Ionix Pharmaceuticals - Molecular Devices
Ionix Pharmaceuticals - Molecular Devices
Ionix Pharmaceuticals - Molecular Devices
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
Using the FLEXstation to<br />
investigate voltage-gated<br />
calcium and sodium<br />
channels<br />
Stuart Ward<br />
<strong>Ionix</strong> <strong>Pharmaceuticals</strong><br />
May 2002<br />
1
Outline of Talk<br />
<strong>Ionix</strong> Profile<br />
Role of FLEXstation in <strong>Molecular</strong><br />
Pharmacology Group<br />
Assays investigating voltage-gated ion<br />
channels:<br />
Calcium<br />
Sodium<br />
2
<strong>Ionix</strong> Profile<br />
Founded July 2001, Cambridge UK<br />
Discovery of novel analgesic drugs<br />
Proprietary drug targets including ion<br />
channels<br />
Drug development to Phase II<br />
3
Role of FLEXstation<br />
To play a key role in assay development<br />
To be used in medium throughput drug<br />
screening<br />
Initially used in the development of<br />
assays to investigate voltage-gated<br />
calcium and sodium channels expressed<br />
in neuroblastoma cell lines<br />
4
Voltage-gated Calcium Channels<br />
IMR32 cells have been used to<br />
investigate calcium channels<br />
Human neuroblastoma<br />
Express L-type and N-type channels<br />
(differentiate for N-type)<br />
Used in low throughput assays<br />
5
Aim<br />
To develop a medium throughput<br />
fluorescence-based assay using IMR32<br />
cells in 96-well plate format<br />
6
IMR32 Cell Culture Conditions<br />
Cells grown in EMEM supplemented with<br />
2mM Glutamine+1%NEAA+10%FBS<br />
Cells were split 1:2/1:3 every 2-3 days<br />
or had the media changed<br />
Differentiate with 1mM dibutyryl cAMP<br />
and 2.5µM bromodeoxyuridine for 8-10<br />
days<br />
7
Adherent vs Suspension Assay<br />
Cells used in suspension<br />
IMR32 cells adhere to surfaces weakly<br />
Changing media every 2-3 days causes<br />
large loss of cells when grown in 96 well<br />
plates<br />
Cells grown in tissue culture flasks<br />
(under differentiating conditions, if<br />
necessary) and then loaded with<br />
fluorescent indicator and assayed in<br />
suspension<br />
8
Fura-2<br />
Fluorescent Indicator<br />
340/380 ratio<br />
Disadvantage: It cannot easily be used with<br />
a FLIPR<br />
Fluo-4 was used in screening assay<br />
Can be used in FLIPR and hence in highthroughput<br />
assays<br />
Data obtained do not require ratio<br />
calculations. Therefore are easily<br />
manipulated<br />
9
Assay Protocol<br />
Detach cells and resuspended in assay<br />
buffer<br />
Incubate with 2.3µM Fluo-4 or 5µM Fura-2,<br />
50mM probenecid for 30min with rotation<br />
Centrifuge and resuspend in assay buffer<br />
Dispense 2x10 5 cells/well<br />
Assay using the FLEXstation. KCl added by<br />
fluidics system to stimulate calcium<br />
channel opening<br />
10
KCl-Induced Calcium Changes In<br />
IMR-32 Cells<br />
2.00<br />
KCl (mM)<br />
1.5<br />
Ratio 340/380<br />
1.75<br />
1.50<br />
50<br />
30<br />
10<br />
5<br />
0<br />
1.25<br />
0 10 20 30<br />
Response<br />
(Area Under Curve)<br />
1.0<br />
0.5<br />
0.0<br />
-0.5<br />
1 10 100<br />
Time (s)<br />
KCl (mM)<br />
11
Calcium Channel Assays<br />
N-Type Assay:<br />
8-10 day differentiated IMR32 cells<br />
Assay run in the presence of nitrendipine or<br />
nicardipine to block L-type component<br />
L-Type Assay (two strategies):<br />
8-10 day differentiated IMR32 cells<br />
Need to block N-type component with<br />
-conotoxin GVIA (expensive option)<br />
Undifferentiated IMR32 cells (preferred<br />
option)<br />
No N-type component<br />
12
1M Nicardipine<br />
+1M -CnTxGVIA<br />
13<br />
Pharmacology of Calcium<br />
Channels in Differentiated<br />
IMR-32 Cells<br />
10.0<br />
7.5<br />
5.0<br />
2.5<br />
Control<br />
1µM Nicardipine<br />
Response<br />
(Area Under Curve)<br />
2.00<br />
1.75<br />
1.50<br />
1.25<br />
1.00<br />
Control<br />
50mM KCl<br />
1µM Nicardipine<br />
1µM Nicardipine<br />
+1µM -CnTxGVIA<br />
0.0<br />
0 10 20 30 40<br />
Time (s)<br />
Ratio 340/380
Measuring Fluo-4 Calcium Responses<br />
150000<br />
FLEXstation Output<br />
Response (RFU)<br />
100000<br />
50000<br />
0<br />
KCl<br />
Basal set to zero<br />
Max-Min<br />
Response<br />
14<br />
-50000<br />
0 10 20 30 40 50<br />
Time (sec)
Increasing CaCl 2 Concentration<br />
in Stimulation Buffer<br />
Increasing CaCl 2 in<br />
assay buffer:<br />
Increasing CaCl 2 in KCl<br />
stimulation solution:<br />
100000<br />
100000<br />
Peak Response<br />
75000<br />
50000<br />
25000<br />
Peak Response<br />
75000<br />
50000<br />
25000<br />
0<br />
1.25 2.50 5.00 10.00<br />
CaCl 2 Concentration (mM)<br />
0<br />
1.25 2.50 5.00 10.00 20.00<br />
CaCl 2 Concentration (mM)<br />
+buffer<br />
+50mM KCl<br />
+buffer<br />
+50mM KCl<br />
15
Assay Validation<br />
Calcium channel blockers were used to validate<br />
the assay<br />
Nimodipine<br />
-Conotoxin-GVIA<br />
Response (% Max)<br />
100<br />
75<br />
50<br />
25<br />
0<br />
Response (% Max)<br />
100<br />
75<br />
50<br />
25<br />
0<br />
0.0001 0.001 0.01 0.1 1 10 100 1000<br />
Nimodipine concentration (M)<br />
N-Type IC 50 = 5.3 M<br />
L-Type IC 50 = 13 nM<br />
1 10 100<br />
-Conotoxin-GVIA concentration<br />
(nM)<br />
N-type IC 50 = 5.5 nM<br />
L-type No Block<br />
16
Calcium Channel Screen<br />
50,000 compound library screened<br />
40 plates/day on FLEXstation<br />
Data analyzed with SOFTmax Pro<br />
17
Assay Plate Layout<br />
Positive<br />
Control<br />
Negative<br />
Control #1<br />
Negative<br />
Control #2<br />
18<br />
80 Test Compounds<br />
(single concentration)
19<br />
SOFTmax PRO Data Analysis
20<br />
SOFTmax PRO Data Analysis
Summary<br />
Developed assay to analyze voltagegated<br />
calcium channels on FLEXstation<br />
Assay now in routine use in screening<br />
laboratory<br />
21
Voltage-gated Sodium Channels<br />
Use of <strong>Molecular</strong> <strong>Devices</strong> Membrane<br />
Potential Assay Kit<br />
Panel of cell lines investigated<br />
Kelly<br />
BE(2)C<br />
SH-SY-5Y<br />
ND7/23<br />
Human neuroblastoma<br />
Human neuroblastoma<br />
Human neuroblastoma<br />
Hybrid of mouse neuroblastoma and rat DRG<br />
22
Experimental Conditions<br />
Neuroblastoma cell lines grown in<br />
96-well plates<br />
Dye loading according to <strong>Molecular</strong><br />
<strong>Devices</strong> protocol<br />
Except that all cells were initially loaded for<br />
60min at 37ºC<br />
Veratridine used to stimulate sodium<br />
channel response<br />
Added by FLEXstation fluidics system<br />
23
Measuring Sodium Channel<br />
Responses<br />
FLEXstation Output<br />
80000<br />
Response (RFU)<br />
60000<br />
40000<br />
Veratridine<br />
Max-Min<br />
Response<br />
20000<br />
0 25 50 75 100<br />
Time (sec)<br />
24
Assay Validation<br />
Responses measured in BE(2)C cells<br />
Veratridine<br />
Tetrodotoxin<br />
(100M Veratridine)<br />
100000<br />
100000<br />
Response (RFU)<br />
75000<br />
50000<br />
25000<br />
Response (RFU)<br />
75000<br />
50000<br />
25000<br />
0<br />
1 10 100 1000<br />
0<br />
0.01 0.1 1 10 100 1000 10000<br />
Veratridine concentration (M)<br />
EC 50 = 13 M<br />
Tetrodotoxin concentration (nM)<br />
IC 50 = 6 nM<br />
25
Assay Validation<br />
Responses measured in SH-SY-5Y cells<br />
Veratridine<br />
Tetrodotoxin<br />
(100M Veratridine)<br />
Response (RFU)<br />
100000<br />
75000<br />
50000<br />
25000<br />
Response (RFU)<br />
100000<br />
75000<br />
50000<br />
25000<br />
0<br />
1 10 100 1000<br />
0<br />
1 10 100 1000<br />
Veratridine Concentration ( M)<br />
Tetrodotoxin Concentration (nM)<br />
EC 50 = 32 M<br />
IC 50 = 38 nM<br />
26
Summary of Data<br />
Cell Line<br />
SH-SY-5Y<br />
ND7/23<br />
Kelly<br />
BE(2)C<br />
EC 50 Veratridine<br />
24µM; n=6<br />
29µM; n=6<br />
31µM; n=6<br />
24µM; n=6<br />
IC 50 Tetrodotoxin<br />
30nM; n=3<br />
11nM; n=3<br />
23nM; n=3<br />
34nM; n=3<br />
27
Assay Optimisation<br />
Dye loading at 25 o C for 30min is better<br />
than at 37 o C for 60min<br />
Assay carried out at 25 o C<br />
Dye concentration can be reduced by<br />
5 fold<br />
28
Summary<br />
Tetrodotoxin sensitive voltage gated<br />
sodium channels have been assayed in<br />
neuroblastoma cell lines using the<br />
<strong>Molecular</strong> <strong>Devices</strong> Membrane Potential<br />
Assay Kit<br />
29
Conclusions<br />
FLEXstation has been successfully used<br />
to…<br />
Develop assays that analyze voltage-gated<br />
calcium and sodium channels expressed in<br />
neuroblastoma cell lines<br />
Carry-out a medium throughput screen on<br />
voltage-gated calcium channels<br />
30
Acknowledgements<br />
<strong>Molecular</strong> Pharmacology Group<br />
Dave Thomas<br />
Stuart Ward<br />
Kathryn Elsegood<br />
Zoe Gladwell<br />
31