new high thoughput screening copy
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High Throughput Screening Assay
By,Manik Bainwad,Mohanlal
HIGH THROUGHPUT SCREENING (HTS)
HIGH THROUGHPUT SCREENING (HTS) is identification of one or more positive candidates extracted from a pool of possible candidates based on specific criteria
It is a drug-discovery process widely used in the pharmaceutical industry
It allows automation to quickly assay the biological or biochemical activity of a large number of compounds
HTS is process by which large nos. of compounds are rapidly tested for their ability to modify the properties of a selected biological target.
Goal is to identify ‘hits’ or ‘leads’
- affect target in desired manner
- active at fairly low concentrations ( more likely to show specificity)
- new structure
It is a useful for discovering ligands for receptors, enzymes, ion-channels or other pharmacological targets, or pharmacologically profiling a cellular or biochemical pathway of interest
DETECTION METHODS IN HTS:• Spectroscopy• Mass Spectrometry• Chromatography• Calorimetry• X-ray diffraction• Microscopy• Radioactive methods
SPECTROSCOPY IN HTS: • Fluorescence Spectroscopy • Total internal reflection fluorescence (TIRF)• Nuclear magnetic resonance (NMR) • Absorption and luminescence • Fourier transformed infrared(FTIR) • Light scattering
CHROMATOGRAPHY IN HTS:•Gas chromatography (GC)•Thin layer chromatography•Liquid chromatography (HPLC)•Ion Exchange chromatography•Reverse phase chromatography•Hydrophobic interaction chromatography•Affinity chromatography
CALORIMETRY IN HTS:• Isothermal titration Calorimetry (ITC)• Differential scanning Calorimetry (DSC)
MICROSCOPY IN HTS:• Scanning Tunnelling Microscopy• Atomic Force Microscopy• Confocal Microscopy
Uses:To screen Micro arrays such as: • DNA chips • RNA chips • Protein chips• To screen for all kind of novel biological active compounds • Natural products
Methodology The heart of the HTS system is a plate, or tray, which
consists of tiny wells where assay reagents and samples are deposited, and their reactions monitored.
The configuration of the plate has changed from 96 wells (in a matrix of 8 rows by 12 columns) to 384, and now to a high - density 1536 - well format, which enables large - scale screening.
Assay reagents may be coated onto the plates or deposited in liquid form together with test samples into the wells.
Both samples and assay reagents may be incubated, and those that interact show signals, which can be detected.
The aim of HTS and UHTS is cost effectiveness and speed of compound scanning
Cell - based assays have become an important test compared with other in vitro assays, as they can provide information about bioavailability, cytotoxicity and effects on biochemical pathway
The enzyme - based and cell - based assay systems consist of receptors or mimetics of receptors (components that mimic active parts of receptors)
Normally the assays are linked to an indicator that shows the ligands – receptor interaction as some form of signal
The advantage of cell - based assays over biochemical assays is that cell - based assays enable the analysis of sample compound activity in an environment that is similar to the one in which a drug would act
It also provides a platform for toxicity studies.
•Home•About NanoCenter•Research•Publications•NanoCenter Labs•People•Collaborators•Industrial Partners•Outreach Activities•News•Opportunities•Contact Us•Home > Center for Nanotechnology and Nanotoxicology > Research > A High Throughput Nanogenotoxicity Assay
A High Throughput Nanogenotoxicity Assay Research Area: NanogenotoxicityFunding Agencies: NIEHS Center at HSPH
•Home•About NanoCenter•Research•Publications•NanoCenter Labs•People•Collaborators•Industrial Partners•Outreach Activities•News•Opportunities•Contact Us•Home > Center for Nanotechnology and Nanotoxicology > Research > A High Throughput Nanogenotoxicity Assay
A High Throughput Nanogenotoxicity Assay Research Area: NanogenotoxicityFunding Agencies: NIEHS Center at HSPH
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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
Biochemical Assay: – These screens use purified proteins, substrates and small compound inhibitors in buffered solutions to produce an optical (fluorescent, luminescent, etc.) readout that monitors enzymatic or binding activity using high throughput platereaders. Small compound inhibitors are ranked on their ability to reduce the protein function, and by extension the optical signal, in this type of screen.
Advantage: Very high throughput, small volume reactions reduce reagent costs and simple readouts. Target of the inhibitor is defined.
Disadvantage: Small compound may not be water soluble, membrane permeable and may be promiscuous or toxic. Single protein inhibition may not cause desired phenotypic change in cells or tissues due to redundant pathways.
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Biochemical AssaysEnzyme 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 (Homogeneous Time-Resolved Fluorescence) 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 Can also be used to detect products of a reaction of interest. Can be used be measure protein-protein interactions.
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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.
These screens small compound that utilize cells plated in 96 or 384 well
plates to produce a visual phenotypic change in the cells which can be
measured.
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)
The three general measurement types :
1. Uniform well readouts These include cell viability assays. These assays usually employ high throughput platereaders to produce their measurements.
2. High-Content Imaging Screens These include small compound as well as RNAi screens and are designed to probe changes to a cellular phenotype (i.e. foci formation screens, nuclear and cellular morphology, localization of proteins, etc). HCI screens employ specialized high content imagers to produce high content pictures which can be used to measure phenotypic changes. They can also provide cell cycle information.
3. Reporter gene systems – These are mostly high throughput FACS (Fluorescence-activated cell sorting) based assays. They employ high throughput FACS to produce readouts of GFP, luciferase, etc. internalized signal.
Advantages: Cell based assays produce phenotypic changes affecting pathways directly associated with disease states and the target protein does not have to be known.
Small compound cell based screens ensure compound solubility, membrane permeability, non-toxic and effectiveness at low therapeutically relevant concentrations.
Disadvantages: Lower throughput than protein based assays, much more technically difficult and much longer duration (days/weeks vs. minutes). Target of inhibitors are not definitively known.
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1) 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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2) 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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3) 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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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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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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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-1Cells
3 days
Cell dispense and drug treatment
3 days
Fixation, Staining and Wash
Acquisition Analysis
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