High throughput screening (HTS) is a widely adopted method in drug discovery that automates the testing of large numbers of compounds to identify potential lead candidates based on various biological activities. It has evolved since the early 1990s from a focus on quantity to an emphasis on quality and specificity, utilizing advanced technologies and equipment to improve screening efficiency and data analysis. Despite challenges in cost and data quality, HTS remains a central and evolving approach in the search for novel drug entities.

1. HIGH THROUGHPUT SCREENING
FOR LEAD DISCOVERY
Ajeesh. V
Department of Pharmacy
School of chemical sciences and pharmacy
Central University of Rajasthan

2. Introduction
 A lead compound is a representative of a compound series with sufficient
potential (as measured by potency, selectivity, pharmacokinetics,
physicochemical properties, toxicity and novelty) to progress to a full drug
development programme.
 High Throughput Screening (HTS) is a method that uses automation and
large data set processing to quickly assay the biological or biochemical
activity of large numbers of compounds, proteins or genes.
 HTS is an approach to drug discovery that has gained widespread
popularity over the last two decades and has become a standard method
for drug discovery in the pharmaceutical industry.

3.  Usually first step in Drug discovery
 It helps to rapidly identify active compounds, antibodies, or genes that
modulate a particular biomolecular pathway.
 HTS assays are used for screening of different types of libraries,
including combinatorial chemistry, genomics, protein, and peptide
libraries.
 It is basically a process of screening and assaying a large number of
biological modulators and effectors against selected and specific
targets.

4. Screening Collection
Screening libraries
(a) Diversity Libraries
(b) Focused Libraries
Ligand-Based Approaches
Structure-Based Approaches
Compound acquisition
Purchasing commercially available compounds is an
important aspect of screening collection enhancement

5. Libraries
 siRNA libraries
providing the best available RNA interference technology with
maximum flexibility
The mouse whole genome contain about 16872 gene targets and the
Human whole-genome contains 18120 gene targets
 microRNA libraries
Human libraries of synthetic microRNA mimics and microRNA inhibitors
 Small Compound libraries
A library of 640 FDA approved drugs (Screen-Well FDA Approved Drug
Library, Enzo Life Sciences) is also available for screening.
The library contains clinically-relevant pharmacophores.

6. Steps in HTS
1st stage screening
 Test optical clarity, abrasion resistance, and adhesion
 Eliminates ~ 90% of samples
2nd stage screening
 Test weather ability, integrity, gloss, and surface smoothness
 ~10% of the samples
Rapidly identify coating samples with desired properties
Candidates for scale up
 Test according to the customer’s specifications

7. Screening Strategies
 Mixtures versus Single Compound Screening
In the early 1990s, the strategy adopted was to synthesize and test compounds
as mixtures
 Full Deck Screening
All compounds in the screening collection are subjected to HTS.
 Focused Screening
Only a subset of the compound collection would be screened. The selection
method could be based on structural diversity, target knowledge, or a
combination of the two.
 Sequential Screening
This consists of either an initial diversity-based or focused screen, followed by
one or more subsequent screens enriched with analogues of the hits.

8. Anatomy of HTS
 It contains
1. Robotic handling systems
2. Liquid handling systems
3. Sensor systems and
4. Control software
 Microplates, each of which consists of an array of wells, are
moved through the system by robotic handling. The wells in the
microplates are filled via the liquid handling systems, and sensors
are used to evaluate the samples in the microplate.
 Control software choreographs the entire process, ensuring
accuracy within the process and repeatability between processes.

9. Equipment
Hamilton STARlet Liquid Handling Workstation
PerkinElmer Envision Multimode Plate Reader
Molecular Devices ImageXpress Micro High-Content
Screening Microscope

10. The introduction of 96-well plates and reduced assay
volumes (50–100 mL) increased screening throughput
from 50 to 7200 compounds per week by end of 1980s
Then 384-well-plate format, enabling screening
capacities of 10–100,000 compounds per day
Recently 1536-well format provides throughput of over
100,000 compounds per day (ultra-HTS)
Latest is 3456-well microplates with a total assay
volume of 1–2 mL per well

11. Detection Methods in HTS
Spectroscopy
Mass Spectrometry
Chromatography
Calorimetry
X-ray diffraction
Microscopy
Radioactive methods
Chromatography
Gas chromatography (GC)
Thin layer chromatography
HPLC
1st stage
•100-200 samples per day
2nd stage
•~10% of the samples
Rapidly identified materials
Candidates for scale up
1st stage
•100-200 samples per day
2nd stage
•~10% of the samples
Rapidly identified materials
Candidates for scale up

12. Ion Exchange chromatography
Reverse phase chromatography
Hydrophobic interaction chromatography
Affinity chromatography
Isothermal titration Calorimetry (ITC)
Differential scanning Calorimetry (DSC)
Isothermal titration Calorimetry (ITC)
Differential scanning Calorimetry (DSC)
Scanning Tunnelling Microscopy
Atomic Force Microscopy
Confocal Microscopy

13. Data analysis
Software's used for data analysis
MetaXpress, including 10 application
modules (Molecular Devices)
AcuityXpress (Molecular Devices)
cellHTS2 (Bioconductor, open-source)
RNAither (Bioconductor, open-source)
CellProfiler (Broad Institute)
Advanced Cell Classifier (ETH Zurich)
Ingenuity Pathway Analysis (Ingenuity Systems)

14. Advantages of HTS
High sensitivity of assay (single molecule detection)
High speed of assay (automation)
Minimization of assay (microtiter plate assay)
Low background signal
Clear message

15. Disadvantages
High cost
Low data quality
Local contamination
Need of relatively pure product

16. Newer method in HTS
High-Throughput, Fluorescence-Based Screening
(a) For screening, expressed proteins are labelled either as
fusions with green fluorescent protein (GFP) or through
translational incorporation of a fluorescent amino acid
derivative, BODIPY-FL-Lysine.
(b) Using fluorescence detection, the entire procedure can
be carried out in approximately 8 h.

17. Applications
In drug discovery
Systematic Study of Mitochondrial Toxicity of
Environmental Chemicals
High-Throughput Screening of Dendrimer-Binding
Drugs
To Fractionate Plant Natural Products for Drug
Discovery

18. Summary
 Since its beginnings in the early 1990s, lead discovery by HTS has
evolved into a mature scientific discipline. Early HTS strategies based on
compound numbers alone have been firmly replaced by an emphasis on
quality and scientific rationale
 The number of HTS-derived drugs has been steadily increasing in recent
years.
 Despite the current limitations and the fact that the overall success of
the HTS paradigm is still debatable, there is no doubt that this is the
most widely applicable lead discovery approach capable of providing a
wide choice of novel chemical entities for a broad range of biological
targets.
 HTS is likely to retain a central role in drug discovery and continue to
provide a focus for major scientific and technological efforts for many
years to come.

19. References
 Text book of Lead Generation Approaches In Drug Discovery
by Zoran Rankovic and Richard Morphy
 Text book of High-Throughput Screening in Drug Discovery
R. Mannhold, H. Kubinyi, and G. Folkers
 Leach AR, Hann MM. The in silico world of virtual libraries.
Drug Discov Today 2000; 5:326–336.
 J. AM. CHEM. SOC. 2010, 132, 13182–13184
 Journal of Natural Products, 2010, Vol. 73, No. 4 753

20. Thank you!