Identification of novel TMEM175 modulators using highthroughput automated patch-clamp and solid-supported
membrane- (SSM-) based electrophysiology platforms
L. Hutchison1, A. Bazzone2, C. Brown1, N. Brinkwirth2, M. Barthmes2, L. Gerrard1, E. Dragicevic2, C. George1, N. Fertig2, D.
Dalrymple1, I. McPhee1, E. and D. Pau1
1SB Drug Discovery, West of Scotland Science Park, Glasgow, Scotland, UK; 2Nanion Technologies, Munich, Germany

1

Contact: elena@nanion.de3

Introduction

TMEM175 activators and antagonists
AB
SBD08533

TMEM175 is a novel, constitutively active ion channel involved inregulating lysosomal pH and autophagy. Mutations in this gene impair
normallysosomal

andmitochondrial

function,

therebyZnCl2Baselineincreasing

4-AP

aggregation of insoluble proteins such as phosphorylated α-synuclein,
CActivation (%)

hampered further investigation into the exact role of TMEM175 in normal

Day 1
Day 2
Day 3

400
200

cell line was characterized using automated patch-clamp and SSM--8

based electrophysiology. We developed and executed robust, high-EC50

8-10minsA2-3mins

3-4mins

B

Success Rate

Z' factor Analysis
1.0100200-7-6-5Log10 [SBD08533] (M)-4-8Day 1
2.056e-006

Day 3
1.624e-006

Day 1
2.829e-006

Day 2
1.947e-006

EC50B

A-7-6-5Log10 [SBD08533] (M)Day 2
2.652e-006

-4

Day 3
5.196e-006

CEC50 SBD08533 (5 µM)
4-AP (1 µM)
Baseline
4-AP (FB)

3-4
mins

400

SBD08533 – A reproducible TMEM175 Activator. Representative current traces (A) and time courses (B)of the hit activator SBD08533 against TMEM175. The application protocol consists of two additions of
extracellular physiological solution for a minimum of 3 minutes each, followed by an application ofSBDD08533 for a minimum of two minutes. ZnCl2 (500 µM) is then applied before the channel is inhibitedby a saturating concentration of 4-AP (1 mM). Normalised concentration response curves (C & D)showing the reproducible activation by SBD0833 tested over several days.

TMEM175 high-throughput automatedpatch clamp screening
Application
protocol.
Theapplication
protocol consists of three additions of
extracellular physiological solution for aminimum of 8 minutes, followed by anapplication of test compound for a minimumof three minutes. ZnCl2 (500 µM) is thenapplied, before the channel is inhibited by asaturating concentration of 4-AP (1 mM).

Day 1Day 2Day 300

lysosomal function and pathological processes. Here, the TMEM175 stable

2600

600

treatment of PD. The lack of specific pharmacological tools has

screening assays, with exceptionally high success rates.

SBD08533 CRC tested over several days

SBD08533 CRC tested over several days

TMEM175 demonstrates significant potential as a key player in thethroughput, and high-content direct electrophysiological intracellular

DActivation (%)

leading to symptoms typical of Parkinson’s Disease (PD). Consequently,

EC50
SBD08533(5 µM)

4-AP(1 µM)

4-AP(FB)

SBD08533 – Antagonist studies. Representative current traces (A), time course (B) and C-R curve (C) ofthe TMEM175 antagonist assay. Activation of TMEM175 by SBD08533 was inhibited in a concentrationdependent manner by reference inhibitor 4-AP.

4Solid-supported membrane- (SSM-)
based TMEM175 screening assay

Z' factor% Success806040200.5Average z' = 0.77Success rate = 82%0.000255075100Assay Number

0125255075100Plate number

125A rapid and robust automated, high-throughput electrophysiology screening assay. SyncroPatch 384,
high-throughput automated electrophysiology system was used to screen a library of 10,000 small
molecules at a single concentration of 10 µM. Average success rate (A) and Z` factor (B) of theTMEM175 HTS assay. The screen yielded an average success rate of 82% (wells passing QC) and anaverage Z’ factor of 0.77.
AB

TMEM175 Activators
TMEM175262522212022928

23161110060525

4200

1950

18400

75

17% Inhibition

600

3

TMEM175
untransfected

100HTS Hit confirmation. % activation of the confirmed TMEM175 Activators (A) and inhibitors (B) against theTMEM175 (blue) and the untransfected HEK (red) cell lines.
ACompound

Activators
EC50 range
3 - 30 µM

Baseline

ZnCl24-AP

BInhibitors
IC50 range
0.7 - 42 µM

Baseline

CompoundNew HTS technology for drug discovery on transporters and channels. The SURFE2R 96SE is a solidsupported membrane- (SSM-) based technology, which allows for measurements of slow andintracellular targets, not accessible in patch clamp in a 96-well format. Parallel target activation inpresence of different compound concentrations reveals IC50 and EC50 values. Lysosomes were purified
from HEK293 cells overexpressing TMEM175, kindly provided by SB Drug Discovery. Lysosomes wereadsorbed to SURFE2R sensors according to Nanion´s standard protocol. 50 mM K+ concentrationgradients were used to trigger K+ flux through TMEM175 at 0 mV and pH 7.6. H+ conductivity assays arepossible; native lysosomal pH gradients may be applied.

27

untransfected

800

1% Activation (above baseline)

1000

TMEM175 Inhibitors

ZnCl24-AP

TMEM175 Hit compound concentration response studies. Representative time courses and C-R curves of
the confirmed TMEM175 Activators (A) and inhibitors (B) against the TMEM175 channel. The hit activators
displayed EC50 values in the range of 3 - 30 µM while the hit inhibitors displayed IC50 values in the rangeof 0.7 – 42 µM.

5Conclusions•TMEM175 stable cell line was characterized using the SyncroPatch 384 automated patch clamp
system and used to develop and execute a high-throughput electrophysiology screening assay.

•This screening campaign successfully identified a number of active compounds with the ability tomodulate TMEM175 in a concentration-dependent manner with EC50/IC50 values in the low
micromolar range.

•The successful development of a TMEM175 electrophysiology assay capable of identifying novel
pharmacological tools will enable investigation of the role of this exciting target in normal
physiology and in disease.

•An assay for TMEM175 on the SURFE2R 96SE was developed was developed and dose-dependent
signal enhancement and inhibition of TMEM175 currents from purified lysosomal membranes of thestable cell line overexpressing TMEM175 were successfully characterized. This approach allows forstable and robust recordings from proteins residing in organellar membranes with a throughput ofup to 10’000 data points per day.

Recent TMEM175 Literature Review Update: Following generation of this data anarticle was published (Hu et al. Cell 2022) in which TMEM175 channel is suggested asa H+ channel rather than a K+ channel at low pH. We are now developing a H+ assay
to be used in conjunction with our K+ assay. Visit our both for more information.