Ionix Pharmaceuticals - Molecular Devices

Using the FLEXstation to
investigate voltage-gated
calcium and sodium
channels
Stuart Ward
Ionix Pharmaceuticals
May 2002
1

Outline of Talk
Ionix Profile
Role of FLEXstation in Molecular
Pharmacology Group
Assays investigating voltage-gated ion
channels:
Calcium
Sodium
2

Ionix Profile
Founded July 2001, Cambridge UK
Discovery of novel analgesic drugs
Proprietary drug targets including ion
channels
Drug development to Phase II
3

Role of FLEXstation
To play a key role in assay development
To be used in medium throughput drug
screening
Initially used in the development of
assays to investigate voltage-gated
calcium and sodium channels expressed
in neuroblastoma cell lines
4

Voltage-gated Calcium Channels
IMR32 cells have been used to
investigate calcium channels
Human neuroblastoma
Express L-type and N-type channels
(differentiate for N-type)
Used in low throughput assays
5

Aim
To develop a medium throughput
fluorescence-based assay using IMR32
cells in 96-well plate format
6

IMR32 Cell Culture Conditions
Cells grown in EMEM supplemented with
2mM Glutamine+1%NEAA+10%FBS
Cells were split 1:2/1:3 every 2-3 days
or had the media changed
Differentiate with 1mM dibutyryl cAMP
and 2.5µM bromodeoxyuridine for 8-10
days
7

Adherent vs Suspension Assay
Cells used in suspension
IMR32 cells adhere to surfaces weakly
Changing media every 2-3 days causes
large loss of cells when grown in 96 well
plates
Cells grown in tissue culture flasks
(under differentiating conditions, if
necessary) and then loaded with
fluorescent indicator and assayed in
suspension
8

Fura-2
Fluorescent Indicator
340/380 ratio
Disadvantage: It cannot easily be used with
a FLIPR
Fluo-4 was used in screening assay
Can be used in FLIPR and hence in highthroughput
assays
Data obtained do not require ratio
calculations. Therefore are easily
manipulated
9

Assay Protocol
Detach cells and resuspended in assay
buffer
Incubate with 2.3µM Fluo-4 or 5µM Fura-2,
50mM probenecid for 30min with rotation
Centrifuge and resuspend in assay buffer
Dispense 2x10 5 cells/well
Assay using the FLEXstation. KCl added by
fluidics system to stimulate calcium
channel opening
10

KCl-Induced Calcium Changes In
IMR-32 Cells
2.00
KCl (mM)
1.5
Ratio 340/380
1.75
1.50
50
30
10
5
0
1.25
0 10 20 30
Response
(Area Under Curve)
1.0
0.5
0.0
-0.5
1 10 100
Time (s)
KCl (mM)
11

Calcium Channel Assays
N-Type Assay:
8-10 day differentiated IMR32 cells
Assay run in the presence of nitrendipine or
nicardipine to block L-type component
L-Type Assay (two strategies):
8-10 day differentiated IMR32 cells
Need to block N-type component with
-conotoxin GVIA (expensive option)
Undifferentiated IMR32 cells (preferred
option)
No N-type component
12

1M Nicardipine
+1M -CnTxGVIA
13
Pharmacology of Calcium
Channels in Differentiated
IMR-32 Cells
10.0
7.5
5.0
2.5
Control
1µM Nicardipine
Response
(Area Under Curve)
2.00
1.75
1.50
1.25
1.00
Control
50mM KCl
1µM Nicardipine
1µM Nicardipine
+1µM -CnTxGVIA
0.0
0 10 20 30 40
Time (s)
Ratio 340/380

Measuring Fluo-4 Calcium Responses
150000
FLEXstation Output
Response (RFU)
100000
50000
0
KCl
Basal set to zero
Max-Min
Response
14
-50000
0 10 20 30 40 50
Time (sec)

Increasing CaCl 2 Concentration
in Stimulation Buffer
Increasing CaCl 2 in
assay buffer:
Increasing CaCl 2 in KCl
stimulation solution:
100000
100000
Peak Response
75000
50000
25000
Peak Response
75000
50000
25000
0
1.25 2.50 5.00 10.00
CaCl 2 Concentration (mM)
0
1.25 2.50 5.00 10.00 20.00
CaCl 2 Concentration (mM)
+buffer
+50mM KCl
+buffer
+50mM KCl
15

Assay Validation
Calcium channel blockers were used to validate
the assay
Nimodipine
-Conotoxin-GVIA
Response (% Max)
100
75
50
25
0
Response (% Max)
100
75
50
25
0
0.0001 0.001 0.01 0.1 1 10 100 1000
Nimodipine concentration (M)
N-Type IC 50 = 5.3 M
L-Type IC 50 = 13 nM
1 10 100
-Conotoxin-GVIA concentration
(nM)
N-type IC 50 = 5.5 nM
L-type No Block
16

Calcium Channel Screen
50,000 compound library screened
40 plates/day on FLEXstation
Data analyzed with SOFTmax Pro
17

Assay Plate Layout
Positive
Control
Negative
Control #1
Negative
Control #2
18
80 Test Compounds
(single concentration)

19
SOFTmax PRO Data Analysis

20
SOFTmax PRO Data Analysis

Summary
Developed assay to analyze voltagegated
calcium channels on FLEXstation
Assay now in routine use in screening
laboratory
21

Voltage-gated Sodium Channels
Use of Molecular Devices Membrane
Potential Assay Kit
Panel of cell lines investigated
Kelly
BE(2)C
SH-SY-5Y
ND7/23
Human neuroblastoma
Human neuroblastoma
Human neuroblastoma
Hybrid of mouse neuroblastoma and rat DRG
22

Experimental Conditions
Neuroblastoma cell lines grown in
96-well plates
Dye loading according to Molecular
Devices protocol
Except that all cells were initially loaded for
60min at 37ºC
Veratridine used to stimulate sodium
channel response
Added by FLEXstation fluidics system
23

Measuring Sodium Channel
Responses
FLEXstation Output
80000
Response (RFU)
60000
40000
Veratridine
Max-Min
Response
20000
0 25 50 75 100
Time (sec)
24

Assay Validation
Responses measured in BE(2)C cells
Veratridine
Tetrodotoxin
(100M Veratridine)
100000
100000
Response (RFU)
75000
50000
25000
Response (RFU)
75000
50000
25000
0
1 10 100 1000
0
0.01 0.1 1 10 100 1000 10000
Veratridine concentration (M)
EC 50 = 13 M
Tetrodotoxin concentration (nM)
IC 50 = 6 nM
25

Assay Validation
Responses measured in SH-SY-5Y cells
Veratridine
Tetrodotoxin
(100M Veratridine)
Response (RFU)
100000
75000
50000
25000
Response (RFU)
100000
75000
50000
25000
0
1 10 100 1000
0
1 10 100 1000
Veratridine Concentration ( M)
Tetrodotoxin Concentration (nM)
EC 50 = 32 M
IC 50 = 38 nM
26

Summary of Data
Cell Line
SH-SY-5Y
ND7/23
Kelly
BE(2)C
EC 50 Veratridine
24µM; n=6
29µM; n=6
31µM; n=6
24µM; n=6
IC 50 Tetrodotoxin
30nM; n=3
11nM; n=3
23nM; n=3
34nM; n=3
27

Assay Optimisation
Dye loading at 25 o C for 30min is better
than at 37 o C for 60min
Assay carried out at 25 o C
Dye concentration can be reduced by
5 fold
28

Summary
Tetrodotoxin sensitive voltage gated
sodium channels have been assayed in
neuroblastoma cell lines using the
Molecular Devices Membrane Potential
Assay Kit
29

Conclusions
FLEXstation has been successfully used
to…
Develop assays that analyze voltage-gated
calcium and sodium channels expressed in
neuroblastoma cell lines
Carry-out a medium throughput screen on
voltage-gated calcium channels
30

Acknowledgements
Molecular Pharmacology Group
Dave Thomas
Stuart Ward
Kathryn Elsegood
Zoe Gladwell
31