the behavior of 5-C10. Of note,
the high receptor affinity of atropine becomes visible by the fact, that it was almost
impossible to induce a second signal for iperoxo after the first application of 10 µM
atropine. To further study the surprising effect of 5-C10, a series of bitopic ligands based
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on acetylcholine as orthosteric building block 6-C4 to 6-C10 (Scheme 2) was investigated.
Figure 3e displays a representative FRET trace of acetylcholine based bitopic ligands and
the bar graph in Figure 3f summarizes the measured FRET-signals. Again a linker length
dependent receptor response was found. Moreover, again inverse FRET-signals for bitopic
ligands of longer chain length were detected. Interestingly, for this compound series a
FRET-signal with similar direction as observed for ACh was never detected.
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2.2.4) Linker fusion at the allosteric building block. In order to find out, why bitopic
ligands with longer linkers produce an inverse FRET signal it was of interest to unravel the
moiety of the ligands contributing to the observed signals. Additionally, the question
arose whether it would be possible to reconstruct the signal derived by different bitopic
ligands by combining the individual components in a fragment-based screening approach
in a single cell. Therefore, the ligands 5-C6 and 5-C10 who showed the most extreme
signals were studied. As shown in Figure 1d the BQCA analogue (3) induces a small
conformational change but the corresponding linker extended iperoxo analogue (1-C6)
did not exhibit a detectable effect at our receptor sensor (Fig. 2c), but 1-C6 binds to the
receptor (Fig. 2d). Both effects are shown in Figure 4a, this time measured at the same
cell. By applying both compounds (3 and 1-C6) at the same time a receptor response was
observed that was on the one hand significantly different to the BQCA response but on
the other hand similar in the maximal signal intensity as reported before for the bitopic
ligand 5-C6 (Fig. 3a), indicating first that it is possible to reconstruct the effect of a bitopic
ligand interacting with a receptor by applying the fragments at the same time to the
receptor sensor, and second that there is cooperativity between the allosteric modulator
BQCA (3) and the ligand 1-C6, which showed high affinity to the orthosteric binding site.
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Comparable experiments were performed with the ligand 5-C10 and its respective
building blocks (Fig. 4b). Interestingly in this case it was impossible to reconstruct the
signal of the bitopic ligand by applying the single fragments, suggesting, that the inverse
signal is likely mechanistically different to the agonistic signal. Interestingly at the M2
mAChR, iperoxo-based bitopic ligands have been shown to bind in at least two different
binding poses31 and one of them was shown to be purely allosteric. This has recently been
further investigated by molecular modeling.34 In an attempt to explain the inverse FRET
signal mechanistically, the set of compounds 4-C4 to 4-C10, consisting of the allosteric
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moiety, was studied. Here a linker moiety of different length and a tertiary amine are
combined to imitate the positively charged amine of the orthosteric ligand (Scheme 2) in
the presence of the allosteric moiety. Figure 4c displays a representative FRET trace
recorded for a single cell that was super-fused with the indicated ligands. Increasing linker
length lead to the appearance of an inverse signal for compound 4-C8 and 4-C10 even in
the absence of the orthosteric moiety similar to the signal observed for compound 5-C10
or 6-C10. This result supports the notion of an alternative second binding pose for the
linker extended bitopic ligands at the M1 receptor, as recently described for the M2
receptor.34 Another hypothesis is the possible binding of the ligands to two allosteric
binding sites in a dimeric receptor.35
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2.3) Concluding remarks. Bitopic ligands are molecular entities, which bind to more than
one pharmacological interesting region of membrane proteins and are thought to have an
impact on further drug development. This study provides a molecular insight of the
interactions between GPCRs and bitopic ligands for a better understanding of ligand
receptor interactions on a molecular level. Here, linker dependent responses of bitopic
ligands at a M1 receptor FRET sensor were investigated. The findings indicate an optimal
linker length of the bitopic ligands for conformational changes at the M1 mAChR. This
optimal linker length is probably different for each receptor subtype and is likely
dependent on the individual receptor architecture. Furthermore, a previously unknown
conformational change for GPCRs induced by bitopic ligands of long linker length was
observed. Furthermore, an understanding of the origin of different conformational
changes of GPCRs is provided. The influences of the reported movements on the
downstream signaling of GPCRs are subjects of current studies. Bitopic ligands were
discussed critically due to the fact, whether the lipophilic linker can pass the aromatic lid
of muscarinic receptors. Here, it could be shown that an increased linker first hampers
the orthoster to induce a conformational change although there is a distinct affinity to the
orthosteric binding region. This phenomenon might be due to a steric clash with the
aromatic lid structure in muscarinic acetylcholine receptors. Moreover, the findings
indicate, that this fact is no longer true for the bitopic analogues indicating that here the
linker is able to interact with the aromatic lid without being sterically hindered.
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3) METHODS
3.1) Chemistry. Melting points were determined with a Stuart melting point apparatus
SMP3 (Bibby Scientific) and are uncorrected. H (400.132 MHz) and 13C (100.613 MHz)
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NMR spectra were recorded on a Bruker AV 400 instrument (Bruker Biospin). As internal
standard, the signals of the deuterated solvents were used (DMSO-d6: H 2.5 ppm, 13C
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39.52 ppm; CDCl3: H 7.26 ppm, 13C 77.16 ppm). Abbreviation for data quoted are: s,
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singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dd, doublet of doublets;
dt, doublet of triplets; tt, triplet of triplets; tq, triplet of quartets. Coupling constants (J)
are given in Hz. TLC analyses were performed on commercial pre-coated plates, silica gel
60 F254 (Macherey-Nagel); spots were further evidenced by spraying with Dragendorff
reagent, 36 for amines. ESI mass spectra of the compounds were obtained on an Agilent
LC/MSD Trap G2445D instrument. Data are reported as mass-to-charge ratio (m/z) of the
corresponding positively charged molecular ions. Microwave assisted reactions were
carried out on a MLS-rotaPREP instrument (Milestone). Chemicals were of analytical
grade and purchased from Aldrich and Merck.
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The purities of the compounds (2-C4, 2-C6, 2-C8, and 2-C10) were determined using
qNMR and were found to be >95% (method see supporting information). Purity of
compounds 1-C2, 1-C6, 1-C7, 1-C8, 1-C9, 1-C10, 5-C4, and 6-C6 were determined using
capillary electrophoresis and were found to be >95% (method see supporting
information). Compounds 1-C4 and 6-C4 (confirming purity >95%) and compounds 5-C8
and 5-C10 (confirming purity >90%) were measured on a HPLC system (Agilent 1100
series system with UV detector) using a C18 reversed-phase (Knauer) (150 x 4.6 mm)
column. The mobile phase (MeOH/phosphate buffer = 70/30) was used at a flow rate of
1.5 mL/min, detecting at 254 nm. The HPLC analyses of compounds 4-C4 (confirming
purity >90%) 4-C6, 4-C8, 4-C10, 6-C8, and 6-C10 (confirming purity >95%) were performed
on a LCMS 2020 Shimadzu. The LCMS-system from Shimadzu Products, contained a DGU-
20A3R degassing unit, a LC20AB liquid chromatograph and a SPD-20A UV/Vis detector.
Mass spectra were obtained by a LCMS 2020. As stationary phase a Synergi 4U fusion-RP
(150 * 4.6 mm) column and as mobile phase a gradient of MeOH/water was used.
Parameters for method: Solvent A: water with 0.1% formic acid, solvent B: MeOH with
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0.1% formic acid. Solvent B from 0% to 90% in 13 min, then 90% for 5 min, from 90% to
5% in 1 min, then 5% for 4 min. The method was performed with a flow rate of 1.0
mL/min. UV detection was measured at 254 nm.
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The compounds 1-C3, 1-C5,12 and 325 as well as 9, 10-C6, 11-C6, and 5-C613 were prepared
according to previously reported procedures. The synthesis of the 4-oxo-quinoline
skeleton 837, 38 was performed in analogy to the Gould-Jacobs procedure, using diethyl 2-
(ethoxymethylene)-malonate for the condensation with 4-fluoroaniline followed by
microwave assisted cyclization in diphenyl ether.22, 23, 24
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3.1.1) General Procedure for the Synthesis of the Iperoxo Analogues 1-C2, 1-C4, 1-C6, 1-
C7, 1-C8, 1-C9, and 1-C10. To a solution of 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-
dimethylbut-2-yn-1-amine11 0.45 g (2.47 mmol) in 10 mL of acetonitrile or chloroform, 5 -
10 equivalents of the corresponding 1-bromoalkane 7-C2 to 7-C10 and a catalytic amount
of KI/K2CO3 (1:1) were added. The mixture was stirred in a sealed container at a
temperature between 55 and 70 °C. The precipitate obtained was filtered and Et2O was
added to complete the precipitation. The solid was washed several times with Et2O, and
dried over P2O5 in vacuo.
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3.1.2) General Procedure for the Synthesis of the Acetylcholine Analogues 2-C4, 2-C6, 2-
C8, and 2-C10. To a solution of 2-(dimethylamino)ethyl acetate 0.50 g (3.81 mmol) in
acetonitrile (10 mL), 2 equiv. of the corresponding 1-bromoalkane 7-C4, 7-C6, 7-C8, and 7-
C10 and a catalytic amount of KI/K2CO3 (1:1) were added. The reaction mixture was
heated in the microwave (500 W, 70 °C) for 4 h. After cooling to room temperature the
surplus of KI/K2CO3 was filtered and the filtrate was evaporated to the half of the volume.
After addition of Et2O, a viscous oil was formed. The solvent was decanted and the
product obtained was dried in vacuo.
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3.1.3) General Procedure for the Synthesis of the Quinolone Analogues 4-C4, 4-C6, 4-C8,
and 4-C10. To a solution of bromoalkyl 4-oxo-quinoline-3-carboxamides 11-C4, 11-C6, 11-
C8, and 11-C10 (0.11 mmol) in acetonitrile (5 mL), trimethylamine (45% in H2O, 0.22
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mmol) was added. The reaction was heated at 40 °C. After completion of the reaction
(5 h) controlled by TLC (CH2Cl2/MeOH = 85:15, Rf = 0.10 – 0.45), the mixture was cooled to
room temperature. The solvent was distilled off. The so obtained solid was crystallized
from acetonitrile, filtered and dried in vacuo.
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3.1.4) General Procedure for the Synthesis of the Quinolone-Iperoxo Hybrids 5-C4, 5-C8,
and 5-C10. To a solution of 1 equiv. of the bromoalkyl 4-oxo-quinoline-3-carboxamides
11-C4, 11-C8, and 11-C10 in 20 mL acetonitrile, 2 equiv. 4-((4,5-dihydroisoxazol-3-yl)oxy)-
N,N-dimethylbut-2-yn-1-amine11 and a catalytic amount of KI/K2CO3 (1:1) were added.
The reaction mixture was heated in the microwave (500 W, 80 °C) for 4 h. The reaction
was monitored by TLC (MeOH/NH4NO2 (0.2 M) = 3:2, Rf = 0.54 – 0.68). After cooling to
room temperature the surplus of KI/K2CO3 was filtered. Et2O was added to the filtrate to
obtain a precipitation. The solid was filtered, washed with Et2O and dried in vacuo.
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3.1.5) General Procedure for the Synthesis of the Quinolone-Acetylcholine Hybrids 6-C4,
6-C6, 6-C8, and 6-C10. To a solution of 1 equiv. of the bromoalkyl 4-oxo-quinoline-3-
carboxamides 11-C4, 11-C8, and 11-C10 in 15 mL acetonitrile, 2 equiv. 2-
(dimethylamino)ethyl acetate and a catalytic amount of KI/K2CO3 (1:1) were added. The
reaction mixture was heated in the microwave (500 W, 80 °C) for 4.0 – 6.5 h. After cooling
to room temperature the surplus of KI/K2CO3 was filtered and Et2O was added to the
filtrate. The solid obtained was filtered, washed with Et2O and dried in vacuo.
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3.2) Pharmacology
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3.2.1) Construction of the hM1 receptor FRET sensor. Muscarinic ACh receptor construct
was C-terminally fused to the enhanced variants of cyan fluorescent protein (eCFP) (BD
Bioscience Clontech) by standard PCR extension overlap technique.39 The amino acid
sequence SR, coding for an Xba I restriction site, was inserted as a linker sequence
between receptor and fluorescent protein. In the third intracellular loop (IL3) an amino
acid motif was introduced, thus the novel sequence reads QG227CCPGCCSGS228E. It
specifically binds the fluorescein arsenical hairpin binder (FlAsH) and codes for a
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restriction site. The construct was cloned into pcDNA3 (Invitrogen) and verified by
sequencing, done by Eurofins Genomics.
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3.2.2) Stable cell line generation. Cells were seeded into a culture dish with a confluency
of 30% three hours before transfecting the cells with the Effectene® reagent ordered
from Quiagene. Reagent concentration and incubation times were applied in accordance
with the manufacturer’s instructions. 24 hours after transfecting the normal culture
medium was replaced by culture medium supplemented with 400 µg mL1 G-418. After
that the medium was refreshed every day until all untransfected cells died. Now the cells
were counted, diluted and applied to 48-well plates resulting in a one cell to well
distribution. This homogenous cell population were characterized with fluorescence
microscopy and were investigated concerning their cDNA content.
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3.2.3) Cell culture. HEK293 cells stably expressing the hM1 receptor FRET sensor were
maintained in DMEM with 4.5 g l 1, 10% (v/v) FCS, 100 U mL1 penicillin, 100 µg mL1
streptomycin sulfate and 2 mM L-glutamine and 200 µg mL 1 G-418. The cells were kept at
37 °C in a humidified 7% CO2 atmosphere and were routinely passaged every two to three
days. Untransfected HEK cells maintained in cell culture medium without G-418.
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3.2.4) FlAsH labeling. A labeling protocol was applied as described previously.15, 40, 41 In
brief, cells were grown to near confluency on Poly-D-lysine coated glass coverslips.
Initially cells were washed with labeling buffer (150 mM NaCl, 10 mM HEPES, 2.5 mM KCl,
4 mM CaCl2, 2mM MgCl2 supplemented with 10 mM glucose (pH 7.3)). After that cells
were incubated with labeling buffer containing 500 nM FlAsH and 12.5 µM
1,2-ethanedithiol (EDT) for 1 h at 37 °C followed by flushing with labeling buffer. To
reduce non-specific FlAsH binding, the cells were incubated for 10 min with labeling
buffer containing 250 µM EDT. After flushing with labeling buffer the cells were held in
cell culture medium.
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3.2.5) Ligand application.The reference ligands were prepared from 1 mM stock solutions
that were stored at -20 °C, taking into consideration, that at least acetylcholine remains
instable in solution.42 Used stock solutions have not been older than a couple of weeks.
Bitopic ligands or analogues were stored at 4 °C and were weighed out directly before the
experiment. Than the ligands were solubilized in measuring buffer (140 mM NaCl, 10 mM
HEPES, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2 (pH 7.3)) to a final concentration of 100 µM.
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3.2.6) Single cell FRET experiments. FRET measurements were performed using a Zeiss
Axiovert 200 inverted microscope endued with a PLAN-Neoflar oil immersion 100-
objective, a dual emission photometric system and a Polychrome IV light source (Till
Photonica) as described previously.15, 14 Experiments were conducted at 25 °C using live
HEK293 cells stably expressing the hM1 receptor FRET-sensor or the previously published
hM3 receptor FRET-sensor26 that were maintained in assay buffer. Single cells were
excited at 436 nm (dichroic 460 nm) with a frequency of 10 Hz. Emitted light was
recorded using 535/30 nm and 480/40 nm emission filters and a DCLP 505 nm beam
splitter for FlAsH and CFP, respectively. FRET was observed as the ratio of FlAsH/CFP,
which was corrected offline for bleed through, direct FlAsH excitation and photo
bleaching using the 2015 version of the Origin software as described recently. To
investigate changes in FRET on ligand addition, cells were continuously super fused with
FRET buffer complemented with various ligands in saturating concentrations as indicated.
Superfusion was done using the ALA-VM8 (ALA Scientific Instruments).
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3.2.7) Data processing. Data are shown as means ± SD for n independent observations.
Fluorescence intensities were acquired using Clampex (Axon Instruments). Statistical
analysis and curve fitting were performed using Origin (OriginLab).
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Supporting Information
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Supporting Information Available: This material is available free of charge via the internet.
More detailed syntheses, elemental analyses data and spectral data of intermediate and
target compounds as well as calcium measurement procedure description and supporting
figures.
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Acknowledgements
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M. Kauk and D. Volpato were supported by the international doctoral college “Receptor
Dynamics: Emerging Paradigms for Novel Drugs” funded within the framework of the Elite
Network of Bavaria. M.C. Alonso Canizal was supported by the Marie Curie Initial Training
Networks (ITN) “WntsApp” grant agreement number 608180. We thank Montana
Molecular and Dr. Anne Marie Quinn for technical support and help to use the dual
sensor.
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Scheme 1: Synthesis of orthosteric M1 agonist agents based on Iperoxo – and Acetylcholine
Analogues.
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Reagents and conditions: (i) KI/K2CO3, CH3CN/CHCl3, 70 °C (48-89%); (ii) KI/K2CO3, CH3CN,
70 °C (microwave) (65-91%).
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Scheme 2: Synthesis of Quinolone Analogues, BQCA/Iperoxo Hybrids and BQCA/ACh Hybrids.
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Reagents and conditions: (i) benzyl chloride, K2CO3, DMF, 80 °C (72%); (ii) 6 N HCl, MeOH, reflux
(77%); (iii) H2N(CH2)nOH, 150 °C (28-33%); (iv) HBr (48%), H2SO4, reflux (73-93%); (v)
trimethylamine, CH3CN, 40 °C (53-96%); (vi) 1, KI/K2CO3, CH3CN, 80 °C (microwave) (37-66%); (vii)
2, KI/K2CO3, CH3CN, 80 °C (microwave) (19-73%).
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