High-throughput screening process are used by today most of the drug discovery industries, this process helps pharmaceutical researches to make drug discovery process faster and also increase the quality and quantity of drugs production. 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

1. ArticleWritten and published at www.worldofchmeicals.com

2.  High-Throughput Screening (HTS) is an approach to drug discovery that
has gained widespread popularity over the past few years and expanded
its applications for the pharmaceutical and biotechnology companies,
university research laboratories et al.
 The technology includes screening of large chemical libraries for activity
against biological targets via the use of automation, miniaturized assays
and large-scale data analysis.
 Since its first advent in the early to mid 1990s, the field of HTS has seen
not only a continuous change in technology and processes, but has gone
through various adaptations to suit industry needs which have given an
edge to drug discovery.
 HTS has now evolved into a mature discipline that is a crucial for the end
to end manufacturing of drugs, namely from the raw material that is the
chemical to the actual drug formation.

3.  In recent years, the industry has witnessed a clear trend in drug
discovery toward rigorous hit validation by the use of orthogonal
readout technologies, label free and biophysical methodologies.
 Today, many experts in the field see HTS at a crossroad with the
need to decide on either higher throughput/more experimentation
or a greater focus on assays of greater physiological relevance,
both of which may lead to higher productivity in pharmaceutical
research & development.
 The aim of high-throughput screening is to accelerate drug
discovery by screening large libraries often composed of hundreds
of thousands of compounds (drug candidates) at a rate that may
exceed 20,000 compounds per week.The following article takes a
look at the evolving technology and applications of the HTS
process.

4.  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.

5.  HTS was invented by Dr GyulaTakatsky 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 available for specialized or
quantitative reverse-transcription polymerase chain reaction assays.
 “Twenty 384-well plates are currently run daily on the AccuriC6 HyperCyt
combination.We could run up to 40 plates in a standard 8 hour workday, over
12,000 compounds”

6.  High-throughput screening in drug discovery
 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) and some other chemical compound, the latter of which
is different for each well across the plate.

7.  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.
Results are expressed as percent activity in comparison to a positive (100
per cent) and a negative (0 per cent) control. Hits are then retested,
usually independently from the first assay.

8.  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.

9.  Detection technologies used in high throughput screening include
time-resolved fluorescence (TR-FRET), fluorescence resonance
energy transfer (FRET), fluorescence polarization, luminescence
and absorbance and require sensitive and versatile multi-mode
microplate readers.
 ProcedureThe 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

10.  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.

11.  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

12.  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, picked out
by micromanipulation and the product
structure, if a peptide, determined by
sequencing on the bead. Non-peptide
structures would need to be identified by one
of the tagging methods.

13.  High-throughput technology can also be put
to use in other areas besides drug
development.
 GenomicsApplications
 DNA Sequencing
 Protein Analysis

14.  Maxim Pharmaceuticals’ HTS laboratory operates a working whole-cell based
screening platform.Their purpose is to screen for inhibitors and inducers of
apoptosis or programmed cell death.The medical justification for inhibitors is
their possible use in preventing unwanted cell death often seen in heart attacks,
strokes and degenerative diseases such as Alzheimer’s and Parkinson’s Disease.
The justification for inducers is primarily to kill cancer cells or cells that do not
have the normal cell death machinery.
 Maxim Pharmaceuticals uses a proprietary substrate that is specific for activated
caspase.The screens are performed in 384 well formats and are whole cell based.
Activated caspase indicates cell death through apoptosis specifically, not just cell
death by necrosis.They screen compound libraries against numerous cancer cell
lines.
 The research flow at Maxim is typical of cell based screening elsewhere. It starts
with compound inventory and solubilization, compound transfer and plate
replication, cell addition, incubation, plate reading, and finally data analysis.

15.  At Maxim the laboratory layout is a combination of track and robotic arm systems. Plates usually
get reagent added with a small volume 96 channel pipettor, compound is added with a 384
pintool head, cells are added with a large volume 96 channel pipettor (all these steps are linked to
stackers, hence this portion is considered a linear setup).
 The plates are manually loaded into an automated incubator. Once the incubation period is
finished, the plates are moved with a robotic arm from the incubator to a large volume 96
channel pipettor, substrate is added, and the plates are moved to a fluorescent plate reader,
stopping by a barcode reader on the way to keep track of what data corresponds to what
compound.The barcodes are then read and the plates are moved back (by the robotic arm) to the
incubator for further incubation.
 Once done incubating a second time, the plates are moved by the arm from the incubator to the
scanner, then to the plate reader for a final read. Once they are done with the final read, they are
essentially trash. A plate sealer has been integrated with the arm so these plates are sealed for
splash-free disposal into a hazardous waste barrel. Once the assay run is complete the robotic
arm uses the pager function to call one of the employees to let them know the assay run has
finished without problems.
 Data analysis is done for the primary screens using a software package to handle the massive
quantities of data generated.The HTS laboratory at Maxim also handles all of the secondary
screens such as Dose Response and Growth Inhibition.The dilutions for these assays are done off-
line using 8 channel pipettors and then the integrated automation is used for the actual assay.
Data from these smaller assays is analyzed using templates in Excel.

16.  HTS has revolutionized compound screening. However, it
has also introduced new challenges to the drug discovery
and development process.
 Just a small fraction of primary hits generated by HTS can
easily overwhelm traditional follow-up testing, therefore
creating new bottlenecks in the drug discovery process.
 Thus, it is now time to explore the application of the high
throughout technologies to those new bottlenecks in the
areas of medicinal chemistry, structural biology,
pharmacology, ADME (Absorption, Distribution,
Metabolism and Excretion) studies and toxicology.

17.  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.

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An Overview, Journal of Applied Pharmaceutical Science 2010, 1 (1), 02-10; Available from-
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compound identification, International Journal of HighThroughput Screening, August 2010Volume 2010:1;
Pages 141 -149- Dove Medical Press Ltd-http://www.dovepress.com/articles.php?article_id=4965
 [4] Paper by Eric Allen, Candace Crogan-Grundy, Jessica Rivers,Tatyana Nisan and Shaoxian Su on “High
throughput screening in drug discovery.” Source for Maxim Pharmaceuticals case study-
http://www.esallen.com/professional/papers_presentations/bioengineering/HTS_paper_draft.pdf
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 To contact the author email: articles@worldofchemicals.com
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