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High Throughput Screening
High Throughput Screening (HTS) is a method that uses automation and large dataset processing to quickly assay the biological or biochemical activity of large numbers of compounds, proteins or genes. Small molecule libraries*
Protein libraries
siRNA/shRNA libraries
cDNA libraries
Lairson – High Throughput Screening
| 2
High Throughput Screening
High Throughput Screening (HTS) is a method that uses automation and large dataset processing to quickly assay the biological or biochemical activity of large numbers of compounds, proteins or genes. Small molecule libraries*
Protein libraries
siRNA/shRNA libraries
cDNA libraries
Lairson – High Throughput Screening
Library Types
Diversity oriented synthesis
Natural products collections
Compounds of known biological activity (e.g., FDA approved drugs, kinase inhibitiors, etc.)
Specific MedChem programs
| 3
High Throughput Screening
High Throughput Screening (HTS) is a method that uses automation and large dataset processing to quickly assay the biological or biochemical activity of large numbers of compounds, proteins or genes. Small molecule libraries*
Protein libraries
siRNA/shRNA libraries
cDNA libraries
Lairson – High Throughput Screening
Library Types
Antibodies
Secreted soluble proteins
etc.
| 4
High Throughput Screening
High Throughput Screening (HTS) is a method that uses automation and large dataset processing to quickly assay the biological or biochemical activity of large numbers of compounds, proteins or genes. Small molecule libraries*
Protein libraries
siRNA/shRNA libraries
cDNA libraries
Lairson – High Throughput Screening
Library Types/Delivery
Genome wide
Gene class (e.g., kinase-wide)
Transient transfection
Viral transduction
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High Throughput Screening
Goal: Identify a molecular structure that:
Selectively binds to and modulates the activity of a
biological target (e.g., a protein) of interest (target-based)
Selectively induces a desired phenotype in a cell
population or organism of interest (phenotype-based)
Lairson – High Throughput Screening
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High Throughput Screening
Identified hits serve as starting points for:
Drug discovery programs
− Medicinal chemistry
− ADMET/PK
− Pharmacology
Chemical biology programs
− Target identification
− Target validation and mechanism of action
Lairson – High Throughput Screening
| 7
HTS - Approaches
Biochemical screens
− Enzymatic assays
− Protein/protein interactions
− Protein stabilization
Cell- and organismal-based screens
− Target-based (e.g., overexpression of GPCR in HEK293T cells)
− Phenotypic-based (e.g., induction of a given factor or process)
Challenge: Need for downstream target identification/validation
Lairson – High Throughput Screening
| 8
HTS – General Steps
1) Assay design, development, optimization and validation
− Access to sufficient quantities of proteins or cells of interest
− Optimization of substrate concentrations or growth conditions
− Optimization of protein concentrations or cell density
− Identification of physiologically relevant controls
− Optimization of end point assay or time course for compound treatment
− Cost considerations
− Dynamic range optimization (limits of detections)
− Miniaturization (96, 384, 1536 well plate formats; microfluidic chips; Z factors)
Lairson – High Throughput Screening
| 9
HTS – General Steps
1) Assay design, development, optimization and validation
Lairson – High Throughput Screening
| 10
HTS – General Steps
2) Screening
− Scale of screen (i.e., library size)
− Library type
Million compound scale
Structurally diverse sub-collections
Known bioactives (e.g., FDA approved drugs, kinase inhibitors)
Natural products
Focused medicinal chemistry
Lairson – High Throughput Screening
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HTS – General Steps
2) Screening
Assumptions:
More compounds = Better chances
Better assays = Better chances
Lairson – High Throughput Screening
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HTS – General Steps
3) Data analysis
− Data normalization and well masking
Lairson – High Throughput Screening
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HTS – General Steps
3) Data analysis
− Data normalization and masking
Lairson – High Throughput Screening
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HTS – General Steps
3) Data analysis
− Data normalization and masking
Lairson – High Throughput Screening
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HTS – General Steps
3) Data analysis
− Hit Selection (Fold change vs. robust z-score; activity of
controls vs. statistical significance)
− Hit Rate (generally ≤ 1%)
Lairson – High Throughput Screening
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HTS – General Steps
4) Confirmation of hits
− Confirmation of primary hits in triplicate and dose response
− Filter assays
Fluorescence
Luminescence
Cytotoxicity
Lairson – High Throughput Screening
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HTS – Assay Formats
Biochemical Assays
− e.g., enzyme inhibition, protein-protein interaction, protein stabilization, DNA/RNA binding
Tend to be robust
Easy to validate and interpret
Complicated assays
Need for significant quantities of pure protein/enzymes/substrates
Hits need to be optimized to facilitate cell permeability, acceptable toxicity profile, etc.
Lairson – High Throughput Screening
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HTS – Assay Formats
Biochemical Assays
− Enzyme assays
Need to optimize enzyme concentration and duration of read
Screen around Km of substrates to facilitate identification of competitive as well as uncompetitive inhibitors
Gain of signal preferred (A + B C; quantify loss of A)
Can use loss of signal, but need to control for artifacts
Fluorescence preferred over absorbance (decreased sensitivity)
Fluoregenic product release (e.g., glycosidases, hydrolases)
Couple to Fluorescent readout (e.g., diaphorase/resorufin coupled)
Lairson – High Throughput Screening
| 19
HTS – Assay Formats
Biochemical Assays
− HTRF assays
Variation of FRET (Forster Resonance Energy Transfer)
Used to measure distances
Distance dependent energy transfer between a donor and acceptor
(Intensity ~ 1/d6; 10-100 Å get a good signal)
Emission spectrum of donor overlaps with the excitation spectrum of
acceptor
Lairson – High Throughput Screening
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HTS – Assay Formats
Biochemical Assays
− HTRF assays
Can be used be measure protein-protein interactions
Lairson – High Throughput Screening
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HTS – Assay Formats
Biochemical Assays
− HTRF assays
Can also be used to detect products of a reaction of interest
Lairson – High Throughput Screening
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HTS – Assay Formats
Biochemical Assays
− HTRF assays
Problems with traditional FRET
− Low relative signal (compared to HTRF)
− High background fluorescence of biomolecules (decreased sensitivity)
Use long lived fluorophores and time resolved detection
Rare earth metals (i.e., lanthanides – Sm, Eu, Tb) complexed to organic molecules have ideal spectral properties
Readout is the ratio of emission signals
Lairson – High Throughput Screening
| 23
HTS – Assay Formats
Biochemical Assays
− Alpha screens (Amplified Luminescence Proximity
Homogeneous Assay)
Bead-based
Generate singlet oxygen on donor bead using 685 nm light
Measure luminescence derived from acceptor bead 4 µsec later
Diffusion limited to 200 nm
If acceptor bead is within 200 nm, luminescence of 520-620 nm
Lairson – High Throughput Screening
| 24
HTS – Assay Formats
Biochemical Assays
− Alpha screens (Amplified Luminescence Proximity
Homogeneous Assay)
Lairson – High Throughput Screening
| 25
HTS – Assay Formats
Biochemical Assays
− Fluorescence Polarization (FP)
Inexpensive
Very useful if you can’t modify substrates, don’t have good
antibodies, looking for binding of small molecule to a protein (e.g,
binding to a receptor - no enzymatic activity)
Can suffer from low signal:noise, requires large differences in MW
Lairson – High Throughput Screening
| 26
HTS – Assay Formats
Biochemical Assays
− Fluorescence Polarization (FP)
Binding of a fluorescent molecule to a macromolecule decreases
rotation/tumbling (relative to fluorescent lifetime) leading to an
increased polarization value (P value) of emitted light
Lairson – High Throughput Screening
| 27
HTS – Assay Formats
Cell-based Assays
− e.g., receptor agonist/antagonist, gene activation/silencing,
cellular differentiation/embryotic development, etc.
Identify cell permeable compounds and obtain cellular toxicity data
Assays can be complicated
Need for significant quantities of relevant cells (phenotypic assays)
Can be expensive
Tend to be lower throughput (phenotypic assays)
Lairson – High Throughput Screening
| 28
HTS – Assay Formats
Cell-based Assays
− FLIPR (Fluorometric Imaging Plate Reader) assays
Plate cell line that overexpresses protein of interest
Incubate with sensing dye, wash
Transfer compounds
Inject reagents simultaneously into microplate
Excite, CCD camera records images over time
Kinetic read of influx
Lairson – High Throughput Screening
| 29
HTS – Assay Formats
Cell-based Assays
− FLIPR (Fluorometric Imaging Plate Reader) assays
Plate cell line overexpressing protein of interest (e.g, calcium channel)
Incubate with sensing dye (e.g., Ionomycin), wash
Transfer compounds
Inject reagents simultaneously into microplate
Excite, CCD camera records images over time
Kinetic read of calicium influx
Lairson – High Throughput Screening
| 30
HTS – Assay Formats
Cell-based Assays
− Reporter-based assays
Transcriptional regulatory region of interest controls the expression
of reporter gene (e.g., GFP, luciferase)
Can be used to look for inducers/repressors of the transcription of a
protein of interest (e.g., a cytokine) or a protein associated with a
given cell state (e.g., a transcription factor, phenotypic assay)
Epigenetics are important (reporter construct vs. knock-in)
Lairson – High Throughput Screening
| 31
HTS – Assay Formats
Cell-based Assays
− Phenotypic assays (Pros and Cons)
Immediate identification of cell-based activity
Not limited by validated target information
Ability to identify novel biology
Ability to obtain sufficient quantities of physiologically-relevant cells
Establishment of a robust physiologically relevant assay
Need for downstream target identification/validation
Lairson – High Throughput Screening
| 32
HTS – Assay Formats
Cell-based Assays
− Phenotypic assays (Considerations)
Maximize physiological relevance
Understanding of known targets/uninteresting hits and potential false positives
Origin of cell type (primary, human vs. rodent), interspecies translation
Throughput (quality vs. quantity)
Appropriate library
Path forward for development (i.e., appropriate secondary assays, in vivo models, etc.)
Lairson – High Throughput Screening
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HTS – Assay Formats
Cell-based Assays
− Phenotypic assays (High content imaging)
Less prone to artifacts
Multi-parametric, obtain lots of information (primary readout(s), cell
cycle, cell health)
Based on immunofluorescent analysis (automated high throughput
microscopy and image analysis)
Need good algorithms (e.g, staining intensity, localization, co-
localization, translocation, morphology, etc.)
Lairson – High Throughput Screening
| 34
HTS – Assay Formats
Cell-based Assays
− Phenotypic assays (Flow cytometry-based)
Good for suspension cells
Multi-parametric, obtain lots of information (up to 9-13 channels, cell
cycle, cell health)
Lairson – High Throughput Screening
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~ 1 Billion THP-1 Cells
3 days
Cell dispense and drug treatment
3 days
Fixation, Staining and Wash
Acquisition Analysis
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Target Identification - Workflow
Lairson – High Throughput Screening
(5)
(4)
(6)
(1)
(3)
(2)
O
NH
O
NH
N3
OO
HN
O
S
NHHN
O
S. Zhu
Anti-biotin Western blot
Parallel Silver Stain
Work flow: (1) Incubate probe with cells (2) Photo-crosslinking (3) Cell lysis (4) Lysate fractionation (5) 2-D SDS-PAGE (6) Proteomics
This strategy has been successfully applied in the identification of the direct molecular targets of multiple small molecules identified by phenotypic screening. Examples : - Stauprimide (NME2): Zhu, S., et al. (2009).
Cell stem cell 4 (5): 416–26 - Kartogenin (FLNA): Johnson, K., et al.
(2012) Science. 336:717-21 - KHS101 (TACC3): Wurdak, H., et al. (2010)
PNAS. 107: 16542-16547
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HTS Case Study – Chemical complementation of Klf4
Goal: Identify small molecules that complement KLF4 activity for iPS cell reprogramming
Approach: Generate mouse strain with fLuc knocked into endogenous Nanog locus (critical since epigenetic reprogramming) and screen Nanog-Luc fibroblasts following transduction with 3 reprogramming factors
Lairson – High Throughput Screening
Lyssiotis et al. (2009) PNAS. 106: 8912.
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HTS Case Study – Chemical complementation of Klf4
Goal: Identify small molecules that complement KLF4 activity for iPS cell reprogramming
Results: Fibroblasts reprogrammed using OSM +Kenpaullone results in the generation of iPS cells (validated by germline transmission)
Lairson – High Throughput Screening
Lyssiotis et al. (2009) PNAS. 106: 8912.
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HTS Case Study – In vitro expansion of HSCs
Goal: Identify small molecules that expand hematopoietic stem cells (HSCs) in vitro
Application: expansion of “matched” cord blood-derived HSCs
Approach: Use primary human HSCs in a HCI-based assay (staining of CD34 following 7 days)
Lairson – High Throughput Screening
Boitano et al. (2010) Science. 329: 1345.
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HTS Case Study – Chemical complementation of Klf4
Results: SR1 induces robust in vitro expansion of HSCs that engraft into mice. Result has translated to the clinic.
Lairson – High Throughput Screening
Boitano et al. (2010) Science. 329: 1345.
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HTS Case Study – Chemical complementation of Klf4
Results: Aryl hydrocarbon receptor (AhR) identified as target from global expression profiling (rare case where this works)
Lairson – High Throughput Screening
Boitano et al. (2010) Science. 329: 1345.
SR1-induced CD34+ cell expansion acts by binding and antagonizing AhR. (A) Percent decrease in CYP1B1 (black squares) and AHRR (black circles) mRNA from mPB CD34+ cells treated with SR1 (solid lines) or LGC006 (dashed lines) for 24 h. (B) Quantification of AhR binding shown in fig. S16. (C) Phenotype of GFP+ cells 8 days after transduction with lentivirus expressing shRNAs targeting AhR. (D) Relative AhR mRNA expression in sorted CD34+GFP+ cells (box in C).
| 42
HTS Case Study – Directed differentiation of ESCs
Goal: Identify small molecules that induce the differentiation of embryonic stem cells (ESCs) toward definitive endoderm lineage
Approach: Use mouse R1 ESCs in a HCI-based assay (staining of Sox17 under basal differentiation conditions)
Lairson – High Throughput Screening
Zhu et al. (2009) Cell Stem Cell. 4: 416.
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HTS Case Study – Directed differentiation of ESCs
Results: Stauprimide induces robust differentiation of human and mouse ESCs towards not only endoderm, but also towards ectoderm and mesoderm lineages
Lairson – High Throughput Screening
Zhu et al. (2009) Cell Stem Cell. 4: 416.
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HTS Case Study – Directed differentiation of ESCs
Results: Affinity-probe used to identify NME2 as the specific protein target. Interaction with NME2 leads to decreased expression of c-Myc.
Lairson – High Throughput Screening
Zhu et al. (2009) Cell Stem Cell. 4: 416.
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HTS Case Study – Directed differentiation of ESCs
Results: Affinity-probe used to identify NME2 as the specific protein target. Interaction with NME2 leads to decreased expression of c-Myc.
Lairson – High Throughput Screening
Zhu et al. (2009) Cell Stem Cell. 4: 416.
| 46
Multiple Sclerosis (MS): remission in MS is largely dependent upon the regenerative process of remyelination
Existing treatments for MS exclusively target immunological mechanisms; however, these invariably fail and disease progresses to a state of chronic demyelination
A promising alternative approach for the development of complementary treatments is the identification of agents that directly stimulate the process of remyelination
Remyelination persists throughout adulthood in the CNS and involves the generation of new myelinating oligodendrocytes (ODs)
A widespread population of proliferating oligodendrocyte precursor cells (OPCs) are the source of the newly formed ODs required for remyelination
Neuron Oligodendrocyte Myelin sheath
Axon
Demyelination
Activation and Migration of OPCs
No remyelination Remyelination
Progressive decline Functional recovery
OPC
Adapted from Franklin, R.J.M and Ffrench- Constant, C. (2008) Nat. Rev. Neurosci. 9: 839.
HTS Case Study – Directed differentiation of OPCs
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Day 2 Day 10 Day 6 DAPI/MBP DAPI/MBP DAPI/MBP
DAPI/MBP DAPI/MBP DAPI/MBP
DMSO
T3
A2B5/ DAPI
Primary rat optic nerve derived OPCs used to develop a high content imaging-based assay for HTS
BF
Rationale: Premyelinating ODs are present in chronic lesions – remyelination is not limited by the abundance or migration of OPCs. Inhibition of OPC differentiation at sites of injury is causative in disease progression.
Chang, A., et al. (2002) N. Engl. J. Med. 346: 165. Wolswijk, G. (1998) J. Neurosci. 18: 601.
HTS Case Study – Directed differentiation of OPCs
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Enprofylline
All Trans Retinoic Acid
DAPI/MBP
ER modulators
ROCK2 Inhibitor DAPI/MBP DAPI/MBP DAPI/MBP
O
O
H
H
HO
OHHN
N N
HN
CH3
O
OCl
S N NHO
O
%MBP- 20% %MBP- 9% %MBP- 14% %MBP-26%
Screen of ~100K diverse drug-like and
bioactive small molecules collection Known modulators of OPC
differentiation identified Novel Scaffolds and Neurotransmitter
receptor modulators identified
HTS Case Study – Directed differentiation of OPCs
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Deshmukh et al. (2013) Nature. Oct 17;502(7471):327-32
HTS Case Study – Directed differentiation of OPCs
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M1/M3 receptor antagonism determined to be required for Benztropine-induce OPC differentiation activity
Evaluation of a broad panel of structurally diverse M1/M3 antagonists indicates that an additional activity is likely involved, as multiple structurally unrelated compounds are inactive
HTS Case Study – Directed differentiation of OPCs
| 51
HTS Case Study – Directed differentiation of OPCs
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Vehicle Benztropine
HTS Case Study – Directed differentiation of OPCs
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Cuprizone diet induces a demyelination program wherein the corpus callosum shows peak demyelination following 6-7 weeks of feeding
By administering drugs at the time when a cuprizone-free diet is reintroduced, the efficacy of promyelinating agents can be examined
HTS Case Study – Directed differentiation of OPCs
| 54
Addition of a suboptimal of Benztropine allows for a 10-fold reduction in FTY-720 dose
Benefit derived from combination of mechanisms (not additive effect of individual mechanisms)
HTS Case Study – Directed differentiation of OPCs
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4-731 2-068 7-012 0-235 7-245 3-005 6-126
4-73
1 2-
068
7-01
2 0-
235
7-24
5 3-
005
6-12
6
EC 50 (µM) : 10 15 1 0.5 0.5 5 5
T3 DMSO
Screen of ~100K drug-like small molecules Seven novel OPC differentiation-inducing scaffolds identified
HTS Case Study – Directed differentiation of OPCs
| 56
MBP, β-actin
2 Scaffolds selected for preclinical development Preliminary medchem studies indicate substantial improvements in
potency and efficacy are achievable relative to benztropine
HTS Case Study – Directed differentiation of OPCs
| 57
• Brain tumors possess a hierarchical cellular organization of heterogeneous cell populations • Tumor- forming capacity and tumor recurrence is attributed to a cancer stem cells (CSCs) • GBM CSCs share several features with neural stem cells including stem-like marker expression (i.e., Nestin, Sox2), the ability to migrate within the brain, the capacity to self- renew and to give rise to more differentiated progeny
GBM CSCs BICs + BMP
DAPI GFAP Tuj1 DAPI GFAP Tuj1
GBM CSCs + BMP4
Wurdak et al. (2010) Cell Stem Cell. 6: 37.
HTS Case Study – Selective inhibition of GBM CSCs
| 58
P/Map2-luciferase
P/Map2 FF luc
2 x 106 small molecules Acumen based
Confirmation (12,000)
StanAs
Astrocytes
Informatics Selectivity Filter of “Toxic” Compounds
00.5
11.5
22.5
33.5
Rel
ativ
e C
aspa
se
3/7
Activ
atio
n
GBM A Astrocyte NPC136 “selectively” toxic
HTS Case Study – Selective inhibition of GBM CSCs
| 59
P/Map2-luciferase
P/Map2 FF luc
2 x 106 small molecules Acumen based
Confirmation (12,000)
StanAs
Astrocytes
Informatics Selectivity Filter of “Toxic” Compounds
136 “selectively” toxic hits
HTS Case Study – Selective inhibition of GBM CSCs
| 60
P/Map2-luciferase
P/Map2 FF luc
2 x 106 small molecules Acumen based
Confirmation (12,000)
StanAs
Astrocytes
Informatics Selectivity Filter of “Toxic” Compounds
00.5
11.5
22.5
33.5
Rel
ativ
e C
aspa
se
3/7
Activ
atio
n
GBM A Astrocyte NPC
136 “selectively” toxic hits
HTS Case Study – Selective inhibition of GBM CSCs
| 61
Caspase 3/7 Activation
8327-1 Series
Cleaved Caspase 3
1 µM
O
O
HN
X
R2
R3 R1
O
• Cell survival, Caspase 3/7 activation and TUNEL assays were used to validate primary toxicity hits
• Primary human astrocytes, hES derived NPCs, and primary human lung fibroblasts were used to determine selectivity
• 8327-1 series was identified as a selective inducer of apoptosis in GBM CSCs
Cleaved Caspase 3
DMSO
Caspase 3 Activation in GBM CSCs
BTIC NPC
HTS Case Study – Selective inhibition of GBM CSCs
| 62
Caspase 3/7 Activation
Cleaved Caspase 3
1 µM
• Cell survival, Caspase 3/7 activation and TUNEL assays were used to validate primary toxicity hits
• Primary human astrocytes, hES derived NPCs, and primary human lung fibroblasts were used to determine selectivity
• 8327-1 series was identified as a selective inducer of apoptosis in GBM CSCs
Cleaved Caspase 3
DMSO
Caspase 3 Activation in GBM CSCs
Aruna 72hrs
Aruna 72hrs
StanA 72 hrs
StanA 72 hrs
DMSO
2.5 uM 1H1 250 nM 1H1
DMSO
HTS Case Study – Selective inhibition of GBM CSCs