tRANSPORtERS - Molecular Devices

FLIPR based assays
for
electrogenic
transporters
Giorgia Michela Stucchi
Axxam
6th International Drug Discovery Product User Meeting
May 21-24, 2002
Half Moon Bay, CA, USA

TOPICS OF THE PRESENTATION
Overview about AXXAM
Overview about Transporters
FLIPR based assays for a. GABA Transporters
Conclusions
b. Glycine Transporters

AXXAM
AXXAM s.r.l. was founded at the 22nd of May 2001 at
Milano
AXXAM is located at the San Raffaele Science Park in
Milano (Italy) and has more than 1400 square metres of fully
equipped lab space
The team consists of 45 people
At the 8th of November 2001 a five years collaboration
agreement (worth of 30 Million Euros) between BAYER AG
and AXXAM was signed

BUSINESS of AXXAM:
From Gene to screen
Target
identification
Target
•Ta
validation
Assay
development
Assay
optimization for
96, 384 or 1536
Functional
analysis
Lead
•Ta
profiling
Protein
expression
•Ta

Transporters
Transporters are involved in different and essential cellular processes
Transporters represent an interesting and growing target class for drug
discovery
Transporter in the genome
Transporter types identified in vitro or in silico
150 categories of transporters classified by Saier
238 transporters in E.Coli
250-500 transporters in yeast
500-700 transporters in Drosophila (5% of genome)
> 1000 transporters in human genome

Transporter families
ion transporters & pumps
- sodium-potassium ATPase
- Organic Anion transporters (OATS)
- Na/Ca exchangers
metabolite transporters
- Amino acids
-Glucose
- Lactate
neurotrasmitter transporters
- Vescicular
-Plasma membrane

GABA transporter family (Na/Cl dependent)
HUGO PRODUCT Substrate (preferred) coupling Locus (human)
SLC6A1 GAT-1 -aminobutiric acid Na + /Cl - 3p25-p24
SLC6A2 NAT1/NET1 norepinephrine Na + /Cl - 16q12.2
SLC6A3 DAT1 dopamine Na + /Cl - 5p15.3
SLC6A4 HTT serotonin Na + /Cl - 17q11.1-q12
SLC6A5 GLYT-2 L-glycin Na + /Cl - 11p15.1-15.2
SLC6A6 TAUT taurin Na + /Cl - 3p26-p24
SLC6A6P pseudogene 21pter-qter
SLC6A7 prot L-proline Na + /Cl - 5q31-q32
SLC6A8 CT1/CRTR creatine Na + /Cl - Xq28
SLC6A9 GLYT-1a to 1c L-glycin Na + /Cl - 1p33
SLC6A10 CT-2 creatine Na + /Cl - 16p11.2
SLC6A11 GAT-3 -aminobutiric acid Na + /Cl - reserved
SLC6A12 BGT-1 -aminobutiric acid, Na + /Cl - 12p13
betaine
SLC6A13 GAT-2 -aminobutiric acid Na + /Cl - 12p13.3
SLC6A14 hATB(0+ neutral, cationic amino acids Na + /Cl - Xq23-q24
SLC6A15 hv7-3/homology reserved
SLC6A16 homology reserved

Pathway for Transporters
Expression profiling
Cloning of the transporter gene
Sequence verification
Functional testing by “patch clamp” analysis
Development of cell based assay for FLIPR
platform

GABA TRANSPORTERS
• belongs to the superfamily of sodium and chloride dependent neurotransmitter
transporters
• Four different GABA transporters (GATs) have been cloned up to now
• GAT-1 and GAT-3 are high affinity neuronal transporters expressed specifically in the CNS
• GAT-2 has a moderate affinity for GABA and is mainly found in glial cells
• GAT-4 low affinity GABA transporter with distribution in glia, liver and kidney
GABA (-aminobutyric acid) is the major inhibitory neurotrasmitter in the CNS.
Termination of GABA transmission is achieved through high affinity GABA transport
in both GABAergic neurons and glial cells
A decrease in the GABAergic transmission has been implicated in the pathophysiology
of several CNS disorders (e.g. epilepsy)

Stoichiometry of GABA transporters
• net positive inward charge transfer
• electrogenic (depolarization)
• voltage sensitive dye (?)

Patch clamp analysis of GABA-induced current in
HEK-293/hGAT-3 cells
HEK-293 mock transfected cells

Voltage sensitive dye and FLIPR
7500
6719
HEK-293/hGAT3
5938
•Fluorescence Counts
5157
4376
3595
2814
2033
1252
471
-310
0 1 2 3 4
HEK-293 mock
100 uM GABA
Time (minutes)

Membrane potential vs fluorescence changes
on FLIPR in HEK-293/hGAT-3
-20
50 M GABA
3000
Membrane Potential (mV)
-22
-24
-26
-28
2400
1200
600
Fluorescence
-30
0
-32
0 10
20 30 40
50 60 70
80
Time (s)

Voltage sensitive dye-FLIPR
GABA Dose-Response
in HEK-293/hGAT3
8500
Fluorescence Counts
7640
6780
5920
5060
4200
3340
2480
1620
760
•buffer
•2.5 M
•5 M
•10 M
•20 M
•40 M
•80 M
•160 M
•320 M
-100
0 1 2 3 4
Time(minutes)

HEK-293/hGAT-3
EC 50 DETERMINATION
Patch clamp vs FLIPR
Fluorescence Change
maximum RFU
10000
8000
6000
4000
2000
0
HEK-293/hGAT3
EC50 = 1.1 uM
10 -7 10 -6 10 -5 10 -4 10 -3
GABA (M)
EC50 = 18 uM

Voltage sensitive dye-FLIPR
-alanine Dose-Response
in HEK-293/hGAT3
RFU
6000
5000
4000
3000
2000
EC50= 184 uM
maximum RFU
1000
Fluorescence Counts
4600
4123
3646
3169
2692
2215
1738
1261
784
307
-170
0
20 40 60 80 100 120 140 160 180 200
Time(seconds)
•buffer
•15 M
•31 M
•62.25 M
•125 M
•250 M
•500 M
•1 mM
•2 mM
0
10 -6 10 -5 10 -4 10 -3 10 -2
-alanine

Patch clamp: GABA-induced current
in HEK-293/rGAT-1

Voltage sensitive dye-FLIPR
GABA Dose Response HEK-293/rGAT1
17000
Fluorescence Counts
15200
13400
11600
9800
8000
6200
4400
2600
800
-1000
0 1 2 3 4
Time(minutes)
• buffer
• 2.5 uM
• 5 uM
• 10 uM
• 20 uM
• 40 uM
• 80 uM
• 160 uM
• 320 uM

HEK-293/rGAT-1
EC 50 DETERMINATION
Patch clamp vs FLIPR
20000
maximun FLU
10000
maximum RFU
0
10 -6 10 -5 10 -4 10 -3
GABA
EC50 = 10.5 M
EC50 = 43.4 M

Raw data GABA dose response
buffer 1 uM 10 uM 50 uM 100 uM 200 uM
HEK-293/hGAT3
HEK-293/rGAT1
Range = ( -2000, 28000 )
• Robust signal
• Good signal:noise
• Good well to well reproducibility
•No wash

Glycine Transporters
Glycine Transporters are members of the Na + /Cl - -dependent family, a group of
integral glycoproteins, with 12 transmembrane domains.
Two glycine transporters subtypes have been identified:
GlyT1
Three isoforms are known: GlyT-1a
GlyT-1b
GlyT-1c
GlyT-1c expression is highly restricted to Brain.
GlyT2
is mainly expressed in spinal cord, brainstem and cerebellum and is present mostly on
neuronal cells.
Many polymorphisms in human GLYT2 has been identified

Glycine 2 mM
Glycine-induced current
in HEK-293/ GlyT2
Glycine 2 mM
Non transfected cells
n=6/6
120
100
current
80
60
GlyT2 transfected cells
n=8/9
40
20
0
10 -5 10 -4 10 -3 10 -2 10 -1
Glycine
EC50=98,21 µM

Voltage sensitive dye-FLIPR
Glycine Dose-Response
HEK-293/hGlyT2
Fluorescence Counts
3200
2870
2540
2210
1880
1550
1220
890
560
230
-100
0 1 2 3 4 5
Time(minutes)
• buffer
• 1 uM
• 10 uM
• 100 uM
• 1 mM
• 10 mM

Glycine 2 mM
Glycine-induced current
in HEK-293/ hGlyT1
EC50 determination by patch clamp
150
o fImax)
%
u rrent( c
100
50
0
EC50= 9.537 uM
-7 -6 -5 -4 -3 -2 -1
Glycine

Voltage sensitive dye-FLIPR
Glycine Dose-Response
HEK-293/hGlyT1
Fluorescence Change (Counts)
1700
1520
1340
1160
980
800
620
440
260
80
-100
0 1 2 3 4 5
Time(minutes)
• buffer
• 1 uM
• 10 uM
• 100 uM
• 1 mM
• 10 mM

Glycine transporters
Fluorescence Counts
3600
3230
2860
2490
2120
1750
1380
1010
640
270
-100
0 1 2 3 4 5
10 mM glycine
Time(minutes)
HEK-293/hGlyT2
HEK-293/hGlyT1
HEK-293

Summary
Voltage sensitive dye and FLIPR can be applied to the study of
electrogenic transporters
Good correlation between patch clamp and FLIPR data for GABA
transporters were demonstrated
Studies on glycine transporters are still on going.
From preliminary data a weaker correlation between patch clamp and
FLIPR data was observed
These results showed the feasibility of FLIPR based HTS screening for
electrogenic transporter modulators

Acknowledgements
Silvia Bovolenta
Sabrina Corazza
Anna Della Bella
Maria Foti
Maria Micheletti
Elisa Redaelli
Frank Hafner (Molecular Devices)