university health network high-throughput screens for
TRANSCRIPT
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High-Throughput Screens for
Identifying Novel Anti-cancer Agents
F-F Liu MD, FRCPCRadiation Oncologist/Senior Scientist
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Outline
1. Introduction
2. Forward Chemical Screen
3. Reverse Screen
4. Conclusions
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HTS
Test large number of molecules simultaneously via miniaturization and automation of assay protocols
1. Drug discoveries
2. Understand biological processes
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Approaches to HTS
1. Reverse screens
2. Forward screens
Reverse Genetics
Mutate Gene
Insert in vivo
Look for phenotype
Screen for chemical binding
Add compound in vivo
Look for phenotype
Reverse Classical Genetics
Reverse Chemical Genetics
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Target Compound Disease
Bcr-Abl Imatinib CML, GIST
Src/Abl Dasatinib Advanced CML
Her1/EGFR Erlonitib NSCLC
Proteasome Bortezomib Multiple Myeloma
Reverse Chemical Genetics
Reverse Chemical Genetics
Forward Genetics
Random mutagenesis
Selectmutant withphenotype
Identified mutated gene
Programmed Cell DeathC. ElegansHorvitz
Cell-CycleS. CerevisiaeHartwell
Embryonic DevelopmentD. MelanogasterNusslein-Volhard
DiscoveryOrganismPI
Programmed Cell DeathC. ElegansHorvitz
Cell-CycleS. CerevisiaeHartwell
Embryonic DevelopmentD. MelanogasterNusslein-Volhard
DiscoveryOrganismPI
Forward Classical Genetics
Forward GeneticsForward
Chemical Genetics
Add 1 compound/well
Plate Cells
Identify protein target
Forward Classical Genetics
Identified mutated gene
Random mutagenesis
Selectmutant withphenotype
Select compound that produces phenotype
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Forward Chemical Genetics
Target Identification
1. Biotin labeling
2. Y3H
3. Micro-array
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Calcium-calcineurin-NFAT signalingFK506
Tor protein nutrient-response signalingRapamycin
Microtubules as cancer targetsPaclitaxel
DiscoveryDrug
Calcium-calcineurin-NFAT signalingFK506
Tor protein nutrient-response signalingRapamycin
Microtubules as cancer targetsPaclitaxel
DiscoveryDrug
Forward Chemical Genetics
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Outline
1. Introduction
2. Forward Chemical Screen
3. Current Reverse Screen
4. Conclusions
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Forward HTS for Head & Neck Cancer
Therapeutics
Day 4(MTS - viability)
Screening ProcedureScreening ProcedureDay 1(Seed Cells)
O
O
N+
O
O
N+
O
ONO2
NH
O OH
O
O
N+
O
O
O
N+
O
NH
Cl
OO
N+
N+
BenzethoniumChloride
Analog 1
Analog 2
Analog 3
Analog 4
Analog 5 O
ON
+
O
O
N+
O
O NO2
NH
OOH
O
O
N+
O
O
O
N+
O
NH
Cl
OO
N+
N+
Benzethonium Chloride
Analog 1
Analog 2
Analog 3
Analog 4
Analog 5
Day 2(Add Compounds)(1/well)
Choose cells that double every 21 h• reduce bias
Screen cancer vs. normal• choose “hit” criteria with caution
Lower dynamic range than other assays, but less manipulation
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LOPAC Library (1280 compounds)Prestwick Library (1120 compounds)
64 hits screened by FaDu cell viability
29 compounds
35 compoundsNIH/3T3 viability
DECREASE
6 compounds
Anti-microbial
23 compounds
NO
YES
Screening ProcedureScreening Procedure
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3 novel 1 studied
6 compounds
5757 viability 1 compound
5 compounds
DECREASE
4 compounds
C666-1 viability 1 compound
DECREASE
Screening ProcedureScreening Procedure
Forward small molecule HTS: 3 existing antimicrobials with novel anticancer properties:
1. Benzethonium Chloride Yip et al, Clin Cancer Res 15, 5557, 2006
2. Alexidine Dihydrochloride Yip et al, Mol Cancer Ther 5, 2234, 2006
3. Cetrimonium Bromide Ito et al, (manuscript under review)
HNC TherapeuticsHNC Therapeutics
Cetrimonium Bromide (CTAB)
Quaternary ammonium compound
Delocalized lipophilic cation Lipophilic; delocalized positive charge Penetrates hydrophobic cellular membranes Accumulates in mitochondria due to negative M
Interacts with mitochondrial H+-ATP synthase
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Anti-Cancer Specificity
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Combination Therapy
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CTAB Induces Apoptosis
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CTAB Induces Apoptosis
CTAB Induces Apoptosis
FaDu: Hypopharyngeal SCCC666-1: NPCGM05757: Primary normal
fibroblast
CTAB Induces Apoptosis
CTAB Inhibits Mitochondrial ATP Synthase Activity
CTAB Reduced Intracellular ATP Levels
M of Cancer vs. Normal Cells
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Anti-Cancer Specificity
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CTAB Ablates In Vivo Tumour-Forming Capacity
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CTAB Reduces Growth of Established Tumours
Proposed mode of action
-
+
-+
M
P
CTAB is concentrated in tumor mitochondria due to M
CTAB-ATP synthase interactions
+
-
Matrix
Outer Mitochondrial Membrane
Inner membrane
Adapted from Wikipedia
H+-gradient across IMM dissipates
M is sensed by PTPM
PTP opening induces MOMP Mitochondrial dysfunction Apoptogenic factors
released Caspase activation
Apoptosis
MOMP
MOMP Apoptosis
Proposed mode of action
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Conclusions
1. Identified a novel mitochondria-mediated apoptogenic anti- cancer agent using a forward HTS.
2. Selective in vitro and in vivo efficacy against HNC models Rooted at the mitochondria M differences between cancer
vs. normal cells
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Outline
1. Introduction
2. Forward Chemical Screen
3. Current Reverse Screen
4. Conclusions
ObjectiveDesign HTS to identify novel genes
that can selectively sensitize cancer cells to radiation when suppressed
Proposed Modifications
1. Target-driven siRNA-based approach
2. Incorporate RT into screen
3. Utilize a more appropriate readout for determining radiosensitization
Adapted from Nature Rev Genetics 7:373, 2006
Readout?
RT +
Kassner; Comb Chem & HTS; 11:175, 2008
Korn & Krause; 11:503, 2007
The multidisciplinary approach of high-content screening (HCS) requires a combination of different expertise.
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Specific Aims
1. Define experimental parameters for target-driven siRNA-based HTS
2. Conduct the screen using siRNA libraries to identify novel radiosensitizing targets
3. Validate and characterize hits
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Specific Aims
1. Define experimental parameters for target-driven siRNA-based HTS
2. Conduct the screen using siRNA libraries to identify novel radiosensitizing targets
3. Validate and characterize hits
siRNA Transfection Parameters
Parameters for 96-well Format
Optimal Condition
Cancer model A549
Forward vs. reverse transfection Reverse
Cell number (507500 cells/well) 750 cells
Transfection reagent volume (0.010.33 l/well)
0.08 l
siRNA concentration (10100 nM/well) 40 nM
Transfection time (2448 h) No difference
Katz et al; Biotechniques; 44:9, 2008
Katz et al; Biotechniques; 44:9, 2008
Automated Clonogenic Assay
Conclusions: Effective in measuring effects of
cytotoxic agents on cancer cells in vitro Limitations
• Low cell # reduces dynamic range• Unable to identify sensitization
Katz et al; Biotechniques; 44:9, 2008
BrdU Cell Proliferation Assay
Bromodeoxyuridine (BrdU) Thymidine analogue Incorporated into newly synthesized DNA
strands of actively proliferating cells (S phase)
Non-radioactive alternative to [3H]-thymidine incorporation
Examines cellular effects with long-term kinetics that are more reflective of therapeutic response
Positive siRNA Control
Day 1 Day 2 Day 3 Day 5 Day 6
Seed cells + siRNA
Transfection
Radiation Treatment
…Add BrdU
Readout
DNA Ligase IV siRNAScrambled siRNA
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Specific Aims
1. Define experimental parameters for target-driven siRNA-based HTS
2. Conduct screen using siRNA libraries to identify novel radiosensitizing targets
3. Validate and characterize hits
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siRNA Libraries
Dharmacon human siARRAY libraries:
4 siRNAs pooled/gene
Protein Kinase siRNA library800 genes
Druggable siRNA library6080 genes
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siRNA Screens
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Specific Aims
1. Define experimental parameters for target-driven siRNA-based HTS
2. Conduct screen using siRNA libraries to identify novel radiosensitizing targets
3. Validate and characterize hits
Validation of siRNA Hits
siRNA library(6880 siRNAs)
137 siRNAsEliminate
Transfect ± RT (0 vs. 2 Gy)
188 siRNAs (2 Gy)
51 confirmed hits Top 15 hits
0 Gy
2 Gy
Decreased surviving fractionIncreased surviving fractionNo effect
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Potential Radiosensitizing Targets
0 2 GyScrambled siRNA
ATM
STK6
123
4
5
6
7
8
9
1011
12
13
Potential Radiosensitizing Targets
Genes #1-15
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Caveat
Off-target Effects (OTE)
1. Up to 30% of sequences can cause phenotypic change, which do not correlate with gene silencing effect.
2. Hence, critical to confirm “hits” using a variety of different approaches.
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Anti-Cancer Specificity
Criteria: ≥40% survival fraction compared to 0 Gy Radiosensitization observed with ≥2 siRNAs
UTSCC 8a UTSCC 42aFaDu3-4
siRNAs/ gene
15 hits (A549)
mRNA Knockdown
#1 #3Scrambled Scrambled
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Confirmed Radiosensitizing Targets
FaDu, UTSCC 8a, and UTSCC 42a:
1. Integrin, alpha V (ITGAV)
2.Gene #2
3.Gene #3
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mRNA Knockdown Kinetics in FaDu
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Western for Protein k/d in FaDu Cells (48 hrs)
40 KDa
GAPDH
Lipo Neg siRNA
Protein #3
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Clonogenic Assay in FaDu cells
Knockdown Gene #3 in FaDu Cells
0
5
10
15
20
25
30
35
0 30 60 90 120 150 180 210 240 270
Time (min)
r-H
2A
X E
xp
res
sio
n (
%)
RT
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Outline
1. Introduction
2. Forward Chemical Screen
3. Current Reverse Screen
4. Conclusions
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Conclusions
1. HTS enable rapid discovery of novel anti-cancer agents.
2. Centrimonium bromide is one such novel mitochondrial-mediated apoptogenic compound.
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Conclusions
3. Experimental parameters need to be carefully defined to conduct RNAi-based radiosensitization screens
4. Identified novel genes with potential radiosensitizing properties
Dr. Mariano Elia Chairin Head & Neck Cancer Research
SLRI Robotics Facility