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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
 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

Target
Target

assay design

assay validation
HTS
engineer
HTS implementation

data capture, storage, and analysis

Decision
Decisionmaking
making
Dr. Maxim Zakhartsev
 Drug discovery today

Analysis of 2000 - year sales of proven target classes

No of Sales Sales per compound

Target class
drugs ($m) ($m)
GPCR 59 40,000 678
Ion channels 14 8,400 597
Proteases 16 8,700 546
Polymerases 12 5,100 425

Dr. Maxim Zakhartsev

 Drug discovery today

disease
target

hit compounds
Drug
develop
lead compounds
ment

candidate drug

drug

drug on market

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

disease
1. Biology & Molecular 1. Phase I
science

2. Bioscience 2. Phase II
Drug
3. Combinatorial chemistry develop 3. Phase III
ment
4. Medical chemistry 4. New Drug Application

drug on market

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

1. Biology & Molecular

disease
science
- target identification
- validate link (target -
Drug
disease)
develop
- manufacture of the ment
target (expression,
purification )

Market examination
- Are the competing
agents already exists ?
- Would be it possible to drug on market
produce a betters drug
at lower price ?
- Patents review

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

1. Biology & Molecular

disease
science
- target identification
- validate link (target -
Drug
disease)
develop
- manufacture of the ment
target (expression,
purification )
Genomics / Proteomics
- Understanding the
function of unknown
proteins and their
interaction in complex cell drug on market
systems

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

2. Bioscience
disease
2.1 Screening
- Assay design
- Assay validation Drug
- Compound library develop
- HTS implementation ment
- Data handling
- Result interpretation

drug on market

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

2. Bioscience
disease
2.2 Confirming biological
activity
- Biochemical-based HTS
- hit compounds Drug
- repeatability develop
- concentration- ment
dependent effect
- Cell-based HTS
- hit the target without
damaging the cell

A compound selected in screening drug on market

should only inhibit the activity of the
target the healthy cell must not be
affected, otherwise an undesired
cytotoxic effect may emerge

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

2. Bioscience
disease
2.3 Determining characteristics
- Pharmacokinetic assays on cell
cultures Drug
- Absorption develop
- Distribution ment
- Metabolism
- Secretion

drug on market

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

2. Bioscience
disease
2.4 Countering resistance
Assays on cell cultures to
determine how long a
compound remains active
Drug
before being broken down develop
- it should reach its
ment
target before being
degraded
- the level of the active
compound in vivo should
remain sufficiently high
for long enough time to
avoid resistance drug on market
development

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

3. Combinatorial chemistry disease

Hit optimization
- arrays of hit compounds
- hit compounds found
in first round are Drug
chemically optimized, develop
providing array of ment
compounds
- hit evaluation and
selection by HTS p lead

HC-R1
Combinatorial
CombinatorialHC-R2
HC-R chemistry and drug on market
chemistry andHC-R3
HTS are closely
HTS are closely
linked!
linked! HC-R4

HC-R
Dr. Maxim Zakhartsev
 Drug discovery today

I. Preclinical research II. Clinical development

4. Medical chemistry
disease
Lead optimization
- arrays of lead compounds
- a lead compound
forms the initial basis Drug
for an extensive
develop
synthesis program
ment
- lead evaluation and
selection by HTS p
Candidate Drug

Computer-Aided Drug Design

drug on market

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

disease 1. Phase I
- initial test on human
- pharmacokinetic
properties & safety
Drug profile
develop
- blood concentration
ment
- healthy volunteers
(20-50)

Phase
PhaseIItrials
trialsare
areproving
proving
that
thataaparticular
particularcompound
compound
drug on market isissafe
safefor
foruse
useininhumans
humans

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

disease 2. Phase II
- test on patients
- medicinal effect
Drug - dosage
develop - side-effect
ment - patients suffering
form the disease
(100-500)

Phase
PhaseII IIhas
hasto
toshow
showthat
that
the compound has the
the compound has the
drug on market intended
intendedmedicinal
medicinaleffect
effect
and
and to determinethe
to determine the
optimal
optimaldosage
dosage

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

disease 3. Phase III

- test on patients
- medicinal effect
Drug - dosage
develop - side-effect
ment - patients suffering
form the disease
(1000s)

Phase
PhaseIIIIIIhas
hasto
toshow
show
that
that the new drug ismore
the new drug is more
efficient
efficientthan
thanthe
thebest
best
drug on market
existing
existingtreatments.
treatments.
Most
Most expensivepart
expensive part

Dr. Maxim Zakhartsev

 Drug discovery today

I. Preclinical research II. Clinical development

disease 4. New Drug Application

- submitted to relevant
registration authorities for
Drug examination and approval
develop
ment

This
Thisphase
phasemay
mayneed
need
up to one year
up to one year

drug on market

Dr. Maxim Zakhartsev

 Another example

Directed protein evolution

HTS

Dr. Maxim Zakhartsev

 HTS is ...

HTS is a main selector of the best compound from the

variety offered by chemical methods.

HTS is a decision maker concerning the direction of

compound development

HTS is the ability to test high numbers of compounds in

multiple screening systems.

HTS

Lead
Target Hit Lead CD Drug
optimization

Dr. Maxim Zakhartsev

 HTS is ...

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.

HTS

Lead
Target Hit Lead CD Drug
optimization

Dr. Maxim Zakhartsev

 HTS is ...

HTS assay methods must be:

1. robust
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

HTS

Lead
Target Hit Lead CD Drug
optimization

Dr. Maxim Zakhartsev

 HTS equipment

Liquid handling Colony picker

Cell sorter HTS robot

Dr. Maxim Zakhartsev

 HTS

Target
Target

assay design

assay validation
HTS
engineer
HTS implementation

data capture, storage, and analysis

Decision
Decisionmaking
making

Dr. Maxim Zakhartsev

 Knowledge requirements for HTS

try An
is ch alyt
em em ica
o ch ist l
Bi ry

Cell biology HTS Statistics

Mo
l
bio ecul r ing
log ar in ee
y g
En

Dr. Maxim Zakhartsev

 Lecture plan

HTS I HTS II
Fall semester Spring semester

Classic HTS New methods and

Drug discovery applications in HTS

Design and implementation of HTS Reporter gene assays

Synthetic chemical libraries RT-PCR
Screening of natural products Microarrays
Separation methods Protein-Protein Interactions
Microplates Two-hybrid system
Enzyme assays Protein Microarrays
Receptor-binding assays Flow Cytometry
ELISA Phage display
Cell-based assays Ribosome display
----------------- In vitro evolution
Physical methods in HTS -----------------
Spectroscopy Microfluidics
Fluorescence
 Milestones

Examinations
Examinations
16-Oct Midterm
?-Dec Final exam

Requirements for final score

% Item
50 Midterm
50 Final examination
100 Final score
 Textbooks
Books in library:
1. Janzen, W.P. (2002). High throughput
screening. Methods and Protocols.
Methods in Molecular Biology, Vol. 190.
Humana Press, Totowa, New Jersey.
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. Jr. (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. John 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