High Throughput Screening

Dr Ranjita Santra (Dhali)Assistant ProfessorDepartment of Clinical & Experimental PharmacologySTM, Kolkata

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 targetSource: Velpandian T, Sharma HP. In: SK Gupta, editor. Drug Sreening Mrthods. 3rd edn. New Delhi :The Health Sciences;2016

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]

Goals2. Identify hits serve as starting points for: • Drug discovery programs

• Medicinal chemistry • Pharmacology

• Chemical biology programs • Target identification • Target validation and mechanism of action

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

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

General Steps

• Target identification and purification

• Target validation

• Assay development (screening of potential compounds against target identified)

• High-throughput library (HTL) screening

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

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

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)

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)

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

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

• Real Time PCR

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

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

Thermal Shift Analysis for HTS Assessment of:1.Role of inhibitor in the binding affinity of

ligand with protein2.Stability of the protein at various pH and salt

conditions3.Ligand screening for lead identificationExamples: Identification of CA enzyme inhibitors in glaucoma/AD/PD/Epilepsy) ER antagonists(Toremefine,tamoxifen) in CryptococcalinfectionsMAP2K4 inhibitor in CA Prostate

Cell based assay

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

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

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)

Others

• HTS PK Studies• HTS-Absorption studies• HTS-Metabolism studies• HTS-CYP Inhibition and Induction studies

• HTS Toxicity studies

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

Data Acquisition Programs

• Programs constructed using LabView• Kaleidagraph

• Synergy Software• Programs constructed using MatLab

• The Mathworks Inc.

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

Schematic of The Combinatorial Factory

Advantages of Combi Chem and High-Throughput Methods

• Automated, reliable processes• Quantitative analysis• Comprehensive database

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

Thank you!Questions or

comments?