High-throughput screening Technology

• Commonly used terms in drug discovery

• High throughput screen: an optimised, miniaturised assay format that

enables the testing of > 100,000 chemically diverse compounds per day.

• Assay: a test system in which biological activity can be detected

• Hit: a molecule with confirmed concentration-dependent activity in a

screen, and known chemical structure. The output of most screens

• Progressible hit: a representative of a compound series with activity

via acceptable mechanism of action and some limited structure-activity
relationship information

• Lead: a compound with potential (as measured by potency, selectivity,

physico-chemical properties, absence of toxicity or novelty) to progress
to a full drug development programme
Drug Discovery Process:
Drug Discovery Process:
•The key steps of drug discovery are:
•research - average 2 to 3 years
•pre-clinical testing - average 1 year
•clinical trial testing (involving human
patients) - average 10 years
•regulatory approval - average 2 years
What is High throughput Screening ?

High throughput screening (HTS) is a tool for early-stage

drug discovery.
• Definition : HTS is process by which large number of
compounds are rapidly tested for their ability to modify the
properties of a selected biological target.
Isolation of active components
History

• HTS was invented by Dr Gyula Takatsky in 1951, who

machined 6 rows of 12 wells in Lucite to make the first
microtiter plate.

• The microtiter plate has further grown to include

standardized 96, 384, 1536 well formats, with additional
3072 well nanoplate formats.

• “Twenty 384-well plates are currently run daily on the Accuri

C6 HyperCytometer combination. We could run up to 40
plates in a standard 8 hour workday, over 12,000 compounds.
• High throughput screening for drug discovery
• Why High throughput screening need arises ?
• FACT 1: recent understanding of disease mechanisms has
dramatically increased no. of protein targets for new drug
treatment
• FACT 2: new technologies have increased the no. of drugs that
can be tested for activity at these targets.
Goal of HTS

Goal is to identify ‘hits’ or ‘leads’

• - affect target in desired manner
• active at fairly low concenteration ( more likely to show
specificity)
• - new structure
• The greater the number and diversity of compounds
screened, the more successful screen is likely to be.
• HTS = 50,000-100,000 compounds screened per day!!!
The majority of drug targets are :

• a) G-protein coupled receptors

• b) nuclear receptors
• c) ion channels
• d) enzymes
Explanation
• High-throughput screening is a method for scientific experimentation especially
used in drug discovery and is relevant to biology and chemistry. This process in
combination with robotics, data processing and control software, liquid handling
devices and sensitive detectors allows a researcher to quickly conduct millions of
chemical, genetic or pharmacological tests.
• High-throughput screening can rapidly identify active compounds, antibodies or
genes which modulate a particular bimolecular pathway. It can be considered - a
process in which batches of compounds are tested for binding activity or biological
activity against target molecules.
• High-throughput screening is a process of screening more compounds against more
targets per unit time, which should generate more hits, which in turn will generate
more leads, subsequently generating more products.
• Various technologies like high-throughput screening defined by the number of
compounds tested to be in the range of 10,000-100,000 per day, ultra high-
throughput screening is defined by screening more than 100,000 data point
generated per day. These two technologies play a vital role in drug discovery to find
new chemical compounds.
Which strategy is best for hit identification?

When a target is identified, a decision has to be made about which

chemicals to screen, in order to identify potential lead compounds.

Random screening :
All possible drug molecules screened against target.
This is simply not possible.
Focussed screening :
A limited number of compounds are pre-selected for screening.
Has proved successful as a hit generation strategy.
Useful when 3D structure of target is known (e.g. crystal structure of a
receptor)
Procedure
High-throughput screening in drug discovery is used to screen :
• Novel biological active compounds
• Natural products
• Combinatorial libraries (Ex: peptides; chemicals)
• Biological libraries
• DNA chips
• RNA chips
• Protein chips
• High-throughput screening’s main lab ware is the microtiter plate.
Modern microplates for high-throughput screening assays are
performed in automation-friendly microtiter plates with a 96, 384,
1536 or 3456 well format. These wells contain experimentally useful
matter, often an aqueous solution of dimethyl sulfoxide (DMSO).
• For most drug discovery labs, the library collection has grown from
400,000 to 1 million or more compounds. The standard paradigms
used to screen these libraries have evolved to automated 384 wells or
higher density single compound test formats.

• Primary screen is designed to rapidly identify hits from compound

libraries. The goals are to minimize the number of false positives and
maximize the number of confirmed hits.

• Depending on the assay, hit rates typically range between 0.1 – 5 per
cent. This number also depends on the cutoff parameters set by the
researchers, as well as the dynamic range of a given assay.

• Primary screens are run in multiplets of single compound

concentrations. Hits are then retested, usually independently from the
first assay.
• If a compound exhibits the same activity, it is coined as confirmed hit,
which proceeds to secondary screens or lead optimization. The results
from lead optimization are used to decide which substances will make
it on to clinical trials.

• The key to high-throughput screening is to develop a test, or assay, in

which binding between a compound and a protein causes some visible
change that can be automatically read by a sensor. Typically the change
is emission of light by a fluorophore in the reaction mixture.

• One way to make this occur is to attach the fluorophore to the target
protein in such a way that its ability to fluoresce is diminished
(quenched) when the protein binds to another molecule. A different
system measures the difference in a particular property of light
(polarization) emitted by bound versus unbound fluorophores. Bound
fluorophores are more highly polarized and this can be detected by
sensors.
•

•
Applications :
• High-throughput technology can also be put to use in
other areas besides drug development.
• Genomics Applications
• DNA Sequencing
• Protein Analysis
Applications