ABCD ABCD - Molecular Devices
ABCD ABCD - Molecular Devices
ABCD ABCD - Molecular Devices
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<strong>ABCD</strong><br />
<strong>Molecular</strong> <strong>Devices</strong> 8th International Drug Discovery<br />
Conference, Berkeley, June 2004<br />
Case study: automated FLIPR screening to identify<br />
agonists for a GPCR target<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
"The direct route to a FLIPR assay"<br />
<strong>ABCD</strong><br />
• compare various Ca kits<br />
• optimize cell number/well<br />
• +/- lid incubations<br />
• 37°/RT incubations<br />
• determine MTP coating<br />
• optimize dye incubation time<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
GPCR X1: assay principle<br />
<strong>ABCD</strong><br />
agonist<br />
GPCR X1<br />
α<br />
IP3<br />
PLC<br />
Fluo[ ] Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Ca 2+ Ca 2+<br />
Ca 2+ Ca 2+<br />
Ca2+<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Protocol for automated assay<br />
<strong>ABCD</strong><br />
• seed 20 ul/well cell suspension (7.000 cells/well) in serum-free<br />
medium in collagen-coated 384-well plate (1 day before)<br />
• add 20 ul/well <strong>Molecular</strong> <strong>Devices</strong> Ca3 Assay Kit<br />
• incubate for 80 min at room temperature (no lid)<br />
• add 20 ul/well Hank's solution<br />
• dilute single-use compound plate, add "high/low" controls to<br />
columns 23/24<br />
• transfer 20 ul/well diluted compound/control solutions from<br />
compound MTP to assay MTP in FLIPR<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Integration of a 2nd FLIPR to an<br />
automated environment<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Integrated FLIPR: overview<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Compound dilution from pierced "single<br />
use plates" (Sciclone)<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Transfer of compound from the dilution<br />
MTP to the assay MTP in the FLIPR<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Sealing of the compound dilution plates<br />
for potential "re-use"<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Primary screening campaign<br />
<strong>ABCD</strong><br />
• 1 FTE provided 140 cell culture [384-well] MTPs/day<br />
• 1 FTE conducted FLIPR screening on these 140 MTPs resulting<br />
in about 54.000 measurements/day (unattended overnight<br />
runs)<br />
• the full primary screening campaign on about 750.000<br />
compounds was finished in 20 assay days (including 4<br />
validation runs)<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Assay statistics<br />
<strong>ABCD</strong><br />
Z' 0.6-0.7<br />
S/B ~11:1<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Quality control software: batch stability<br />
over half an assay day<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Quality control software: batch statistics<br />
<strong>ABCD</strong><br />
200%CTL: full activation of GPCR X1 by reference agonist<br />
100%CTL: no activation of GPCR X1 using buffer<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Primary screen<br />
<strong>ABCD</strong><br />
100 % CTL: inactive compound<br />
200 % CTL: reference agonist stimulated<br />
Hit threshold<br />
• ~750.000 compounds<br />
• Hit threshold >150 %CTL<br />
• ~23000 primary hits (hit rate of 3.1 %)<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Hit confirmation<br />
<strong>ABCD</strong><br />
compound number<br />
3000<br />
2500<br />
2000<br />
1500<br />
1000<br />
500<br />
confirmed hits run 1<br />
primary hit rate: 3.1 %<br />
hits retested twice: ~23000<br />
confirmed hits: 21000<br />
confirmation rate: 90 %<br />
0<br />
< 0 100 200 300 400 500 600 700<br />
%CTL<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Counterscreen 1<br />
<strong>ABCD</strong><br />
I 0 hit<br />
α<br />
PLC<br />
Fluo[ ] Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Ca 2+ Ca 2+<br />
Ca 2+ Ca 2+<br />
Ca2+<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Host cell counterscreen: no compound<br />
effect<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Host cell counterscreen: autofluorescent<br />
compound<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Host cell counterscreen: non-specific Carelease<br />
agonist<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Correlation of the two CHO counter<br />
screen runs<br />
<strong>ABCD</strong><br />
5000<br />
compound number<br />
4000<br />
3000<br />
2000<br />
1000<br />
~16000 specific hits, run 1<br />
0<br />
< 0 100 200 300 400 500 600<br />
%CTL<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Counterscreen 2<br />
<strong>ABCD</strong><br />
I 0 hit<br />
antagonist<br />
α<br />
PLC<br />
Fluo[ ] Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Fluo[ ]<br />
Ca 2+ Ca 2+<br />
Ca 2+ Ca 2+<br />
Ca2+<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Correlation of two counter screen runs in the<br />
presence of excess reference antagonist<br />
<strong>ABCD</strong><br />
compound number<br />
4000<br />
3500<br />
3000<br />
2500<br />
2000<br />
1500<br />
1000<br />
500<br />
~16000 specific hits, run 1<br />
0<br />
< 0 100 200 300 400 500 600<br />
% CTL<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Correlation of non-specific hits from the<br />
antagonist versus the CHO counter screen<br />
<strong>ABCD</strong><br />
>90 % of the CHO-active hits are also active despite the presence of reference antagonist,<br />
which confirms their non-specific character<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
EC50 determination/selectivity<br />
<strong>ABCD</strong><br />
• 7652 compounds underwent EC50 testing at the target molecule,<br />
GPCR X1 receptor<br />
• the same compounds underwent EC50 testing at the closest<br />
homologues: GPCR X2 and GPCR X3<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
GPCR X1 agonist<br />
<strong>ABCD</strong><br />
EC50 0.457 ug/ml<br />
EMax 198 %CTL<br />
R 0.970<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
GPCR X1 super agonist<br />
<strong>ABCD</strong><br />
EC50 0.918 ug/ml<br />
EMax 253 %CTL<br />
R 0.977<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
GPCR X1 partial agonist<br />
<strong>ABCD</strong><br />
EC50 0.051 ug/ml<br />
EMax 177 %CTL<br />
R 0.966<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
GPCR X1 incomplete dose response<br />
<strong>ABCD</strong><br />
EC50 11.5 ug/ml<br />
EMax 234 %CTL<br />
R 0.952<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
GPCR X1 no dose response curve fit<br />
<strong>ABCD</strong><br />
EC50<br />
EMax<br />
R<br />
Selectivity profile of "potent and efficient"<br />
GPCR X1 hits vs GPCR X2/X3<br />
<strong>ABCD</strong><br />
EC 50 (X1) [µM]<br />
No Effect on Subtype<br />
X2<br />
No Effect on<br />
Subtype X3<br />
Selectivity_X2<br />
Selectivity_X3<br />
No Effect on Subtypes X2<br />
or X3<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Functional secondary assays for the FLIPR<br />
format: cAMP-AlphaScreen<br />
<strong>ABCD</strong><br />
GPCR<br />
α γ<br />
β<br />
ATP<br />
adenylate<br />
cyclase<br />
↑ Fsk<br />
cAMP<br />
680 nm<br />
520-620 nm<br />
biotin-cAMP<br />
Acceptor-Anti-cAMP<br />
Donor-SA<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Validate cAMP-AlphaScreen for Gs and Gi<br />
pathway in GPCR X1-transfected CHO cells<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Functional secondary assays for the FLIPR<br />
format: IP3-AlphaScreen<br />
<strong>ABCD</strong><br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
Validate IP3-AlphaScreen for Gq pathway<br />
in GPCR X1 transfected CHO cells<br />
<strong>ABCD</strong><br />
35000<br />
30000<br />
10000<br />
cells/well<br />
172.000 units<br />
Counts AlphaQuest<br />
25000<br />
20000<br />
15000<br />
10000<br />
5000<br />
0<br />
2500<br />
cells/well<br />
7500<br />
cells/well<br />
5000<br />
cells/well<br />
1 2 3 4 5<br />
controls<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery<br />
Thanks to the "Kappadag" gang<br />
<strong>ABCD</strong><br />
Thank you for your attention!<br />
Ralf Heilker<br />
Dep. of Integrated Lead Discovery
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