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