Lecture 11 High Throughput Screening.pdf

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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

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Protein libraries


cDNA libraries


Library Types

Diversity oriented synthesis

Natural products collections

Compounds of known biological activity (e.g., FDA approved drugs, kinase inhibitiors, etc.)

Specific MedChem programs

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Protein libraries


cDNA libraries


Library Types

Antibodies

Secreted soluble proteins

etc.

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Protein libraries


cDNA libraries


Library Types/Delivery

Genome wide

Gene class (e.g., kinase-wide)

Transient transfection

Viral transduction

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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)


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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


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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


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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)


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1) Assay design, development, optimization and validation


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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


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2) Screening

Assumptions:

More compounds = Better chances

Better assays = Better chances


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3) Data analysis

− Data normalization and well masking


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3) Data analysis

− Data normalization and masking


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3) Data analysis

− Data normalization and masking


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3) Data analysis

− Hit Selection (Fold change vs. robust z-score; activity of

controls vs. statistical significance)

− Hit Rate (generally ≤ 1%)


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4) Confirmation of hits

− Confirmation of primary hits in triplicate and dose response

− Filter assays

Fluorescence

Luminescence

Cytotoxicity


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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.


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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)


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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


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Biochemical Assays

− HTRF assays

Can be used be measure protein-protein interactions


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Biochemical Assays

− HTRF assays

Can also be used to detect products of a reaction of interest


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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


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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


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Biochemical Assays

− Alpha screens (Amplified Luminescence Proximity

Homogeneous Assay)


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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


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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


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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)


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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


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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


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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)


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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


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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.)


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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.)


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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)


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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


(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


Lyssiotis et al. (2009) PNAS. 106: 8912.

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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)


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)


Boitano et al. (2010) Science. 329: 1345.

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Results: SR1 induces robust in vitro expansion of HSCs that engraft into mice. Result has translated to the clinic.



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Results: Aryl hydrocarbon receptor (AhR) identified as target from global expression profiling (rare case where this works)



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).

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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)


Zhu et al. (2009) Cell Stem Cell. 4: 416.

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Results: Stauprimide induces robust differentiation of human and mouse ESCs towards not only endoderm, but also towards ectoderm and mesoderm lineages



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Results: Affinity-probe used to identify NME2 as the specific protein target. Interaction with NME2 leads to decreased expression of c-Myc.



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Results: Affinity-probe used to identify NME2 as the specific protein target. Interaction with NME2 leads to decreased expression of c-Myc.



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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.


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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


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Deshmukh et al. (2013) Nature. Oct 17;502(7471):327-32


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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


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Vehicle Benztropine


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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


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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)


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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


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MBP, β-actin

2 Scaffolds selected for preclinical development Preliminary medchem studies indicate substantial improvements in

potency and efficacy are achievable relative to benztropine


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• 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

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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


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P/Map2-luciferase

P/Map2 FF luc



StanAs

Astrocytes


136 “selectively” toxic hits


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P/Map2-luciferase

P/Map2 FF luc



StanAs

Astrocytes


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


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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


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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


Lecture 11 High Throughput Screening.pdf

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