Screening for Cell Death
A Comparison of Methods
Lisa McWilliams,
HTS, Discovery Sciences, AstraZeneca;
ELRIG Research and Innovation 2015
18/03/2015
Importance of cytotoxicity screening
Within AstraZeneca we are performing an increasing number of
cell based assays where cell toxicity can be a confounding activity
Cell-based screening now accounts for about half of all HTS
screens carried out in AstraZeneca.
Toxicity is also a key reason for failure in the clinic and it is
desirable to annotate clusters from HTS screens to alert projects
as to which clusters may have a toxic liability.
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Lisa McWilliams | 18th March 2015
Discovery Sciences | Global HTS Centre
Screening cascade
48 h incubation
24 h incubation
Orthogonal Assay
CellToxTM Green Assay
Primary HTS
CellTiter-Blue®
Assay
Hit Confirmation
CellTiter-Blue®
Assay
4.4% active rate
90% Hit Conf.
Concentration
Response
CellTiter-Blue® Assay
86.8% active rate
Artefact screen (2)
Fluorescent quench Assay
Orthogonal Assay
MultiCyt 4-Plex Apoptosis
Screening Kit
0.08% active rate
Promega
CellTiter-Blue®
Cell Viability
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Lisa McWilliams | 18th March 2015
Promega
CellToxTM Green
Cytotoxicity
Intellicyt iQue platform
Multicyt Apoptosis kit
Apoptosis
Discovery Sciences | Global HTS Centre
Assessing agreement of different assays
CellTiter-Blue® vs CellToxTM Green
•
Group of compounds (red circle
in graph A and B) showing
higher potency when cell
viability measured by CellTiterBlue® compared with other
cytotoxicity assays
•
Difference seen may be due to
cytostatic activity of
these
compounds
•
Data in graph C show that there
is good agreement between a
measure of membrane integrity
(CellToxTM Green) and markers
of apoptosis (flow cytometry 4plex assay).
A
Caspase vs CellTiter-Blue®
B
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Lisa McWilliams | 18th March 2015
Caspase vs CellToxTM Green
C
Discovery Sciences | Global HTS Centre
Use of multiple methods leads to more reliable
assessment of compound toxicity
SN 1059862729
Cytotoxic
Cytostatic
25
C aspase
L iv e _ D e a d
E ffe c t
%% Effect
% Effect
0
M ito D a m a
-2 5
A n n e x in
-5 0
Q uench
A la m a rB lu
-7 5
C e llT o x G
-1 0 0
-1 2 5
-9
-7
25
C aspase
0
-2 5
-2 5
-7 5
% E ffe c t
0
-5 0
Quench
-4
L iv e _ D e a d
M ito D a m a g e
M ito D a m a g e
-9
A la m a rB lu e
-7 5
-8
-1 2 5
A n n e x in
Q uench
-5 0
C e llT o x G re e n
Q uench
A la m a rB lu e
C e llT o x G re e n
-1 0 0
-1 2 5
C aspase
L iv e _ D e a d
A n n e x in
-1 0 0
Lisa McWilliams | 18th March 2015
-5
SN 0208654166
25
5
-6
S N 0 2 0 8 6 5 4 Log[conc]
1 6 6 Conc
Log[conc]
ffe c t
% EEffect
%
-8
-7
-6
Conc
Log[conc]
-9
-8
-7
-5
-4
-6
Discovery Sciences | Global HTS Centre
-5
-4
Further annotation is essential
CellTiter Blue® assay results in a significant number of potential
false positives and should not be used in isolation to determine
cell toxicity.
It is recommended that a measure of membrane integrity is used
routinely as it appears more reliable to assess compound toxicity.
We anticipate that this annotation of our screening collection will
allow better decision making early in cascades especially where
cellular screening systems are used.
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Lisa McWilliams | 18th March 2015
Discovery Sciences | Global HTS Centre