Englerin A as a novel potent activator of TRPC4 and TRPC5 ... · PDF filediet Sukumar et al 2012 ... The Max Plank Institute, Dortmund, Germany Herbert Waldmann ... (–)-Englerin
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Aurora Ion Channel Retreat 2015
(–)-Englerin A as a novel potent activator of TRPC4 and TRPC5
channels Hannah J. Gaunt
David Beech Lab School of Medicine University of Leeds
Introduction to TRP channels
• Non-selective cation channels • Ubiquitously expressed throughout
the human body • 7 families of TRP channels
• C = ‘canonical’ • 6 membrane spanning domains with
the pore loop between S5 and S6
• Often expressed as heteromeric channels
• Lack of specific small molecule
modulators
Introduction to TRP channels
TRP channel activation by
natural chemicals – TRPC?
TRPV1 TRPM8 TRPA1 TRPC-
Ca2+/Na+ Ca2+/Na+ Ca2+/Na+ Ca2+/Na+
? capsaicin
heat menthol
cold
wasabi
mustard oil
cinnamon
Inhibition of TRPC5 by an omega-
3 fatty acid
α-Linolenic acid (ALA) is an essential omega-3 fatty acid found in seeds, nuts and many common vegetable oils.
0 200 400 600
1.5
2.0
2.5
3.0
lino.
Gd3+
Ca
2+
i (F
ra
tio
)
Time (s)
lino./control
control
0
25
50
75
100
125
lino. (mole/L)
0 50101Gd
3+
res
po
ns
e (
% c
on
tro
l)
0.1
diet
Sukumar et al 2012 Circ Res
Inhibition of TRPC5 by a chemical
extract from ginger
A traditional Chinese medicine for cardiovascular complaints
Naylor et al - In preparation
0 1 2 3 4 5
0
15000
30000
45000
60000
gal.
veh.
100M Gd3+
Ca
2+
i (F
, A
U)
Time (min)
TRPC5 (tet+)
0.1 1 100.0
0.5
1.0
TRPC5 (tet+)
no
rm. to
ma
x r
es
po
ns
e
Galangin conc. (M)
The discovery of (-)-Englerin A
(-)-Englerin A is an extract from Phyllanthus engleri
Heinrich Engler (1844-1930) German botanist
(-)-Englerin A
Renal cell carcinoma
61,560 new cases in USA (2015) The most common type of kidney cancer 7th most common cancer in UK Resistant to chemotherapy Often asymptomatic until it spreads to other organs
Naveen Vasudev Consultant in Medical Oncology
Sebastian Trainor Registrar in Medical Oncology
The ‘Englerin team’
Synthesis and target
identification
(-)-Englerin A
A498 = Renal cell carcinoma cell line
1 10 100 1000
0
20
40
60
GI50
59 nM
WS
T1 a
bs. (%
decre
ase)
(-)EA (nM)
A498
Akbulut Gaunt et al 2015 Angewandte Chemie Int Ed
0 120 2401.5
2.0
2.5
HEK (Tet-)
HEK-TRPC4 (Tet+)
3 nM (-)EA
Ca
2+
i (
F r
ati
o)
Time (s)
0 1 2 3 4 51
2
3
4
HEK-TRPC4
0 Ca2+
e
1.5 Ca 2+
e
Time (min)
Ca
2+
i (
F r
ati
o)
100 nM (-)EA
0 1 2 3 4 51
2
3
4
100 nM (-)EA
HEK-TRPC4
1 M (+)EA
Time (min)
Ca
2+
i (
F r
ati
o)
(-)EA / (+)EA
-100 -50 50 100
-1
1
2
HEK-TRPC4
(-)EA
I (n
A)
V (mV)
veh.
(-)EA
Potent stereo-selective
activator of TRPC4
(-)-Englerin A evokes calcium entry in HEK cells overexpressing TRPC4
Extremely efficacious activator
Prof. Katsuhiko Muraki
Aichi Gakuin University
0 2 4 6 8 10
-5
0
5
10
HEK C4 100 nM (-)EA
20 M La3+ +100 mV
I (n
A)
Time (min)
-100 mV
0.1 1 10 100
0
1
2
3
HEK-TRPC4
(-)EA (nM)
C
a2+
i (
F r
ati
o)
EC50 11.2 nM
0.1 1 10 100
0
1
2
3
HEK-TRPC5
C
a2+
i (
F r
ati
o)
(-)EA (nM)
EC50 7.6 nM
Activates TRPC4 and TRPC5
with nanomolar potency
Specific TRPC4/5 channel
activator
0.1 1 10 100
0
1
2
3
(-)EA (nM)
C
a2+
i (
F r
ati
o)
HEK-TRPC6
0.1 1 10 100
0
1
2
3
(-)EA (nM)
C
a2+
i (
F r
ati
o)
HEK-TRPM2No effect on
TRPC6
TRPM2
TRPC3
TRPV4
Orai1 SOCE
Orai3
NaV1.5
KV4.3
KV1.5
KCNQ
hERG
CaV1.2
Kir2.1
HCN4
• No effect on 14 other ion channels or 59 GPCRs • No binders detected in unbiased proteomic screening
-100 -50 50 100
-0.2
0.2
(-)E
A-e
vo
ked
I (n
A)
V (mV)
HEK C4
0 10 20 30 40 50-1
0
1
2
HEK-TRPC4
10
100100100100
-100 mV
+100 mV
ML 5 M
(-)EA (nM)
10
1000
I (n
A)
Time (min)
100
Powerful activation
independent of G-proteins
Outside-out patch configuration
ML204 TRPC4/5 channel inhibitor
TRPC4 IC50 = 0.99 μM TRPC5 IC50 = 9.2 μM
0 10 20 30
-0.2
0.0
0.2
0.4
HEK-TRPC4
ML 5 M
100
I (n
A)
Time (min)
(-)EA (nM) 100100
-100 mV
+100 mV
-100 -50 50 100
-0.2
0.2
0.4
(-)EAe+i
HEK-TRPC4
(-)EAi
I (n
A)
V (mV)
(-)EAe+i
Extracellular effect
Outside-out patch configuration (-)-Englerin A present in the intracellular pipette solution throughout
0 100 200 300
1.6
1.8
2.0
Time (s)
(-)-EA
Ca
2+
i (
F r
ati
o)
A498 (-)-EA or vehicle
vehicle
0.0
0.1
0.2
0.3
(+)-EA(-)-EA
C
a2
+
i (
F r
ati
o)
veh.
*
A498 NS
0.0
0.1
0.2
0.3
+--++
ML204
*
NSA498
C
a2
+
i (
F r
ati
o)
(-)-EA -
0.1 1 10 100 1000
0.0
0.2
0.4
0.6
(-)-EA (nM)
C
a2
+
i (
F r
ati
o)
A498
(-)-Englerin A acts potently to
evoke calcium entry in A498
cells
EC50 9.3 nM
0 2 4 6 8
-0.2
0.0
0.2
0.4
0.6
0.8A498
-100 mV
+100 mV
5 M ML204
I (n
A)
Time (min)
100 nM (-)EA
-100 -50 50 100
-0.2
0.2
0.4
0.6
(-)EA
A498
veh.
I (n
A)
V (mV)
(-)EA
-100 -50 50 100-0.2
0.2
0.4
0.6
(-)EA
HEK C4+C1
veh.I
(nA
)
V (mV)
(-)EA
0 2 4 6 8
-0.2
0.0
0.2
0.4
0.6
0.8100 nM (-)EA
5 M ML204
-100 mV
+100 mV
I (n
A)
Time (min)
HEK C4+C1
C1 C4
The native channels act
remarkably like heteromers
-100 -50 50 100
-0.2
0.2
0.4
(-)EAe+i
HEK-TRPC4
(-)EAi
I (n
A)
V (mV)
(-)EAe+i
TRPC1 and TRPC4 are involved
in the mechanism of cell death
Blocking antibodies siRNA
0
20
40
60
A498
anti-
TRPC1
anti-
TRPC4
cont.
*
WS
T1
ab
s.
(% d
ec
rea
se
)
*
cont.
Melanie Ludlow Hannah Gaunt
0
10
20
30
40
50
60
*
A498
siTRPC1siTRPC4 cont.W
ST
1 a
bs
. (%
de
cre
ase
)cont.
*
Melanie Ludlow, Hannah Gaunt, Katsuhiko Muraki
0.0
0.5
1.0
1.5
2.0
-40
-20
0
20
40
60
80
Pro
tein
(T
RP
C1
+4 /
-acti
n)
Hs5
78T
A49
8
786-
O
UO-3
1
ACHN
UM
RC2
HT-2
9
HeL
a
HUVEC
Hs5
78T
A49
8
786-
O
UO-3
1
ACHN
UM
RC2
HT-2
9
HeL
a
HUVEC
Ce
ll d
eath
(%
)
HEK29
3
0 2 4 6
-0.4
0.0
0.4
0.8
1.2
HT-29
-100 mV
+100 mV
5 M ML204
I (n
A)
Time (min)
100 nM (-)EA
Cell death Protein
HT-29 colorectal cancer cell line
RCC RCC
Emerging correlation TRPC1/4 expression, cell death and ionic current
Ionic current
6 8 10 12 14
1
2
3vehicle
A498 x
en
og
raft
Tu
mo
ur
exp
an
sio
n
days treatment
(-)EA
Naveen Vasudev, Hannah Gaunt
• A498 cells were subcutaneously implanted on the left flanks of 8 week old SCID beige mice
• 2 mg / kg (-)-EA or DMSO
treatment (n=4 each)
• Intraperitoneal injection daily
In-vivo
Summary
(-)-Englerin A selectively activates TRPC4 and TRPC5 channels with high potency and efficacy
Acts on the extracellular side of the membrane independent of G-protein
activation
TRPC1/4 protein expression correlates with the susceptibility to (-)-Englerin A evoked cell death
(-)-Englerin A activated TRPC1/4 channels are involved in its mechanism of cell
death
Implications:
1. A potential route to a renal cell carcinoma therapy
2. A channel activator in screens for TRPC4/5 inhibitors
3. For understanding TRPC4/5 channel biology
The University of Leeds, UK David Beech Katsuhiko Muraki Naveen Vasudev Melanie Ludlow Mohamed Amer Alexander Bruns Sebastian Trainor Nicholas Moss
The Max Plank Institute, Dortmund, Germany Herbert Waldmann Yasemin Akbulut Slava Ziegler
Thank you!
Freie Universität Berlin Germany Mathias Christmann
Out of Africa:
a natural born killer
TRPC4 activators as selective chemotherapy “magic bullets” for renal cell carcinoma !?
Hannah Gaunt bs10hg@leeds.ac.uk
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