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RESEARCH PAPER
Yoda1 analogue (Dooku1) which antagonizes
Yoda1-evoked activation of Piezo1 and aortic
relaxation
Correspondence Professor David J Beech, Leeds Institute of Cardiovascular and Metabolic Medicine, LIGHT Building, Clarendon Way,
School of Medicine, University of Leeds, Leeds LS2 9JT, UK. E-mail: d.j.beech@leeds.ac.uk
Dr Richard Foster, School of Chemistry, University of Leeds, Leeds LS2 9JT, UK. E-mail: r.foster@leeds.ac.uk
Received 9 October 2017; Revised 14 February 2018; Accepted 14 February 2018
Elizabeth L Evans
1,
*,KevinCuthbertson
2,
*, Naima Endesh
1,
*, Baptiste Rode
1
,NicolaMBlythe
1
,
Adam J Hyman
1
, Sally J Hall
1
, Hannah J Gaunt
1
,MelanieJLudlow
1
,RichardFoster
2
and David J Beech
1
1
Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds, UK, and
2
School of Chemistry, University of
Leeds, Leeds, UK
*Equal contributors.
BACKGROUND AND PURPOSE
The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist,
but the pharmacology of these channels is otherwise limited.
EXPERIMENTAL APPROACH
Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca
2+
and Tl
+
measurements were made in HEK 293 or CHO
cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made
from rings of mouse thoracic aorta.
KEY RESULTS
Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1.
The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1-induced Ca
2+
-entry with IC
50
sof1.3μM (HEK 293 cells) and
1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca
2+
ele-
vation or store-operated Ca
2+
entry in HEK 293 cells or Ca
2+
entry through TRPV4 or TRPC4 channels overexpressed in CHO and
HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings, which was mediated by an endothelium- and NO-
dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1.
CONCLUSION AND IMPLICATIONS
Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible, and the existence of a specificchemicalinteractionsite
is suggested with distinct binding and efficacy domains.
Abbreviations
SBS, standard bath solution; TRP, transient receptor potential
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
British Journal of
Pharmacology
British Journal of Pharmacology (2018) 175 1744–1759 1744
DOI:10.1111/bph.14188 © 2018 The Authors. British Journal of Pharmacology
published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.
Introduction
Piezo1 protein is important for mechanical force sensing
and its transduction in higher organisms (Coste et al., 2010;
Ranade et al., 2015; Wu et al., 2016). It assembles as a trimer
with a propeller-like structure around a central ion pore,
which is permeable to the cations Na
+
,K
+
and Ca
2+
(Coste
et al., 2012; 2015; Ge et al., 2015; Guo and MacKinnon,
2017; Saotome et al., 2017; Wu et al., 2017; Zhao et al.,
2018). Mechanical forces that include membrane tension
and laminar flowareabletoactivatethechannel(Coste
et al., 2010; Li et al., 2014; Lewis and Grandl, 2015; Syeda
et al., 2016).
Roles of Piezo1 have been identified in embryonic vascu-
lar maturation, BP regulation, physical performance,
hypertension-dependent arterial structural remodelling,
urinary osmoregulation, epithelial homeostasis and axonal
growth (Li et al., 2014; Ranade et al., 2014; Cahalan et al.,
2015; Retailleau et al., 2015; Koser et al., 2016; Martins
et al., 2016; Gudipaty et al., 2017; Rode et al., 2017). In
addition, pathological significance of Piezo1 has been
suggested in humans. Gain of function mutations have been
linked to a form of haemolytic anaemia (hereditary
stomatocytosis), and loss of function mutations have been
linked to autosomal recessive congenital lymphatic dyspla-
sia (Zarychanski et al., 2012; Albuisson et al., 2013; Andolfo
et al., 2013; Bae et al., 2013; Fotiou et al., 2015; Lukacs
et al., 2015).
Piezo1 pharmacology is in its infancy. Inhibitors of the
channel are limited to generic inhibitors of the ion pore
(Gd
3+
and ruthenium red)andthespidertoxinGsMTx4,
which inhibits a range of mechanosensitive ion channels
and may act indirectly via the lipid bilayer (Drew et al.,
2002; Suchyna et al., 2004; Bowman et al., 2007; Bae et al.,
2011). The first chemical activator of the channel, Yoda1,
was discovered in 2015 through high-throughput screening
(Syeda et al., 2015). Yoda1 is a useful research tool, not faith-
fully mimicking mechanical stimulation of the channels but
facilitating study of Piezo1 channels from a practical
perspective without the need for mechanical stimulation
and importantly lacking effect on Piezo2 channels
(Cahalan et al., 2015; Lukacs et al., 2015; Wang et al., 2016;
Rode et al., 2017).
As a step towards improved understanding of Piezo1 and
the development of more and better Piezo1 modulators, in-
creased knowledge on the structure–activity relationship for
Yoda1 activation of Piezo1 would be helpful. Here, we ad-
dressed this knowledge gap by synthesizing Yoda1 analogues
and testing them against the Piezo1 channel, other channels
and vascular contractile function.
Methods
Piezo1 tetracycline-inducible HEK 293 cell line
HEK T-REx
™
cells, which overexpress Piezo1 upon induction
with tetracycline, were made as described in Rode et al.
(2017). Expression was induced by treating the cells for 24 h
with 10 ng·mL
1
tetracycline (Sigma) and analysed by quan-
titative RT-PCR and Western blots.
Cell culture
HEK 293 cells stably expressing tetracycline-regulated human
Piezo1 were utilized as described above. HEK 293 cells stably
expressing tetracycline-regulated human TRPC4 have been
described previously (Akbulut et al., 2015). For the
TRPC4-expressing cells, selection was achieved by including
400 μg·mL
1
zeocin and 5 μg·mL
1
blasticidininthecell
medium. To induce expression, cells were incubated with
1μg·mL
1
tetracycline for 24 h prior to experiments. All
HEK 293 cells were maintained in DMEM (Invitrogen, Paisley,
UK) supplemented with 10% FCS (Sigma-Aldrich) and 1%
penicillin/streptomycin (Sigma-Aldrich).
HUVECs purchased from Lonza were maintained in En-
dothelial Cell Basal Medium. This media was supplemented
with a bullet kit (Cell Media and Bullet Kit, Lonza) contain-
ing the growth factors: 10 ng·mL
1
VEGF, 5 ng·mL
1
hu-
man basic FGF, 1 μg·mL
1
hydrocortisone, 50 ng·mL
1
gentamicin, 50 ng·mL
1
amphotericin B and 10 μg·mL
1
heparin, in addition to 2% FCS (Sigma). HUVECs were
passaged 2–6times.
CHO K1 cells stably expressing human TRPV4 were
maintained in Ham’s F12 (ThermoFisher Scientific) in the
presence of 1 mg·mL
1
G418 (Sigma-Aldrich).
All cells were grown at 37°C and in 5% CO
2
in a humidi-
fied incubator.
RT-PCR
Total RNA was extracted using TRI reagent (Sigma-Aldrich).
Five hundred nanograms of total RNA was used for reverse
transcription using the Reverse Transcription System
(Promega). Real-time PCR was conducted using an Applied
Biosystems 7500 Real-Time PCR system with intron spanning
primers and Taqman probe for human Fam38A (Piezo1)
(Hs00207230_m1) and GAPDH (Hs99999905_m1) (Applied
Biosystems).
Western blot for Piezo1 protein
Cells were harvested in lysis buffer [10 mM Tris (pH 7.4),
150 mM NaCl, 0.5 mM EDTA and 0.5% Nonidet P40
substitute] containing protease and phosphatase inhibitor
cocktails (Sigma). Equal amounts of protein were loaded
onto a 4–20% gradient gel (BioRad) and resolved by
electrophoresis. Samples were transferred to PVDF
membranes and labelled overnight with BEEC-4 (1:1000,
Cambridge Biosciences). HRP-donkey anti-rabbit secondary
antibody (Jackson ImmunoResearch Laboratories) and
SuperSignal Femto detection reagents (Pierce) were used
for visualization.
Intracellular Ca
2+
measurements
HEK 293 and CHO cells were plated in poly-d-lysine coated
96-well plates (Corning, NY,USA)andHUVECsinclear96-
well plates (Corning, NY, USA) at a confluence of 90%, 24 h
before experimentation. Cells were incubated with 2 μM
fura-2-AM (Molecular Probes
™
)or4μMfluo-4-AM (for TRPV4
expressing CHO cells), in the presence of 0.01% pluronic acid
(Thermo Fisher Scientific) in standard bath solution (SBS) for
1 h at 37°C. For recordings with fluo-4, 2.5 mM probenecid
(Sigma Aldrich) was included in the SBS throughout the
experiment. Cells were washed with SBS for 30 min at room
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1745
temperature. If inhibitors were being tested, these were added
at this time, immediately following an SBS wash and main-
tained during the rest of the experiment. Measurements were
made at room temperature on a 96-well fluorescence plate
reader (FlexStation, Molecular Devices, Sunnyvale, CA, USA)
controlled by Softmax Pro software v5.4.5. For recordings
using fura-2, the change (Δ) in intracellular calcium was indi-
cated as the ratio of fura-2 emission (510 nm) intensities for
340 and 380 nm excitation. For recordings using fluo-4, the
dye was excited at 485 nm and emitted light collected at
525 nm, and measurements are shown as absolute fluores-
cence in arbitrary units. The SBS contained (mM): 130 NaCl,
5 KCl, 8 D-glucose, 10 HEPES, 1.2 MgCl
2
,1.5CaCl
2
and the
pH was titrated to 7.4 with NaOH. For the Ca
2+
add-back ex-
periments, Ca
2+
free SBS was used (without CaCl
2
), and Ca
2+
add-back was 0.3 mM. For the washout experiments, inhibi-
tors were washed 3 times with SBS immediately prior to
recording.
FluxOR
™
intracellular Tl
+
(thallium ion)
measurements
Induced (Tet+) and non-induced (Tet) Piezo1 HEK 293 cells
were plated in poly-d-lysine coated 96-well plates (Corning,
NY, USA) and HUVECs in clear 96-well plates (Corning, NY,
USA) at a confluence of 90%, 24 h before experimentation.
Cells were loaded with FluxOR dye for 1 h at room tempera-
ture, before being transferred to assay buffer for 20 min. If
inhibitors were being tested, these were added at this time
and maintained throughout the experiment. Cells were
stimulated with a Tl
+
-containing K
+
-free solution according
to the manufacturer’s instructions (Molecular Probes).
Measurements were made at room temperature on a 96-well
fluorescence plate reader (FlexStation, Molecular Devices,
Sunnyvale, CA, USA) controlled by Softmax Pro software
v5.4.5. FluxOR was excited at 485 nm, emitted light collected
at 520 nm, and measurements were expressed as a ratio
increase over baseline (F/F
0
).
Chemical synthesis of Yoda1 analogues
Analogues of Yoda1 were synthesized using three general
synthetic approaches: 11 compounds [2a-2 k] were synthe-
sized using a one-step procedure (Supporting Information
Figure S1), compounds 7a and 7b using a four-step procedure
(Supporting Information Figure S2) and compound 11 using
a separate four-step procedure (Supporting Information-
Figure S3). All chemicals synthesized were purified by
column chromatography or trituration and determined as
>97% pure by
1
H NMR (proton NMR) and
13
CNMR(car-
bon-13 NMR). Synthetic and analytical details are reported
in the Supporting Information.
Animals
Twelve to sixteen week-old, wild-type male C57BL/6 mice
were used for experiments. All mice were housed in GM500
individually ventilated cages (Animal Care Systems) at 21°C,
50–70% humidity and with a 12 h alternating light/dark
cycle. They had ad libitum access to RM1 diet (SpecialDiet
Services, Witham, UK) with bedding from Pure’oCell
(Datesand, Manchester, UK). All animal experiments were
authorized by the University of Leeds Animal Ethics
Committee and the UK Home Office. Animal studies are re-
ported in compliance with the ARRIVE guidelines (Kilkenny
et al., 2010; McGrath and Lilley, 2015).
Aorta contraction studies
Thewiremyographtechniqueusingvesselsfrommiceis
regarded as a useful model for studying vascular reactivity
(Outzen et al., 2015). Animals were killed by CO
2
inhalation,
according to Schedule 1 procedure approved by the UK Home
Office. Thoracic aorta was dissected out and immediately
placed into ice-cold Krebs solution (125 mM NaCl, 3.8 mM
KCl, 1.2 mM CaCl
2
,25mMNaHCO
3
,1.2mMKH
2
PO
4
,
1.5 mM MgSO
4
,0.02mMEDTAand8mMD-glucose,
pH 7.4). Connective tissue and fat were carefully removed un-
der a dissection microscope. Segments, 1 mm long, were
mounted in an isometric wire myograph system (Multi Wire
Myograph System, 620 M, Danish Myo Technology) with
two 40 μm diameter stainless steel wires, bathed in Krebs
solution at 37°C and bubbled with 95% O
2
,5%CO
2.
The seg-
ment was then stretched stepwise to its optimum resting ten-
sion to a 90% equivalent transmural pressure of 100 mmHg
and equilibrated for 1 h prior to experiments. The stretch
was approximately equal to that expected at diastolic BP
(Rode et al., 2017).
Data and statistical analysis
The data and statistical analysis comply with the recom-
mendations on experimental design and analysis in phar-
macology (Curtis et al., 2015). OriginPro 2015 (OriginLab,
Northampton, MA, USA) was used for all data analysis.
Averaged data are presented as mean ± SEM, where nrepre-
sents the number of independent experiments for a given
result and Nindicates the total number of replicates within
the independent experiments. Technical replicates were
used to improve the confidence in data from independent
experiments. In order to compare the pharmacological
activity of Yoda1 analogues, data were normalized to the
response of Yoda1 (agonist experiments) or the response
of Yoda1 following pretreatment with vehicle only (inhibi-
tor experiments). Data subjected to statistical analysis
contained at least five independent experiments (n). For
comparisons between two sets of data, Student’st-tests were
used. For multiple comparisons, one-way ANOVA was used
with Tukey’spost hoc test. P<0.05 was deemed significant.
For IC
50
determination, data were normalized to the vehicle
controls (DMSO), and curves were fitted using the Hill1
(Origin Pro 2015) equation. The analogues were novel,
and so, their initial testing occurred without knowledge of
what effects might occur. Later in the study, analogues were
blinded for aorta contraction experiments and used in
random order. Randomization and blinding were not other-
wise used.
Materials
Unless stated otherwise, all commercially available chemicals
were purchased from Sigma-Aldrich. Stocks of chemicals were
reconstituted in DMSO and stored at 20°C unless stated oth-
erwise. Fura-2-AM and fluo-4-AM (Molecular Probes) were
dissolved at 1 mM. Pluronic acid F-127 was stored at 10%
w
.
v
-1
in DMSO at room temperature. Probenecid was freshly
prepared in 0.5 M NaOH and diluted 1:200 in SBS to give a
E L Evans et al.
1746 British Journal of Pharmacology (2018) 175 1744–1759
working concentration of 2.5 mM. Yoda1 (Tocris) was stored
at 10 mM. All Yoda1 analogues were synthesized and purified
(for more information, see Supporting Information) and pre-
pared as 10 mM stock solutions. Stock solutions were diluted
1:500 in the recording solution to give a final working con-
centration of 0.02% DMSO. Thapsigargin and 4α-
phorbol 12, 13-didecanoate were stored as 5 and 10 mM
stocks respectively. (-)-Englerin A was prepared as a
10 mM stock solution and stored at 80°C. In experiments,
(-)-Englerin A was used in SBS containing 0.01% pluronic acid
as a dispersing agent to minimize aggregation of compound.
Phenylephrine was stored at 100 mM in an aqueous solu-
tion. ATP wasstoredat10mMinanaqueousstocksolution.
U46619 was stored as a 10 mM stock in water. SIN-1 was
stored as a 20 mM stock. BEEC-4 anti-Piezo1 antiserum, di-
luted 1:1000 for experiments, was generated by Cambridge
Biosciences in rabbits by presentation of the Piezo1 peptide
DLAKGGTVEYANEKHMLALA.
Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked
to corresponding entries in http://www.guidetophar-
macology.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 2017/2018 (Alexander et al.,
2017a,b).
Results
2,6-Dichlorophenyl ring of Yoda1 is important
for Piezo1 activity
We synthesized a series of Yoda1 analogues, focusing on sim-
ple modifications to the 2,6-dichlorophenyl ring (Figure 1A).
To reliably study the effects of Yoda1 analogues on
overexpressed Piezo1 channels, we stably incorporated
tetracycline-inducible human Piezo1 expression in HEK
293 T-REx
™
cells. These cells, hereby referred to as Piezo1
T-REx cells, showed Piezo1 expression after tetracycline
induction but not without induction (Figure 1B, C) and
displayed dose-dependent Ca
2+
entry in response to
Yoda1, in comparison with normal HEK 293 T-REx
™
cells
(without Piezo1 incorporation) that showed no response
(Figure 1D, E). The Yoda1 analogues were screened at
10 μM for their ability to cause Ca
2+
entry in these Piezo1
T-REx cells and compared with the Ca
2+
entry caused by
thesameconcentrationofYoda1(Figure1F).Allofthe
structural changes caused Piezo1 activation to be lost or
mostly lost, with all compounds showing less than 30% ac-
tivation compared with Yoda1 (Figure 1F).
The analogues were also screened for their ability to
inhibit the Yoda1 response (Figure 1G). Each analogue was
pre-incubated with the cells for 30 min at 10 μM, prior to
the application of 2 μM Yoda1 in the continued presence of
the analogue. Pre-incubation with these analogues did not
affect the Ca
2+
entryevokedbyYoda1,apartfrom2g which
caused inhibition. These data suggest that the 2,6-
dichlorophenyl moiety of Yoda1 is essential for interacting
with the Piezo1 channel. Only analogue 2g had any effect,
showing a slight inhibitory effect but little agonist effect; it
is chemically similar to Yoda1 but with one fluorine replacing
one chlorine.
Identification of a Yoda1 analogue which
antagonizes Yoda1
To further investigate the structure–activity relationship of
Yoda1, we synthesized analogues of the pyrazine group
(Figure 2A). Similarly, these analogues were tested at 10 μM
for their ability to cause Ca
2+
entry in Piezo1 T-REx cells, com-
pared with Yoda1 (Figure 2B, C). Modification to the pyrazine
ring significantly reduced activity in comparison with Yoda1,
but analogue 7a reached 50% of Yoda1 activity (Figure 2B, C).
We then synthesized analogues of the thiadiazole group
(Figure 2D) and tested these in the same manner (Figure 2E, F).
Analogues containing an oxadiazole in place of a thiadiazole
were also less active, but analogue 11, the most similar in
structure to Yoda1, showed ~70% activity (Figure 2E, F).
These data suggest that the ability of Yoda1 to activate
Piezo1 channels is dependent on very specificstructuralre-
quirements but that changes to the pyrazine and thiadiazole
groups can be tolerated.
To investigate whether these analogues could inhibit
Yoda1 activity, we pre-incubated cells with analogues and
then tested Yoda1 (Figure 3A–G). The Yoda1 response was re-
duced by all analogues (Figure 3G). Analogues 2i (Figure 3A),
2j (Figure 3B), 7a (Figure 3D), 7b (Figure 3E) and 11 (Figure3F)
also had agonist activity, as shown by the elevated baseline
Ca
2+
signal compared with vehicle (DMSO) control. In con-
trast, analogue 2k (Figure 3C) inhibited the Yoda1 response
without changing the baseline and so lacked agonist activity
(Figure 3C). Analogue 2k was found to cause concentration-
dependent inhibition of Yoda1-induced Ca
2+
entry with an
IC
50
value of 1.30 μM (Figure 3H). Inhibition was incom-
plete at 10 μM, but higher concentrations of 2k were not in-
vestigated because of solubility limitations. Recovery from
the inhibitory effect of 2k occurred after its washout
(Figure 3I). The inhibitory effect of 2k was not significantly
differentat37°Ccomparedwithroomtemperature(Figure3
J, K). The data suggest that 2k is an antagonist of Yoda1 that
lacks agonist capability. We named 2k, Dooku1.
Dooku1 (analogue 2k) has selectivity for Piezo1
Pretreatment with 10 μM Dooku1 had no effect on endoge-
nous Ca
2+
release in native HEK 293 cells in response to
20 μM ATP (Figure 4A). Dooku1 (10 μM) had no effect on
store-operated Ca
2+
entry in HEK 293 cells: the Ca
2+
addback
response after intracellular Ca
2+
store depletion by 2 μM
thapsigargin (Figure 4B). Dooku1 (10 μM) had no effect on
Ca
2+
entry through TRPV4 channels overexpressed in CHO
cells and activated by 4αPDD (Figure 4C) or on Ca
2+
entry
through TRPC4 channels overexpressed in T-REx
™
HEK
293 cells and activated by 100 nM (-)-Englerin A (EA)
(Figure 4D). The data suggest selectivity of Dooku1 for
Piezo1 channels.
Dooku1 does not inhibit constitutive Piezo1
activity
To investigate whether the effect of Dooku1 depends on
Yoda1, we took advantage of constitutive Piezo1 channel
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1747
activity observed in the Piezo1 T-REx cells (Rode et al., 2017).
The activity can be detected using an intracellular thallium
(Tl
+
)sensitiveFluxOR
™
indicator dye whereby Tl
+
influx acts
as a surrogate for Na
+
influx (Rode et al., 2017). Cells were
maintained in a Tl
+
free solution until 2 μMTl
+
was added ex-
tracellularly 30 s into the recording, and the resulting eleva-
tion of intracellular Tl
+
was detected. To ensure that
constitutive Piezo1 channel activity was being represented
in this assay, we compared the rate of Tl
+
entry in
tetracycline-induced (Tet+) Piezo1 overexpressing cells to
control cells to which tetracycline was not added (Tet)
(Figure 5A, B). The initial rate of Tl
+
entry in the Tet + cells
was nearly double that of control Tetcells (Figure 5A, B).
Pretreatment with Dooku1 did not reduce constitutive
Piezo1 channel activity as shown by comparing the DMSO
and Dooku1 DMSO data (Figure 5C, D). Yoda1 increased the
rate of Tl
+
entry by ~2.5-fold, and this effect was inhibited
by 10 μM Dooku1 as shown by comparing the Yoda1 and
Dooku1 Yoda1 data (Figure 5C, D). These data suggest that
Dooku1 has no effect on constitutive Piezo1 channel activity
and therefore that its effect depends on the presence of
Yoda1.
Figure 1
The 2,6-dichlorophenyl group of Yoda1 is required for activation of Piezo1. (A) Structures of Yoda1 and analogues. Structural variation to Yoda1 is
highlighted by the box outline. (B) Western blot of control T-REx and Piezo1 T-REx cells with anti-Piezo1 antibody, confirming Piezo1 expression
(predicted size, 286 kDa). (C) Real-time PCR of Piezo1 mRNA levels relative to GAPDH mRNA in T-REx and Piezo1 T-REx cells. Error bars indicate
SEM (n= 3). (D and E) FlexStation intracellular Ca
2+
measurement data for T-REx cells (D) and Piezo1 T-REx cells (E) exposed to Yoda1 at the spec-
ified concentrations or exposed to the vehicle only (DMSO). (F) (Left) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells ex-
posed to 10 μM2e or exposed to vehicle only (DMSO). Error bars indicate SEM (N= 3). (Right) Summary for experiments of the type shown on the
left measured between 40–60 s after Yoda1 analogue application, expressed as a % of the 10 μM Yoda1 response. Each data point represents a
value from an independent experiment with mean values and error bars representing SEM indicated in black (n= 5). (G) (Left) FlexStation intra-
cellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 2 μM Yoda1 after pretreatment with 10 μM2e or vehicle only (DMSO). Error
bars indicate SEM (N= 3). (Right) Summary for experiments of the type shown on the left, as for (F, right) except data are expressed as a % of
the Yoda1 response when pretreated with vehicle only (DMSO) (n=5;2b,2c,2e,2g,2h,n=7;2a,2d,2f).
E L Evans et al.
1748 British Journal of Pharmacology (2018) 175 1744–1759
Dooku1 inhibits endogenous Yoda1-activated
channels
The above studies were on overexpressed Piezo1 channels. To
investigate the relevance to endogenous Piezo1 channels, we
studied HUVECs that robustly express endogenous Piezo1
channels (Li et al., 2014) and display a Piezo1-dependent
Yoda1 response (Rode et al., 2017). Similar to observations
in Piezo1 T-REx cells (Figure 3C), Dooku1 did not evoke
Ca
2+
entry (Figure 6A). Dooku1 was however able to inhibit
the Yoda1 response in HUVECs (Figures 6B, C). Dooku1 had
a concentration-dependent inhibitory effect against Yoda1-
induced Ca
2+
entry in HUVECs, acting with an IC
50
of
1.49 μM (Figure 6D), which was comparable with the value
in Piezo1 T-REx cells even though its maximum effect was less
(Figure 3H). These data suggest that Dooku1 is also an antag-
onist of Yoda1-induced Piezo1 channels in endothelial cells.
To investigate the reason for reduced Dooku1 effect against
the endogenous Yoda1-activated channel, we compared the
concentration-effect curves of Yoda1 in HUVECs (Figure 6E)
and Piezo1 T-REx cells (Figure 6F). Yoda1 had increased
potencyinHUVECswithanEC
50
of 0.23 μM, compared with
2.51 μM in Piezo1 T-REx cells, suggesting that greater Yoda1
potencyinHUVECsmayexplainthesmallereffectofDooku1
in HUVECs.
Yoda1 causes endothelium-dependent and NO-
dependent relaxation of aorta
To investigate physiological responses, we made isometric
tension recordings from isolated murine thoracic aorta rings.
Yoda1 had no effect in the absence of phenylephrine (PE),
whichisanagonistofα
1
-adrenoreceptors (Figure 7A). Rings
contracted in response to PE (Figure 7B) and Yoda1 caused
concentration-dependent relaxation following this pre-
contraction, with an estimated EC
50
of 2.3 μM(Figure7B).
Endothelium-denudation abolished the Yoda1 response but
didnotaffectthePEresponse(Figure7C,D).Responseto
ACh was a positive control for functional endothelium, and
this response was present in endothelium-intact rings but
Figure 2
Changes to the pyrazine ring or replacing the thiadiazole with an oxadiazole give rise to less active analogues. (A) Structures of Yoda1 and
analogues with changes to the pyrazine ring. Structural variation to Yoda1 is highlighted by the box outline. (B) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 10 μM7a or exposed to vehicle only (DMSO). Error bars indicate SEM (N= 3). (C) Summary
for experiments of the type shown in (B) measured between 40–60 s after Yoda1 analogue application, expressed as a % of the 10 μMYoda1
response. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated in
black (n= 5). (D) Structures of Yoda1 analogues with an oxadiazole. Structural variation to Yoda1 is highlighted by the box outline. (E) FlexStation
intracellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 10 μM2j or exposed to vehicle only (DMSO). Error bars indicate SEM
(N= 3). (F) Summary for experiments of the type shown in (E), as for (C).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1749
Figure 3
Yoda1 analogues are able to inhibit Yoda1-induced Piezo1 activity. (A–F) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells
exposed to 2 μM Yoda1 after pretreatment with 10 μM2i (A), 2j (B), 2k (C), 7a (D), 7b (E), 11 (F) or vehicle only (DMSO). Error bars indicate
SEM (N= 3). (G) Summary for experiments of the type shown in (A–F) measured between 40–60 s after Yoda1 analogue application, expressed
as a % of the Yoda1 response when pretreated with vehicle only (DMSO). Each data point represents a value from an independent experiment
with mean values and error bars representing SEM indicated in black (n= 5). (H) Mean data for the type of experiment shown in (C) with cells
pretreated with indicated concentrations of 2k. Expressed as a % of the Yoda1 response when pretreated with vehicle only (DMSO). The fitted
curve is the Hill equation with IC
50
1.30 μM(n= 5). (I) Summary of intracellular Ca
2+
measurement data (as for G) for Tet + Piezo1 T-REx cells
exposed to 2 μM Yoda1, following pretreatment with 10 μM2k or vehicle only (DMSO); 2k was washed out before the recording (n= 5). (J)
As for (C) but conducted at 37°C. (K) Summary for experiments of the type shown in (J) (n=5).
E L Evans et al.
1750 British Journal of Pharmacology (2018) 175 1744–1759
absent in endothelium-denuded rings (Figure 7C). The data
suggest that Yoda1 causes endothelium-dependent relaxa-
tion in mouse thoracic aorta.
To determine whether the relaxation caused by Yoda1
was dependent on NOS, we exposed rings to NOS inhibitor,
Nω-nitro-L-arginine methyl ester (L-NAME). L-NAME
Figure 5
Dooku1 does not affect Piezo1 constitutive activity (A) Intracellular Tl
+
measurement data using FluxOR for Tet + Piezo1 T-REx cells or control Tet
cells exposed to extracellular Tl
+
. The FluxOR measurements are displayed as the fluorescence intensity (F) divided by the initial fluorescence in-
tensity (F
0
). Error bars indicate SEM (N= 3). (B) Summary for experiments of the type shown in (A) measured between 0–30 s after Tl
+
application,
normalized to rate of change of F in the Tetresponse. Each data point represents a value from an independent experiment with mean values and
error bars representing SEM indicated in black (n= 5). (C) Intracellular Tl
+
measurement data for Tet + Piezo1 T-REx cells exposed to extracellular
Tl
+
and 5 μM Yoda1 or vehicle (DMSO), following pretreatment with 10 μM Dooku1 or vehicle only (DMSO). Error bars indicate SEM (N= 3). (D)
Summary for experiments of the type shown in (C), as for (B) except data are normalized to the rate of change of the vehicle only (DMSO) control
condition (n=5).
Figure 4
Selectivity of Dooku1. Ca
2+
indicator dyes were fura-2 (A, B, D) or fluo-4 (C). Experiments conducted in native HEK 293 cells (A, B), CHO cells over-
expressing TRPV4 (C) or HEK 293 cells overexpressing TRPC4 (D). Intracellular Ca
2+
measurement data for cells exposed to 20 μM ATP (A), 0.3 mM
Ca
2+
addback (B), 5 μM4α-phorbol 12,13-didecanoate (4α-PDD) (C) or 100 nM (-)-Englerin A (EA) (D) following pretreatment with DMSO or
10 μM Dooku1 (left). Error bars indicate SEM (N= 3). Summary for experiments of the type shown on the left measured between 10–30 s (A),
60–90 s (B), 220–240s(C)or20–60 s (D) after treatment application and normalized to the peak amplitude values for the vehicle only (DMSO)
pretreatment condition (right). Each data point represents a value from an independent experiment with mean values and error bars representing
SEM indicated in black (n=5).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1751
prevented Yoda1-induced and ACh-induced relaxation
(Figure 7E, F). The data suggest that Yoda1 causes
endothelium-dependent relaxation in mouse thoracic aorta
by stimulating NO production via the endothelium.
Dooku1 inhibits Yoda1-induced relaxation of
aorta
To determine if Dooku1 inhibits relaxation caused by Yoda1,
aortic rings were pre-incubated with 10 μMDooku1for
20 min. Dooku1 strongly suppressed the Yoda1-induced
relaxation (Figure 8A–C). To characterize this phenomenon
in more detail, we tested four further Yoda1 analogues in
the aorta assay. The selected analogues showed various
abilities to inhibit Yoda1 responses in Piezo1 T-REx cells:
analogues 2e (no activation and no inhibition) (Figure 1),
2g (slight activation and slight inhibition) (Figure 1), 7b
(slight activation and partial inhibition) (Figures 2 and 3)
and 11 (slight activation and partial inhibition) (Figures 2
and 3). Analogue 2e had no effect (Figure 8D–F). 2g,7b and
Figure 6
Dooku1 is effective against the endogenous Piezo1 channel. (A) Intracellular Ca
2+
in HUVECs after exposure to 10 μM Dooku1 or vehicle only
(DMSO). Error bars indicate SEM (N= 3). (B) Intracellular Ca
2+
in HUVECs after exposure to 2 μM Yoda1 after pretreatment with 10 μM Dooku1
or vehicle only (DMSO). Error bars indicate SEM (N= 3). (C) Summary for experiments of the type shown in (B) measured 40–60 s after Yoda1
application and normalized to peak amplitudes for the vehicle only group. Each data point represents a value from an independent experiment
with mean values and error bars representing SEM indicated in black (n= 7). (D) Mean data for the type of experiment shown in (B) with cells
pretreated with indicated concentrations of Dooku1. Data are expressed as a % of the Yoda1 response when pretreated with vehicle only (DMSO).
The fitted curve is the Hill equation with IC
50
1.49 μM(n= 5). (E, F) Mean intracellular Ca
2+
for HUVECs (E) or Piezo1 T-REx cells (F) exposed to the
indicated concentrations of Yoda1. The fitted curve is the Hill equation with EC
50
of 0.23 μM(E)and2.51μM(F)(n=3).
E L Evans et al.
1752 British Journal of Pharmacology (2018) 175 1744–1759
11 in contrast suppressed the Yoda1-induced relaxation
(Figure 8G–K). Moreover, the ability of these analogues to
inhibit Yoda1-induced relaxation correlated with inhibition
of Yoda1-induced Ca
2+
entry (Figure 8L). The data suggest
strong efficacy of Dooku1 as an inhibitor of Yoda1-induced
aortic relaxation that is mediated through disruption of
Yoda1-induced Piezo1 channel activity.
Dooku1 is selective for Yoda1-induced
relaxation but partially inhibits agonist
contractile responses
AnalysisofthePEresponseinthepresenceofDooku1
revealed significant inhibition without effect on baseline
tension (Figure 9A, B). To determine whether Dooku1’s
inhibition of PE-induced contraction was specifictothis
contractile agent, we also tested the effect of Dooku1
against contraction induced by U46619, a Tx A
2
mimetic.
Aortic rings were pre-contracted with 0.1 μM U46619
(Figure 9C, D). Addition of Dooku1 caused partial relaxation
(Figure 9D, E). In contrast, Dooku1 had no effect on relaxa-
tion evoked by ACh (1 μM) or the NO donor SIN-1 (10 μM)
(Figure9F,G).InvestigationofthePEresponseinthe
presence of the other four Yoda1 analogues revealed no
inhibitory effect (Figure 10). The data suggest that Dooku1
selectively inhibits Yoda1-induced relaxation but also
partially inhibits receptor-mediated agonist responses via
unknown mechanisms.
Discussion and conclusions
This study has provided insight into the structure–activity
relationships for Piezo1 channel activation by Yoda1 with
the goal of generating new tools for investigating Piezo1
channel function. Through this research, we have identified
and named Dooku1, an inhibitor of Yoda1-induced Piezo1
channel activity that strongly inhibits Yoda1-induced
Figure 7
Yoda1-induced relaxation in mouse thoracic aorta is endothelium- and NO-dependent. (A) Isometric tension recording from aorta exposed to the
indicated concentrations of Yoda1. (B) (left) As for (A) but following pre-constriction with 0.3 μM phenylephrine (PE). (Right) Mean data for ex-
periments of the type shown on the left expressed as % relaxation. The fitted curve is the Hill equation with EC
50
of 2.3 μM(n= 5). (C) Isometric
tension recording of aorta pre-constricted with PE and exposed to 5 μM Yoda1 (left) or 5 μM ACh control (middle and right) with the endothelial
layer removed (left and middle) or intact (right). (D) Summary data for experiments of the type shown in (B and C, left) expressed as % relaxation
evoked by Yoda1 (left) or the response to PE (right) in the presence (EC+) or absence (EC) of the endothelial cell layer. Each data point represents
a value from an independent experiment with mean values and error bars representing SEM indicated by the black lines (n= 5). (E) As for (C) but
following pre-incubation with 100 μMN
ω-
nitro-L-arginine methyl ester (L-NAME). (F) As for (D) but for experiments of the type shown in (E).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1753
relaxation of aorta. The data suggest that Dooku1 may
compete with Yoda1 at a binding site or act allosterically at
another site to reduce the binding or efficacy of Yoda1.
During the discovery of Yoda1, the 2,6-dichlorophenyl
group of the compound was highlighted as important with
particular reference to the chlorine atoms (Syeda et al.,
Figure 8
Dooku1 inhibits Yoda1-induced dilation in aorta. (A–K) Isometric tension data from mouse thoracic aorta with intact endothelium. (A) Pre-con-
stricted with PE and exposed to 5 μM Yoda1. (B) As for (A) but following 30 min pre-incubation with 10 μM Dooku1. (C) Summary data for ex-
periments of the type shown in (A, B) expressed as % relaxation evoked by Yoda1. Each data point represents a value from an independent
experiment with mean values and error bars representing SEM indicated by the black lines (n= 7). (D–F) (G–I) As for (A–C) but following pre-in-
cubation with 10 μM2e (D–F) or 7b (G–I) (n= 5 on F, I). (J, K) As for (C) but following pre-incubation with 10 μM2g (J) or 11 (K) (n= 5). (L)
Comparison of the mean % inhibition of Yoda1-induced relaxation in mouse thoracic aorta and the mean % inhibition of Yoda1-induced Ca
2+
entry by the five compounds: 2e,2g,Dooku1,7b and 11.Thepointsarefit to a straight line with Pearson’s correlation coefficient of 0.78.
E L Evans et al.
1754 British Journal of Pharmacology (2018) 175 1744–1759
2015). Our findings support this conclusion and add new
knowledge by demonstrating that small changes to this
group result in complete loss of Piezo1 channel activation.
Removing one of the chlorine atoms [2b] or altering the posi-
tion of the chlorine atom around the ring [2c/2d] abolished
activity. Replacing one or both of the chlorine atoms with
fluorine [2a/g] also abolished activity implying that both
chlorine atoms are important for activity and may interact
with Piezo1 in a chlorine specific manner, potentially via a
σ-hole interaction, such as a halogen-pi bond. The
4-methoxyphenyl [2e] and 4-nitrophenyl [2f]analogues
were also inactive. Investigating the inhibitory potential of
the compounds showed that all but 2g,whichisthemost
similar in structure to Yoda1, were ineffective at inhibiting
Yoda1 activity.
Piezo1 channel activation by analogues with modifica-
tion to the pyrazine group was less than that of Yoda1, with
the most successful analogue, compound 7a,inwhichthe
pyrazine was replaced with a 3-pyridyl group, exhibiting
50% of the activity of Yoda1. This demonstrates the impor-
tance of the nitrogen atom in the 2-position of the pyrazine
ring, with loss of this nitrogen resulting in a 50% drop of
activity. The remaining two compounds from the series, the
phenyl [2i] and 2-pyrrolyl [7b] analogues, were less active
Figure 9
Specificity of Dooku1 in aorta. All experiments were performed on mouse thoracic aorta with intact endothelium. (A, B) Summary data for exper-
iments of the type shown in Figure 8A, B, expressed as the response to PE (A) or resting tension (B) before and after pre-incubation with 10 μM
Dooku1. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated by
the black lines (n= 7). (C) Aorta were pre-constricted with 0.1 μM U46619 and treated consecutively with DMSO, 1 μMAChand10μMSIN-
1. (D) As for C but pretreated with Dooku1 instead of DMSO. (E–G) Summary data for experiments of the type shown in (C, D) expressed as %
of the effect of Dooku1 on the contraction by U46619 (E) or % relaxation evoked by ACh (F) or SIN-1 (G) before and after pre-incubation with
10 μM Dooku1. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated
by the black lines (n=5).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1755
than 7a that suggests that the presence of the nitrogen atom
at the 3-position of the pyridine ring in 7a is also contribut-
ing to Piezo1 activation, supporting our understanding of
the importance of the nitrogen atom at the equivalent posi-
tion on the pyrazine ring of Yoda1 to activity. We next inves-
tigated replacement of the central thiadiazole ring by an
oxadiazole [11]. This change was largely tolerated with the
new compound demonstrating 70% of the activity of Yoda1.
The other two compounds from the series were less active,
although the data for the 2-pyridyl analogue [2j] were inter-
esting in that the partial activity observed for the analogue
suggests that the position of the nitrogen atom on the pyri-
dine contributes to activity, reinforcing the importance of
the equivalent Non the pyrazine ring of Yoda1 to activity.
Investigation into the inhibitory potential of this set of
left-hand and middle ring-modified analogues provided
Figure 10
Lack of effect of other Yoda1 analogues on PE-induced contraction. Summary data for experiments of the type shown in Figure 8 D–E, G–H
expressed as resting tension (left) or the response to PE (right) following pre-incubation with 10 μM2e (A), 2g (B), 7b (C) and 11 (D). Each data
point represents a value from an independent experiment with mean values and error bars representing SEM indicated by the black lines (n=5).
E L Evans et al.
1756 British Journal of Pharmacology (2018) 175 1744–1759
compounds with potential promise of being pharmacologi-
cal tools. All of the compounds from the series had the abil-
ity to reduce Ca
2+
entry evoked by Yoda1 by at least 40%,
andasmuchas75%inthecaseof2j. However, most of
these compounds exhibited partial agonist activity. The
most promising compound, 2k (Dooku1) effectively
reduced Yoda1 activity by 60%, without causing any
activation and was a strong inhibitor of the Yoda1 response
in the physiological setting of murine aortic rings. This
shows that the pyrazine ring can be replaced to identify
compounds, which do not activate the channel but do in-
hibit the Yoda1 response. It appears that analogues lacking
the 2,6-dichlorophenyl group do not activate the channels
or inhibit Yoda1 whereas pyrazine-modified analogues show
reduced activation and ability to inhibit Yoda1. Therefore,
the di-chloro group seems to be critical for binding while
the pyrazine group is less important for binding but key
for channel activation.
Currently, the only available inhibitors of Piezo1 activity
are not selective for Piezo1 (Drew et al., 2002; Bae et al.,
2011). Dooku1 is also not perfect as it does not directly block
the channels, but it is a new tool compound that is useful for
Piezo1 characterization studies. It antagonizes the action of
Yoda1 and could facilitate understanding of an important
small-molecule binding site on or near to Piezo1 channels.
Without agonist activity, Dooku1 effectively inhibits Yoda1-
induced Piezo1 activity. It does so without disturbing several
Ca
2+
handling events in the cell or affecting other aortic
relaxing agents. Although these data suggest specificity of
Dooku1 for Piezo1 channels, further studies to address this
point are warranted, especially given the inhibitory effect of
Dooku1 against PE and U46619-induced contractions of aor-
tic rings that might reflect a Piezo1 mechanism or some other
unknown effect of Dooku1. It is possible that Dooku1 may be
acting on Piezo1 in smooth muscle cells of the vessel,
partially inhibiting contraction. This assumes that the
channels become activated via a Yoda1-like mechanism
during contraction. Piezo1 was found not be required for
normal myogenic tone (Retailleau et al., 2015), and so, a
non-Piezo1 target of Dooku1 should be considered.
Dooku1 only has activity against Yoda1-induced and not
constitutive Piezo1 channel activity. Such an effect is consis-
tent with Dooku1 acting at the same or a similar site to Yoda1
and thereby occluding access of Yoda1 to its agonist binding
site. The reversibility of Dooku1 is consistent with the
reversibility of Yoda1 (Rocio Servin-Vences et al., 2017). It
would be good to investigate if the Dooku1 effect is consis-
tent with competitive antagonism, but solubility limitations
of the compounds prevented construction of appropriate
concentration–response curves. The inability of Dooku1 to
have any effect on constitutive activity suggests that the
mechanism of background channel activity is different to
that of chemical activation with Yoda1.
Dooku1 partially inhibited Yoda1 in HUVECs but
strongly inhibited it in aorta (Figure 6D cf. Figure 8C). We ini-
tially speculated that the difference was due to the higher
temperature of the contraction studies (37°C cf. room
temperature), but the Dooku1 effect was not significantly
temperature dependent (Figure 3K). An alternative explana-
tion might be that Ca
2+
entry is not directly proportional to
NO production, so that partial inhibition of Yoda-1 induced
Ca
2+
entry is sufficient to inhibit most of the relaxation in-
duced by Yoda1. Another divergence was that Yoda1 was
more potent in HUVECs than Piezo1 T-REx cells, showing a
difference between native and over-expressed Piezo1 chan-
nels (Figure 6E, F). We speculate that this difference reflected
a higher basal state of activity of the channels in endothelial
cells, as described previously (Rode et al., 2017), making the
channels more sensitive to Yoda1 because they are better
primed for opening.
In summary, this study has provided important insight
into the structure–activity relationships of Yoda1 and sup-
ported the concept of a specific chemical binding site on or
in close proximity to Piezo1 channels. It has also revealed
the discovery of a useful tool compound, Dooku1, which ef-
fectively antagonizes Yoda1-induced Piezo1 channel activity,
distinguishing it from constitutive Piezo1 channel activity.
The complete role of Piezo1 in vascular biology is still being
established, but the protein may have significant clinical
interest with emerging roles in genetic disease, BP control,
hypertension-induced arterial remodelling and exercise ca-
pacity (Retailleau et al., 2015; Wang et al., 2016; Rode et al.,
2017). As yet, it is not clear whether activating or inhibiting
this channel may be advantageous, but increasing our
pharmacological knowledge, alongside our physiological
knowledge of Piezo1 will be essential if therapeutic potential
of this protein is to be harnessed in the future. Learning more
about Piezo1 channel interactions with small-molecules
promises to be an important aspect of the overall effort to
understand Piezo1 biology.
Acknowledgements
This work was supported by research grants from the
Wellcome Trust, Medical Research Council and British Heart
Foundation and a studentship from the BBSRC for A.J.H.
and the Libyan Government for N.E.
Author contributions
E.L.E., K.C., N.E., B.R., N.M.B., A.J.H., S.J.H., H.J.G. and M.J.L.
performedexperimentsanddataanalysis.E.L.E.,K.C.,D.J.B.
and R.F. designed the research. D.J.B. and R.F. raised funds
to support the work. E.L.E., K.C., R.F. and D.J.B. co-wrote
the paper.
Conflict of interest
The authors declare no conflicts of interest.
Declaration of transparency and
scientificrigour
This Declaration acknowledges that this paper adheres to the
principles for transparent reporting and scientificrigourof
preclinical research recommended by funding agencies,
publishers and other organisations engaged with supporting
research.
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1757
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Supporting Information
Additional Supporting Information may be found online in
the supporting information tab for this article.
https://doi.org/10.1111/bph.14188
Figure S1 The general thiol alkylation reaction used to pro-
duce 11 compounds.
Figure S2 General synthetic route towards 7a and 7b. 2,6-
dichlorobenzyl chloride (3) is first converted to the thiol 4
followed by an SNAr to give 5, which is then brominated to
give 6 ready for a Suzuki cross-coupling to give the desired
products 7a-b.
Figure S3 Synthetic route for 10. 2,6-dichlorobenzyl chlo-
ride (3) is first converted to the thiol 4 ready for a reaction
with CDI and hydrazine to afford 8. Compound 8 is then uti-
lized in an amide coupling with 9 using EDCI to produce 10
ready for a cyclising-condensation reaction to afford 11.
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 1744–1759 1759
