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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