( )-Englerin A as a novel potent activator of TRPC4 and TRPC5 channels

Transcription

1 Aurora Ion Channel Retreat 215 ( )-Englerin A as a novel potent activator of TRPC4 and TRPC5 channels Hannah J. Gaunt David Beech Lab School of Medicine University of Leeds

2 Introduction to TRP channels 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

3 TRP channel activation by natural chemicals TRPC? capsaicin heat menthol cold wasabi mustard oil cinnamon? TRPV1 TRPM8 TRPA1 TRPC- Ca 2+ /Na + Ca 2+ /Na + Ca 2+ /Na + Ca 2+ /Na +

4 Gd 3+ response (% control) (F ratio) 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 lino./control Gd 3+ control Ca 2+ i lino. Sukumar et al 212 Circ Res Time (s) diet lino. ( mole/l)

5 norm. to max response Inhibition of TRPC5 by a chemical extract from ginger A traditional Chinese medicine for cardiovascular complaints TRPC5 (tet+) 6 1 M Gd 3+ Ca 2+ i (F, AU) 45 veh gal Time (min) TRPC5 (tet+) Galangin conc. ( M) Naylor et al - In preparation

6 The discovery of (-)-Englerin A (-)-Englerin A is an extract from Phyllanthus engleri Heinrich Engler ( ) German botanist

7 (-)-Englerin A

8 Renal cell carcinoma 61,56 new cases in USA (215) The most common type of kidney cancer 7 th 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

9 WST1 abs. (% decrease) The Englerin team Synthesis and target identification A498 = Renal cell carcinoma cell line A GI 5 59 nm (-)-Englerin A (-)EA (nm) Akbulut Gaunt et al 215 Angewandte Chemie Int Ed

10 I (na) Ca 2+ (F ratio) i Ca 2+ (F ratio) i Ca 2+ (F ratio) i Potent stereo-selective activator of TRPC nm (-)EA HEK-TRPC4 (Tet+) HEK-TRPC4 1 nm (-)EA Ca 2+ e HEK-TRPC4 (-)EA / (+)EA 4 1 nm (-)EA HEK (Tet-) Time (s) 2 Ca 2+ e Time (min) 2 1 M (+)EA Time (min) HEK-TRPC4 2 (-)EA 1 veh V (mv) -1 (-)EA (-)-Englerin A evokes calcium entry in HEK cells overexpressing TRPC4

11 I (na) Extremely efficacious activator HEK C4 1 1 nm (-)EA Prof. Katsuhiko Muraki Aichi Gakuin University M La mv -1 mv Time (min)

12 Ca 2+ ( F ratio) i Ca 2+ ( F ratio) i Activates TRPC4 and TRPC5 with nanomolar potency HEK-TRPC4 HEK-TRPC5 3 2 EC nm 3 2 EC nm (-)EA (nm) (-)EA (nm)

13 Ca 2+ ( F ratio) i Ca 2+ ( F ratio) i Specific TRPC4/5 channel activator HEK-TRPC (-)EA (nm) 1 HEK-TRPM (-)EA (nm) No effect on 14 other ion channels or 59 GPCRs No binders detected in unbiased proteomic screening No effect on TRPC6 TRPM2 TRPC3 TRPV4 Orai1 SOCE Orai3 Na V 1.5 K V 4.3 K V 1.5 KCNQ herg Ca V 1.2 Kir2.1 HCN4

14 I (na) (-)EA-evoked I (na) Powerful activation independent of G-proteins Outside-out patch configuration ML24 TRPC4/5 channel inhibitor TRPC4 IC 5 =.99 μm TRPC5 IC 5 = 9.2 μm HEK-TRPC (-)EA (nm) ML 5 M HEK C mv -1 mv Time (min) V (mv) -.2

15 I (na) I (na) Extracellular effect Outside-out patch configuration (-)-Englerin A present in the intracellular pipette solution throughout HEK-TRPC4.4 1 (-)EA (nm) 1 1 ML 5 M HEK-TRPC4.4 (-)EA e+i.2 +1 mv.2. (-)EA i Time (min) -1 mv V (mv) -.2 (-)EA e+i

16 Ca 2+ ( F ratio) i Ca 2+ ( F ratio) i Ca 2+ (F ratio) i Ca 2+ ( F ratio) i (-)-Englerin A acts potently to evoke calcium entry in A498 cells A (-)-EA or vehicle (-)-EA vehicle Time (s) A veh. NS * (-)-EA (+)-EA A (-)-EA (nm) A (-)-EA - ML24 - NS + - EC nm * + +

17 I (na) I (na) I (na) I (na) I (na) The native channels act remarkably like heteromers A nm (-)EA 5 M ML24 +1 mv -1 mv Time (min) A (-)EA veh (-)EA -.2 V (mv) HEK-TRPC4.4 (-)EA e+i.2 (-)EA i V (mv) -.2 (-)EA e+i HEK C4+C nm (-)EA 5 M ML24 +1 mv HEK C4+C (-)EA veh. C4 C mv Time (min) V (mv) (-)EA

18 WST1 abs. (% decrease) WST1 abs. (% decrease) TRPC1 and TRPC4 are involved in the mechanism of cell death Blocking antibodies sirna A498 A * * * * cont. anti- TRPC4 cont. anti- TRPC1 cont. sitrpc4 cont. sitrpc1 Hannah Gaunt Melanie Ludlow

19 Cell death (%) Protein (TRPC1+4 / -actin) I (na) Emerging correlation TRPC1/4 expression, cell death and ionic current Cell death Protein Ionic current HT nm (-)EA 5 M ML mv -2-4 HEK293 HUVEC HeLa HT-29 UMRC2 ACHN RCC UO O A498 Hs578T.5. RCC HUVEC HeLa HT-29 UMRC2 ACHN UO O A498 Hs578T Melanie Ludlow, Hannah Gaunt, Katsuhiko Muraki. -1 mv Time (min) HT-29 colorectal cancer cell line

20 A498 xenograft Tumour expansion In-vivo 3 vehicle 2 1 (-)EA days treatment 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 Naveen Vasudev, Hannah Gaunt

21 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

22 Thank you! 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 Freie Universität Berlin Germany Mathias Christmann

23 Out of Africa: a natural born killer TRPC4 activators as selective chemotherapy magic bullets for renal cell carcinoma!? Hannah Gaunt bs1hg@leeds.ac.uk