pharmaceutical

1. 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
SCREENING IN BIOMEDICAL
RESEARCH AND PHARMACOLOGICAL

2. 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

3. Well shape

4. ELISA

5. Microplate Reader
taken from Synergy

6. Reusing plate
taken from © 2014 Kinesis Ltd.
taken from Labotal Scientific Equipment (1997) LTD

7. Materials
•
polyprolyene- for wide change in temperature
environment
•
polycrabonate- disposable,
•
Cyclo-olefins

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

9. 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

10. taken from RNAi @ NIH Chemical Genomics Center

11. 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

12. 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!!!

13. taken from Pete the Blogger

14. !
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
c) ion channels
d) enzymes
(est total >150)
(est total 1000)
(est total uncertain)

15. 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

16. Fluorescent Polarization
Assay
taken from GlycoForum

17. Aequorin assay

18. Luciferase Reporter Gene Assay
from Thermo Scientific

19. 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

20. •
Ligand based- logic based
rules feature substructures
and chemical properties of
the candidate ligands
•
Structure based- virtually
docking candidate ligands to
the receptors

21. The End