High Throughput Screening (HTS) involves the automated screening of large libraries of chemical compounds against biological targets at a rapid pace. HTS uses robotics, miniaturization, and automation to screen 50,000-100,000 compounds per week. The goals of HTS are to identify compounds that selectively bind and modulate biological targets of interest. Hits identified through primary screening are then evaluated further through secondary assays, SAR analysis, and in vitro/in vivo studies. HTS accelerates drug discovery by allowing for the rapid evaluation of large compound libraries.

1. High Throughput Screening
Dr Ranjita Santra (Dhali)
Assistant Professor
Department of Clinical & Experimental Pharmacology
STM, Kolkata

2. What is High Throughput Screening (HTS) ?
• The last two decades have seen astonishing
innovations in technology that have helped the
manual low speed screening to evolve into an
automated, microprocessor controlled robotic
process called High Throughput Screening (HTS)
• HTS - A synergy of chemistry, biology,
engineering and informatics
• Screening of over 50,000-1,00,000
compounds/week
• Screening against the validated biological target
Source: Velpandian T, Sharma HP. In: SK Gupta, editor. Drug Sreening Mrthods. 3rd
edn. New Delhi :The Health Sciences;2016

3. Goals
1. Identify a molecular structure that:
• Selectively binds to and modulates the
activity of a biological target (e.g., a
protein) of interest [target-based]
• Selectively induces a desired
phenotype in a cell population or
organism of interest [phenotype-based]

4. Goals
2. Identify hits serve as starting points
for:
• Drug discovery programs
• Medicinal chemistry
• Pharmacology
• Chemical biology programs
• Target identification
• Target validation and mechanism of action

5. Applications of HTS
• Drug discovery
• Toxicology (in silico toxicology methods)
• Screening of different types of libraries
• Combinatorial Chemistry
• Genomics
• Protein libraries (Antibodies, secreted
soluble protiens)
• Peptide libraries

6. HTS in Drug Discovery
• Accelerates drug discovery by screening large
compound libraries at a rate that may exceed a
few thousand compounds per day or per week
• Parallel and combinatorial chemical synthesis
generates a vast number of novel compounds
• Characterization of metabolic, pharmacokinetic
and toxicological data about new drugs

7. General Steps
• Target identification and purification
• Target validation
• Assay development (screening of potential
compounds against target identified)
• High-throughput library (HTL) screening

8. General Steps
• Lead optimization
• Confirmation of ‘hits’
Hit = A molecule with confirmed activity from primary
HTS assay with a good profile in secondary assays
and with confirmed structure
Lead = Hit series exhibiting the Structure Activity
Relationship(SAR) and demonstrating activities
both in vitro and in vivo

9. The Array
• Polycarbonate base
film, 0.5-mm thick
• 6x8 array of
coatings
• each sample is 2-5-
μm thick and 10-mm
in diameter
• Flexible silicon
rubber template to
create wells

10. In Vitro interaction studies
• Chemical microarray
• Chemical -protein interactions
• Enzyme activity inhibition
• Target identification
• Signal pathway elucidation
• Chip based microarray
• Microfluidics helps in mixing of samples
with the targets bound in the chip
• Final signals read by analyzers
(Fluoroscent/simple UV/visible signals)

11. Other microarrays
• Cytokine based kits
• Angiogenesis responsive biomolecular kits
• Transcription factor analysis kits
Common principle: Biological samples bind to
the Abs bound to the array membrane,
incubation, binding to the secondary Abs and
final tagging of secondary Abs with the signal
emitting molecules (e.g. dyes)

12. Fluorescence tecniques in HTS
• Fluorescence (FU) based detection of the compounds,
analytes or genetic materials
• Luminescence-based binding assays - core of
interaction of ligand with the biological compartment
• Detection systems - Multimode readers(screening a
single reaction well for different modes e.g. UV, visible
to FU etc.)
• FU microscopes - Identification:
• Entry of fluorescent tagged compound at desired site
• Drug effect on a biomolecule / cellular component

13. Fluorescent techniques in drug
discovery
• Fluorescence Anisotropy(FA)/
Fluorescence Polarization(FP)
• Time Resolved FA (TRFA)
• Fluorescence Correlation
Spectroscopy(FCS)
• Fluorescence Resonance Energy
Transfer(FRET)
• TR-FRET
• Fluorescence microscopy

14. Areas of Application
• Receptor-ligand binding assay
• Screening of chemical compounds
• Drug -receptor, drug-enzyme interaction
• Tissue structural analysis
• Gene expression analysis
• Cellular component analysis

15. Thermal Shift Analysis for HTS
• Identifies ligand interaction with the
proteins
• Fluorescent dye (e.g. sypro orange)
associates and dissociates from the
protein with change in the temperature of
the reaction
• Principle: Binding of ligand to protein,
stabilizing or destabilizing its structure,
final change in the Tm of the protein

16. Thermal Shift Analysis for HTS
Assessment of:
1.Role of inhibitor in the binding affinity of
ligand with protein
2.Stability of the protein at various pH and salt
conditions
3.Ligand screening for lead identification
Examples: Identification of CA enzyme inhibitors in
glaucoma/AD/PD/Epilepsy)
ER antagonists(Toremefine,tamoxifen) in Cryptococcal
infections
MAP2K4 inhibitor in CA Prostate

17. Cell based assay

18. In Vivo Techniques
• Vascular mapping
• Tissue perfusion studies
• Imaging vasculature of brain
• Angiograms of eye
• GIT imaging using endoscopic optics
• Imaging tumours, atherosclerosis,
apoptosis, osteoblastosis

19. Virtual screening/In Silico drug
development
• Indirect approach using advanced
computer technology to screen newer
compounds based on virtual
coordinates of receptors and signals by
CAMD
• 3D molecular structure of the receptor
and the ligands for docking
• For lead optimization

20. Unconventional rapid pharmacological
and toxicity testing models
• Zebrafish (angiogenic and anti-
angiogenic effects, heart regeneration)
and Gold fish
• Chick (Anti-cataract activity on lens)
• Nematode C. elegans (growth
retardation with caffeine)

21. Others
• HTS PK Studies
• HTS-Absorption studies
• HTS-Metabolism studies
• HTS-CYP Inhibition and Induction studies
• HTS Toxicity studies

22. HTS in the analysis of toxins
• Identification of Marine toxins :
Palytoxins, ciguatoxins, cyclic amines
and tetrodotoxins
• Concern for sea food and sea water
• Steps by EC and EFSA in this regard

23. Data Acquisition Programs
• Programs constructed using LabView
• Kaleidagraph
• Synergy Software
• Programs constructed using MatLab
• The Mathworks Inc.

24. Combinatorial Chemistry
• Miniaturization and automation yield high-
throughput experimentation
• Decreased sample size
• Processes almost completely automated
• Quantitative analysis of data
• All steps linked to central database
• Self-updating processes
• Easy to use
• Reliable and versatile
• Combinatorial factory: Larger scale testing to
meet industry specifications

25. Schematic of The Combinatorial
Factory

26. Advantages of Combi Chem and
High-Throughput Methods
• Automated, reliable processes
• Quantitative analysis
• Comprehensive database

27. Advantages of Combi Chem and
High-Throughput Methods
• Automated, reliable processes
• Quantitative analysis
• Comprehensive database
• Test more samples in shorter period of
time
• Minimize cost per sample
• Faster

28. Thank you!
Questions or
comments?