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High Throughput Screening

(HTS) Technology
by Dr. Helge Weingart
How to reach me
Dr. Helge Weingart
University Lecturer
Research II, Room 92
Phone: 3581
h.weingart@jacobs-university.de
Plan for today
1. What is HTS?
2. Examples
3. General HTS equipment
4. Lecture plan
5. Milestones
6. Textbooks
Dr . Maxim Zak har tsev
HTS
technological approach (set of analytical instruments and
methods) to proceed huge amount of samples to assess it for
the certain criteria and chose the best one among thousands
today an important part of scientific research and drug discovery
assay design
assay validation
HTS implementation
data capture, storage, and analysis
HTS
engineer
Target
Target
Decision making
Decision making
Drug discovery today
425 5,100 12 Polymerases
546 8,700 16 Proteases
597 8,400 14 Ion channels
678 40,000 59 GPCR
Sales per compound
($m)
Sales
($m)
No of
drugs
Target class
Analysis of 2000 - year sales of proven target classes
Dr . Maxim Zak har tsev
Dr . Maxim Zak har tsev
Drug discovery today
target
hit compounds
drug
lead compounds
candidate drug
disease
drug on market
Drug
develop
ment
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
1. Biology & Molecular
science
3. Combinatorial chemistry
4. Medical chemistry
2. Bioscience
2. Phase II
3. Phase III
4. New Drug Application
1. Phase I
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- manufacture of the
target (expression,
purification )
- target identification
- validate link (target -
disease)
1. Biology & Molecular
science
- Would be it possible to
produce a betters drug
at lower price ?
- Patents review
- Are the competing
agents already exists ?
Market examination
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- manufacture of the
target (expression,
purification )
- target identification
- validate link (target -
disease)
1. Biology & Molecular
science
- Understanding the
function of unknown
proteins and their
interaction in complex cell
systems
Genomics / Proteomics
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
2. Bioscience
2.1 Screening
- Data handling
- HTS implementation
- Assay validation
- Compound library
- Result interpretation
- Assay design
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- hit compounds
- Cell-based HTS HTS
- concentration-
dependent effect
- repeatability
- Biochemical-based HTS HTS
- hit the target without
damaging the cell
2.2 Confirming biological
activity
2. Bioscience
A compound selected in screening
should only inhibit the activity of the
target the healthy cell must not be
affected, otherwise an undesired
cytotoxic effect may emerge
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- Metabolism
- Absorption
- Secretion
- Distribution
- Pharmacokinetic assays on cell
cultures
2.3 Determining characteristics
2. Bioscience
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- it should reach its
target before being
degraded
- the level of the active
compound in vivo should
remain sufficiently high
for long enough time to
avoid resistance
development
Assays on cell cultures to
determine how long a
compound remains active
before being broken down
2.4 Countering resistance
2. Bioscience
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
Hit optimization
- hit compounds found
in first round are
chemically optimized,
providing array of
compounds
- hit evaluation and
selection by HTS HTS lead lead
- arrays of hit compounds
3. Combinatorial chemistry
HC-R
HC-R
1
HC-R
2
HC-R
3
HC-R
4
HC-R
Combinatorial
chemistry and
HTS are closely
linked!
Combinatorial
chemistry and
HTS are closely
linked!
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- a lead compound
forms the initial basis
for an extensive
synthesis program
- lead evaluation and
selection by HTS HTS
Candidate Drug Candidate Drug
- arrays of lead compounds
Lead optimization
4. Medical chemistry
Computer-Aided Drug Design
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- blood concentration
- pharmacokinetic
properties & safety
profile
- healthy volunteers
(20-50)
- initial test on human
1. Phase I
Phase I trials are proving
that a particular compound
is safe for use in humans
Phase I trials are proving
that a particular compound
is safe for use in humans
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- dosage
- side-effect
- medicinal effect
- patients suffering
form the disease
(100-500)
- test on patients
2. Phase II
Phase II has to show that
the compound has the
intended medicinal effect
and to determine the
optimal dosage
Phase II has to show that
the compound has the
intended medicinal effect
and to determine the
optimal dosage
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- dosage
- side-effect
- medicinal effect
- patients suffering
form the disease
(1000s)
- test on patients
3. Phase III
Phase III has to show
that the new drug is more
efficient than the best
existing treatments.
Most expensive part
Phase III has to show
that the new drug is more
efficient than the best
existing treatments.
Most expensive part
Dr . Maxim Zak har tsev
Drug discovery today
disease
drug on market
Drug
develop
ment
I. Preclinical research II. Clinical development
- submitted to relevant
registration authorities for
examination and approval
4. New Drug Application
This phase may need
up to one year
This phase may need
up to one year
Dr . Maxim Zak har tsev
Another example
Directed protein evolution
HTS HTS
Dr . Maxim Zak har tsev
HTS is ...
HTS HTS is a main selector of the best compound from the
variety offered by chemical methods.
Hit Lead
Lead
optimization
CD Target
HTS
Drug
Dr . Maxim Zak har tsev
HTS HTS is a decision maker concerning the direction of
compound development
HTS HTS is the ability to test high numbers of compounds in
multiple screening systems.
HTS is ...
Dr . Maxim Zak har tsev
Main features are:
1. The ability to make critical and well-founded decisions
regarding future research.
2. The time from identifying the first active compound to selecting
a candidate drug that can be developed towards clinical trials
is expensive, and must be kept as brief as possible.
Hit Lead
Lead
optimization
CD Target
HTS
Drug
HTS is ...
HTS HTS assay methods must be:
Dr . Maxim Zak har tsev
2. replicable and generate the same values repeatedly
3. must not be excessively time-consuming
4. should be cheap
5. confer the opportunity of a high throughput of assays
6. results must be as relevant as possible
1. robust
Hit Lead
Lead
optimization
CD Target
HTS
Drug
HTS equipment
Colony picker Liquid handling
Cell sorter HTS robot
Dr . Maxim Zak har tsev
HTS
assay design
assay validation
HTS implementation
data capture, storage, and analysis
HTS
engineer
Target
Target
Decision making
Decision making
Dr . Maxim Zak har tsev
Knowledge requirements for HTS
B
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h
e
m
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y
Cell biology
M
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Statistics
HTS
Dr . Maxim Zak har tsev
Lecture plan
HTS I
Fall semester
HTS II
Spring semester
Classic HTS
Drug discovery
New methods and
applications in HTS
Reporter gene assays
RT-PCR
Microarrays
Protein-Protein Interactions
Two-hybrid system
Protein Microarrays
Flow Cytometry
Phage display
Ribosome display
In vitro evolution
-----------------
Microfluidics
Design and implementation of HTS
Synthetic chemical libraries
Screening of natural products
Separation methods
Microplates
Enzyme assays
Receptor-binding assays
ELISA
Cell-based assays
-----------------
Physical methods in HTS
Spectroscopy
Fluorescence
Milestones
Final exam ?-Dec
Midterm 16-Oct
Requirements for final score
Final score 100
Final examination 50
Midterm 50
Item %
Examinations
Examinations
Textbooks
Books in library:
1. J anzen, W.P. (2002). High throughput
screening. Methods and Protocols.
Methods in Molecular Biology, Vol. 190.
Humana Press, Totowa, New J ersey.
2. Devlin, J .P. (1997). High throughput
screening: the discovery of bioactive
substances. Marcel Dekker, Inc., New York.
3. Eisenthal, R. and Danson, M.J . (2002).
Enzyme assays: a practical approach. 2nd
ed. Oxford University Press, Oxford, UK.
4. Hagemeyer, A., Strasser, R. and Volpe,
A.F. J r. (2004). High-throughput screening
in chemical catalysis. Wiley-VCH,
Weinheim.
5. Atta-urRahman, Choudhary, M.I., and
Thomsen, W.J . (2001). Bioassay
techniques for drug development. Taylor &
Francis, Boca Raton.
Books on reserve:
1. Voet, D. and Voet, J . G. (2004).
Biochemistry. 3rd ed. J ohn Wiley & Sons,
Inc.
2. Holme, D.J . and Peck, H. (1998). Analytical
biochemistry. 3rd ed. Pearson Education
Limited, Essex, New York.
3. Freifelder, D. (1982). Physical biochemistry:
applications to biochemistry and molecular
biology. 2nd ed. W.H. Freeman, San
Francisco, USA.
4. Harris, D.C. (2003). Quantitative chemical
analysis. 6th ed. W.H. Freeman and Co.,
New York, NY.
High throughput screening
finding the needle in a haystack