ARTICLES
CEM100TN00). The CHO cell line, which stably expresses the rat mGlu2 receptor,
was a generous gift from S. Nakanishi (Kyoto University). CHO-M1 cells were
maintained in Ham’s F12 media (Life Technologies) supplemented with 10% (v/v)
fetal bovine serum (FBS) (Life Technologies), 1% (v/v) penicillin-streptomycin
(10.000 units ml–1 (Life Technologies)) and 0.25 mg ml–1 G418 (Life Technologies).
HEK293 cells that endogenously express the β2 adrenergic receptor and CHO-mGlu2
receptor cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)
(Life Technologies) supplemented with 10% (v/v) FBS or dialysed FBS (Life
Technologies), respectively, and 1% (v/v) penicillin-streptomycin (10.000 units ml–1
(Life Technologies)). CHO-mGlu2 media was also supplemented with 1% (v/v)
L-proline. GS-22A cells, derived from HEK293 purchased from ACTT, were cultured
which are complex cyclic depsipeptide natural products, and
thereby provide the first total synthesis of the complete bioactive
structures of YM-254890 and FR900359. A combination of
solution-phase synthesis for depsipeptide building blocks and
solid-phase approaches was used to generate both YM-254890
and FR900359, which provided a confirmation of the original
structural assignment of the corresponding natural products. Here
we generated sufficient material for pharmacological characteriz-
ation and, gratifyingly, a comparison of the inhibitory activities of
synthetic FR900359 with the isolated natural product showed iden- in DMEM supplemented with 10% (v/v) FBS. All the cells were kept at 37 °C in a
humidified atmosphere (95% air and 5% CO2).
tical IC50 values (Fig. 3a and Table 1), which confirms that synthetic
FR900359 is pharmacologically equivalent to the natural product.
Functional assays. The IP1 HTRF assay (Cisbio) was used to quantify Gq/11
In previous studies using the natural products, it was shown that
signalling activity, and the cAMP dynamic 2 assay (Cisbio) was used to quantify the
Gs and Gi signalling. G protein inhibitors were dissolved in 20 mM DMSO stock
YM-254890 and FR900359 inhibit Gq-mediated signalling with
IC50 values in the range 0.03–0.20 µM (refs 12,18,21,42), which cor- solutions, diluted in assay buffer (Hanks’ balanced salt solution buffer, 20 mM
HEPES pH 7.4, 1 mM CaCl2, 1 mM MgCl2) and added to a 384-well Optiplate
(PerkinElmer) in triplicate unless stated otherwise. Subconfluent cells were detached
from the culture dish with non-enzymatic cell-dissociation solution (Sigma).
relates very well with our results (Table 1). Here we also performed
the first head-to-head comparison of the two compounds and
revealed that FR900359 is threefold more potent than YM-254890.
The preparation of derivatives YM-385780 and YM-385781 was
motivated by previous studies showing that YM-385781 was almost
equipotent to YM-25489019 and, consequently, may provide an
attractive alternative starting point for Gq inhibitor synthesis.
However, although YM-385781 was synthesized in slightly fewer
steps, we demonstrated that in our hands it is ∼16-fold less potent
IP1 assay of the M1 receptor. CHO-M1 cells were resuspended in assay buffer
supplemented with 0.2% bovine serum albumin (BSA) to achieve a cell density of
2 million cells ml–1. G protein inhibitors and cells (10.000 cells per well) were
incubated at 37 °C for one hour. After the incubation, carbamoylcholine chloride
(carbachol (Sigma)) was dissolved in assay buffer supplemented with 200 mM LiCl
(final concentration of LiCl in the assay was 20 mM). Carbachol was added to each
well at a final concentration of 3 µM (∼EC80 (drug concentration that induces 80% of
than YM-254890. The N-MeDha functionality prompts a different the maximal response)) and the plate was incubated at 37 °C for one hour followed
by 15 minutes of incubation at room temperature. The detection solution was
prepared as follows: IP1 conjugate and lysis buffer (Cisbio) + 2.5% anti-IP1 cryptate
Tb conjugate (Cisbio) + 2.5% D-myo-IP1-d2 conjugate (Cisbio).
binding conformation relative to that of the hydrogenated depsipep-
tides (Supplementary Fig. 7). This conformational change can influ-
ence the potency in two ways, either by affecting the cell
permeability or the G protein binding directly.
cAMP assay. HEK293 cells or CHO-mGlu2 cells were resuspended in assay buffer
supplemented with 0.2% BSA and 100 µM 3-isobutyl-1-methylxanthine (Sigma) to
achieve a cell density of 1 million cells ml–1. G protein inhibitors and cells (5.000 cells
per well) were incubated at 37 °C for one hour. To stimulate Gs signalling,
isoproterenol bitartrate salt (Sigma) was dissolved in the assay buffer and added to
each well containing HEK293 cells at a final concentration of 17 nM (∼EC80). For Gi
activity, L-glutamate (Sigma) was dissolved in assay buffer supplemented with
We took advantage of the versatility of the synthetic method-
ology and designed and synthesized ten new analogues of
YM-254890, which constitute two small, systematic SAR studies,
and examined either the backbone modifications (29–33 (Fig. 2c))
or changes in the key β-HyLeu moieties (34–38 (Fig. 2d)). This out-
lined key moieties in retaining potent Gq activity and demonstrated forskolin (Sigma) (the final concentration of forskolin in the assay was 15 µM) and
added to each well containing CHO-mGlu2 cells at a final concentration of 16 µM
(∼EC80). The plate was incubated at room temperature for 30 minutes on a plate
shaker. The detection solution was prepared as follows: cAMP conjugate and lysis
buffer (Cisbio) + 2.5% anti-cAMP cryptate conjugate (Cisbio) + 2.5% cAMP-d2
that, in general, even very small changes in the structure can have
substantial pharmacological effects, but also that certain modifi-
cations do not compromise activity too much. All the compounds
were also evaluated for their effect on Gs- and Gi-mediated signal-
ling, and they showed only weak or no activity, in line with the
previously established Gq selectivity of YM-254890 and FR900359.
Finally, YM-254890 was examined for its effect on the two
most-prevalent oncogenic Gα11 mutations, R183C and Q209L.
YM-254890 fully inhibited one of these mutants, Gα11-R183C,
but was ineffective on Gα11-Q209L. This selectivity profile, which
has been shown previously for these mutations in Gαq (refs 12,24),
could be related to the distinct locations of the two mutations rela-
tive to YM-254890 (Fig. 3b) and/or to differences in the mechan-
isms by which they modulate G protein function12.
Collectively, these efforts not only provide access to selective Gq
inhibitors, YM-254890 and FR900359, but also allow ample oppor-
tunities to perform further SAR studies, which could optimize the
pharmacological and physicochemical properties of these com-
pounds. Moreover, YM-254890 and FR900359 may be used as
synthetic templates for the structure-based design of highly war-
ranted inhibitors for other G protein classes, as well as being
explored as templates for drug design directed towards the treatment
of various forms of cancer.
conjugate (Cisbio). For both assays, the detection solution was added to the plate
(10 µl per well) and the plate was incubated in the dark for one hour at room
temperature. The plate was read on an EnVision Multilabel Reader (PerkinElmer
Life and Analytical Sciences), with excitation at 340 nm and measurements of
emission at 615 and 665 nm. The fluorescence resonance energy transfer ratios
(665/615 nm) were converted into IP1 or cAMP concentrations, respectively, by
interpolating values from an IP1 or cAMP standard curve.
Measuring the effects of Gα11 mutants. HEK293 cells were transfected transiently
in 96-well plates with pcDNA3.1-derived plasmids that encoded human Gα11-WT
(100 ng per well), Gα11-R183C (100 ng per well) or Gα11-Q209L (10 ng per well)
using Fugene HD. Two days later, the cells were treated with different concentrations
of YM-254890 for 30 minutes in the presence of LiCl and then analysed for IP1
content (IP-One HTRF assay kit (Cisbio)), as described above.
Received 2 November 2015; accepted 16 June 2016;
published online 25 July 2016
References
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Methods
Chemical synthesis. Detailed procedures for the synthesis of all the compounds and
their characterization are provided in the Supplementary Information. The cell lines
CHO-k1, native HEK293 and GS-22A tested negative for mycoplasma infection in
our lab, and the mGluR2-CHO cell lines were verified pharmacologically in our lab.
Cell culturing. CHO-k1 cells that stably express the muscarinic M1 receptor were
purchased from the cDNA Resource Center (www.cdna.org, catalogue No.
6
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