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

                                                                                                                                                                                 

| 13

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.

| 15

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)

| 16

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.

| 17

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

| 18

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

| 19

Biochemical Assays

Alpha screens (Amplified Luminescence Proximity

Homogeneous Assay)

| 20

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

| 21

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

| 22

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.

| 25

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

| 26

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)

| 27

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

| 28

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.)

| 29

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.)

| 34

Phenotypic assays (Flow cytometry-based) Good for suspension cells

Multi-parametric, obtain lots of information (up to 9-13

channels, cell cycle, cell health)

| 35

~ 1 Billion THP-1Cells

3 days

Cell dispense and drug treatment

3 days

Fixation, Staining and Wash

Acquisition Analysis

THANK YOU...