M I C R O P L AT E A S S AY S : T H E K E Y P L AT F O R M F O R H I G H T H R O U G H P U T S C R E E N I N G I N B I O M E D I C A L R E S E A R C H A N D P H A R M A C O L O G I C A L

Microtiter Plate

• develop in 1990s

• 2:3 rectangle matrix with wells

• 6, 24, 96, 384, 1536 wells

• used for Enzyme-linked Immunosorbent Assay, High Throughput Screening, storage

• Flat-shaped, U-shaped, V-shaped

Well shape

ELISA

Microplate Reader

taken from Synergy

Reusing plate

taken from Labotal Scientific Equipment (1997) LTD

taken from © 2014 Kinesis Ltd.

Materials

• polyprolyene- for wide change in temperature environment

• polycrabonate- disposable,

• Cyclo-olefins

High Throughput Screening

an optimized, miniaturized assay format that enables the testing of > 100,000 chemically diverse compounds per day.

Biotechnology Research Institute The Hong Kong University of Science and Technology

• TCMs, including those for treatment of Alzheimer’s disease, neuro-protection, stroke, and sleep disorders

taken from RNAi @ NIH Chemical Genomics Center

History of Developing a Drug!

Drug discovery

Initial characterisation

Pre-clinical trials

Regulatory approval sought to start trials in humans

Clinical trials Phases I, II, III

Submission of marketing/manufacturing

authorization application to regulatory authorities

Regulatory authorities review information and grant (or refuse) licences

Product goes on sale

Post-marketing surveillance

!

Library of compounds

In vitro screening: human/animal receptor/enzyme assay; reporter system

Hits/lead

Biochemical, tissue or animal model of function

lead

Animal model of therapeutic target

ADME, formulation, acute toxicology

High throughput screening for drug discovery high throughput screening (HTS) is number one tool for early-stage drug discovery

!HTS is process by which large numbers of compounds are rapidly tested for their ability to modify the properties of a selected biological target. Goal is to identify ‘hits’ or ‘leads’ - affect target in desired manner - active at fairly low concs (∴ more likely to show specificity) - new structure !!HTS = 50,000-100,000 cpds screenings per day!!!

taken from Pete the Blogger

!Aim of screening is to find progressible hits, not to discover the lead molecule itself !!The majority of drug targets are a) G-protein coupled 7 TM receptors (est total 5000) b) nuclear receptors (est total >150) c) ion channels (est total 1000) d) enzymes (est total uncertain)

Assays Definition - a test system in which biological activity can be detected

• Aequorin assays

• Colorimetric assays

• Membrane potential assays

• Intracellular calcium flux assays

• Fluorometric assays

• Luciferase reporter gene assays

• Fluorescent and radiolabeled ligand binding assays

Fluorescent Polarization Assay

taken from GlycoForum

Aequorin assay

Luciferase Reporter Gene Assay

from Thermo Scientific

Virtual Screening

• computational technique used in drug discovery to search libraries of small molecules in order to identify those structures which are most likely to bind to a drug target, typically a protein receptor or enzyme

• Ligand based- logic based rules feature substructures and chemical properties of the candidate ligands

• Structure based- virtually docking candidate ligands to the receptors

The End