GENERALLY

1. By :Waqar Ahmad Bamkhelvi
Session: 2011-2016
University Of Swabi
00923015820152
High-throughput
screening Technology

2. •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

4. Drug Discovery Process:

6. 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

7. 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.

9. 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.

13. •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.

14. 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!!!

15. The majority of drug targets are :
• a) G-protein coupled receptors
• b) nuclear receptors
• c) ion channels
• d) enzymes

19. 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.

23. 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)

24. 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).

25. • 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.

26. • 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.
• In combination with bioinformatics, it allows potential drugs to be quickly and
efficiently screened to find candidates that should be explored in more detail.
Initial screening of these compounds for their binding ability is the job for high-
throughput screening.
• 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.

27. •
•

28. Procedure
The sets of compounds produced by combinatorial chemistry are
generally referred to as libraries, which depending on how the solid-
phase is handled, may be either mixtures or individual compounds.
There are a range of options for testing the libraries in a biological
assay.
Test mixture in solution
Test individual compounds in solution
Test compounds on the beads

29. Test mixture in solution :
• All the compounds are cleaved from the beads and tested in solution.
• If activity in a pharmacological screen is observed, it
is difficult to find out which compounds are active. To identify the
most active component, it is necessary to resynthesize the
compounds individually and thereby find the most potent. This
iterative process of resynthesis and screening is one of the most
simple and successful methods for identifying active compounds
from libraries.

30. Test individual compounds in solution
• A second method is to separate the beads manually into individual
wells and cleave the compounds from the solid-phase. These
compounds can now be tested as individual entities.
•Test compounds on the beads :
• A third method for screening is testing on the beads, using a
colorimetric or fluorescent assay technique. If there are active
compounds, the appropriate beads can be selected by color or
fluorescence.

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

34. Applications

35. Conclusion :
• HTS is became an effective technique and competitive
with the latest, upcoming related technologies in the market. The
growing importance of this process is cost effectiveness of drug-
discovery and development, operating processes for development of
homogeneous, fluorescence-based assays in reduced formats.
• The usage of 384, 1536 and 3456 wells density plates and robotics
made the HTS process through which compounds can be screened
more than 100,000 data points per day.
• The number of higher density plates used in the drug-discovery
process is inversely proportional to the samples required for the
process; thereby it reducing the initial setup costs.
• The combination with robotics, data processing and control software,
liquid handling devices, TR-FRET, FRET, Fluorescence polarization
techniques has added a significant valued to each data point
generated by high throughput screens.

36. •Thanks For All Of
You.