Download Combinatorial Chemistry and High Throughput Screening

Combinatorial Chemistry and High Throughput Screening
1. The conceptual stage, a historical review
2. Types of combinatorial libraries
3. Solid phase and liquid phase combinatorial synthesis
4. Bioactivity Detection / High throughput screening
Combinatorial Chemistry-a historical review
1. Solid-phase synthesis of peptides
2. Solid-phase synthesis of oligodeoxyribonucleotides
3. Traditional vs. combinatorial synthesis:
Mario Geysen: Multipin technique
Richard Houghton: Tea bag technique
Ronald Frank: SPOT technique
John Ellman: First combinatorial synthesis of small
heterocyclic compounds
4. Basic concepts of combinatorial chemistry
5. Combinatorial chemistry in drug development
1. Solid-phase Peptide Synthesis
(SPPS) by Boc method
SPPS: developed by Merrifield, a Nobel
laureate.
Solid-phase synthesis vs.
traditional solution synthesis:
1.
Easy product purification,
especially suitable for
multistep synthesis
2.
Heterogeneous reaction, reactions are
slower and less efficient than
the solution reactions.
Solid-phase Peptide Synthesis (SPPS) by Fmoc method
O
H 2N--AA
N
H
H
N
O
FmocNH-AA-COOH
DCC, HBTU
SPPS
O
H
X
O
O
N
H
+ CO2 + RNH2
FmocHN-AA--AA
O
X
20% piperidine
TFA
O
N
H
FmocNH
2
Fmoc deprotection
x
Rn
R2
N
H
O
H
N
O
X
R1
N
Fmoc method
Chemical synthesis of DNA oligos
Step 1: Terminal deprotection: a few min
CCl3COOH(TCA) or CHCl2COOH(DCA)
OMe
MeO
DMTr
Step 2
Step 2: Coupling: 2-3min
Step 3: Oxidation: I2, a few min
Step 4: Capping: to block unreacted 5’-OH
Ac2O, lutidine, N-methylimidazole, THF
Deprotection: 30% NH4OH, 5h at 50oC
Purification: HPLC, electrophoresis,
ion-exchange catridge, etc.
Step 3
Synthetic cycle
Multipin technique for
parallel synthesis of peptides
Geysen, HM; Rodda, HM; Mason, TJ. The delineation of peptides able to mimick assembled
epitopes. In Synthetic Peptides as Antigens. Ciba Foundation Symposium 119; Porter, R.,
Wheelan, J., Eds.; Wiley: New York, 1986; pp131-149
Washing and deprotection
in a big pan
Coupling: use 96-well plate
Protected AA building
blocks in each well depends on
the sequence of the target
peptide on the pin it
Corresponds to.
Tea bag technique for parallel synthesis of peptides
Richard A. Houghten, General method for the rapid solid-phase synthesis of large numbers of
Peptides: specificity of antigen-antibody interaction at the level of individual amino acids.
PNAS USA 1985, 82, 5131-5135
Problems:
1970s
one 13 mer peptide: 2 weeks,
20 31-residue analogues of β-endorphin, several months, full time
1980-1984
As a faculty at Scripps, synthesize 600 peptides/yr with 5 synthesizer
Washing and deprotection: done together
Coupling:
Sort the bags in to groups
according to the AA to be coupled.
Each group is coupled separately
SPOT technique for parallel synthesis of oligonucleotides
Ronald Frank et al. A new general approach for the simultaneous chemical synthesis of large
Number of oligonucleotides: segmental solid supports. Nucl. Acids Res. 1983, 11, 4365-4377
Filter disc method: one oligo per disc
Washing, deprotection, and oxidation:
done together
Coupling:
Put the discs into four different vials
containing dA, dC, dT, dG building blocks
accoding to the oligo sequence of each disc.
SPOT method: one oligo per disc
Washing, deprotection, and oxidation:
done together
Coupling:
Pipette coupling solution
containing dA, dC, dT,
dG building blocks
onto the discs
according to
the sequence.
Automated
SPOT system
Traditional synthesis of diazepines
First combinatorial synthesis of small heterocycle compouds
Jonathan Ellman et al. A general and expedient method for the solid-phase synthesis of 1,4benzodiazepine. J. Am. Chem. Soc. 1992, 114, 10997-10998
192 compounds: from three variable substituents R1, R2, R3,
Traditional vs Parallel Synthesis
Combinatorial chemistry:
generation of more than one structurally related organic compounds
by parallel synthesis
薄层色谱、
质谱
Combinatorial Chemistry
…
… an
an integral
integral part
part of
of the
the drug
drug research
research and
and development
development process
process
Identification of a
biological target
Development of an assay
High-throughput
screening
Hit
Lead-Structure
Lead-Optimization
Development
Random Libraries
Combinatorial Chemistry
Focused Libraries
Combinatorial Chemistry
Some basic considerations:
The Diversity Problem
Diversity:
Differences of the compounds in a library with
respect to: chemical structure, stereochemistry,
lipophilicity, electrostatic potential and other
physicochemical parameters
Hypothesis:
The higher the diversity of a library is, the higher is
the probability to find active compounds within that
library (concept of maximum diversity)
Combinatorial Chemistry
Unlimited !
Sources
Sources of
of Chemical
Chemical Diversity:
Diversity:
8
H, F, Cl
-NH2, -CN,
-SMe
6
H
N
H2N
O
N
N
N
N H3C
N
N
N
H3C
H
N
N
O
H2N
N
HO O
HO
O
N
H
H2N
N
N
HO
N
O
O
O
-OH
or
prodrugs
e.g.. Ester
H2N
H2N
N
X
N
N
X
N, CH,
N
H3C
N
N
H
N
N
H3C N
H2N
H2N
N
H3C N
No variation
allowed
N
N
N
F, H,
CH3, OH,
Vinyl,Ethinyl
X
N
NH2
H2N
-NH2, CF3
H2N H
O
20
N
H
N
N, C-H, C-F
C-Cl, -CH3,
-CF3, -OMe,
-O-CHF2,
Vinyl,
Ethinyl
Hal
7
F
N
CH3
H
CH3
F
10
O
S
CH3
20 x 10 x 7 x 3 x 8 x 6
= 201,600
2
CH3
O
CH3
O
CH3
N
CH3
5
>3
Combinatorial Chemistry
Some
Some basic
basic considerations
considerations
by Schreiber et al.
Diversity (or universal) vs. focus library
Combinatorial Chemistry
Some
Some basic
basic considerations:
considerations:
The Template Idea (the structures of privilege)
benzodiazepine as a scaffold for library building
O
N
N
HO2C
Me
Me
O
O
N
O
O
N
N
O
oxazepame
psychopharmakological
S
H
N
OH
Cl
Me
N
N
H
N
N
H
MK-329 (CCK-A antagonist)
gastrointestinal
N
ras-farnesylase-inhibitor
cancer
Automated derivatisation of basic pharmacophoric units
Examples of Scaffold Libraries
O
NR1R2
R3
NH
O
O
N
Y
R3
H
z
O O
ArQule
25,600
solution phase
azomethin ylide
and chalcone
N
Houghten
102,459 members
solid phase
N
N
HN
X
N
R2
N
R2
O
S.L. Schreiber
2.18 million members
solid phase
derived from shikimic acid
R4
R3
N
R3
NH2
H
O
ArQule
12,800 members
soluton synthesis
chalcone and
acetoacetamides
R4
N
O
O
R3
R4
R1
R1
R2
O
N
R2
O O
ArQule
25,600
solution phase
chalcone, isatins
and L-proline
R1
N
R2
ArQule
7,680 members
chalcone and
aminobenzimidazoles
Combinatorial Chemistry
Some
Some basic
basic considerations:
considerations:
Structure
F
O
N
H
N
The Template Idea
Name
Indication
Mechanism
Diflufenican
Herbicide
Carotine-Biosynthesis
Dithiopyr
Herbicide
Mitosis
Imazapyr
Herbicide
Biosynthesis of
branched
amino acids
Nicosulfuron
Herbicide
Biosynthesis of
branched
amino acids
Imidacloprid
Insecticide
Agonist
acetylcholine
receptor
Haloxyfop
Herbicide
Acetyl-CoAcarboxylase
Chlorfluazuron
Insecticide
Chitin-Biosynthesis
Mevastatin
Lipid Lowering
HMG-CoAreductase
Adalat
Blood Pressure
Ca-Channel-Blocker
F
O
CF3
O
O
MeS
SMe
F2CH
Example of privileged structures
N
CF3
CO2H
N
N
Me
N
H
O
O
Commercial Products with
a pyridine substructure
NMe2 OMe
N
SO2 N
NH
N
OMe
O
N
N
N
H
NO2
N
Cl
CO2H
N
O
O
CF3
Me
Cl
F
Cl
O
F
N
H
H
N
Cl
O
CF3
N
O
Cl
F
OH OH
CO2Me
MeO
N
NO2
CO2Me
MeO2C
Me
N
H
Me
Combinatorial Chemistry
Strategy:
Strategy:
Hit
Hitfinding
finding
Random Library
Lead
Leadfinding
finding
HTS: First Hit
Targeted Library
Lead Structure
O
S R1
O
O
H2N
R1
O
H2N
HN
O
N
H
S
O
H2N
N
H
HN
R2
S
O
R2
HN
R3
Cl
5000 compounds
S
O
EC50: 10 µM
F
500 compounds
Final Optimization Program
EC50 20 nM
References:
Combinatorial Peptide and Nonpeptide Libraries, A Handbook, Ed.
Gunther Jung, 1996, VCH, Weinheim, New York, Basel
Combinatorial Chemistry-Synthesis, Analysis, Screening, Ed. Gunther
Jung, Wiley-VCH Verlag GmbH, Weinheim, 1999
M. Lebl, Parallel personal comments on “classical” papers in combinatorial
Chemistry. J. Comb. Chem. 1999, 1, 3-24
D. Hudson. Matrix assisted synthetic transformation: a mosaic of diverse
Contributions. I. The pattern emerges. J. Comb. Chem. 1999, 1, 333-360
D. Hudson. Matrix assisted synthetic transformation: a mosaic of diverse
Contributions. II. The pattern is completed. J. Comb. Chem. 1999, 1, 403-457
Comprehensive survey of combinatorial library synthesis: 1998, 1999, 2000,
And 2001, 2002, 2004, 2005, 2006, J. Comb. Chem.
Solid Phase Organic Synthesis
Functional group, directly attached to the solid support
anchor
starting
material
X
solid
support
linker
Bifunctional spacer molecules which contain on one
end a functional group for attachment to the solid
support and on the other end a selectively cleavable
group onto which the starting molecule is attached
Solid Phase Organic Synthesis
Polymeric Carriers
Parallel Synthesis of Single Compounds
divide-couple-and-recombine strategy ( = split and mix)
tea-bag method
radiofrequence-coding
laser-coding
parallel synthesizer
multipin synthesis
spot synthesis
photolithografic techniques
Fully
Fully Automated
Automated Synthesizers
Synthesizers
Magellan: a high end synthesizer, using pin technology
A newly designed heater/agitator with removable reaction block.
Unique resin wash system using a vacuum aspiration
system and fast 4 probe addition of up to 8 solvents streamlines the wash procedure.
Dual function vacuum aspiration system collects waste or
cleaved product.
Aliquoting of cleaved product into a variety of vessels
for library storage/screening.
Separation of wastee products to avoid potential
adverse side reactions
All components of the system are bar-coded to ensure data integrity
Liquid handling based on the successful Guardian range of robotic sample
processors, with independent single and four-way probes, ensures safe,
reliable and fast fluid handling.
Intuitive Windows based software with direct interface to MS Excel allows
easy creation of reaction sequences for the most complex chemistries
Modular, scaleable system with full computer networking enables any
number of Magellan synthesisers and heater/agitator units to work in
harmony to increase productivity
Combinatorial Chemistry
Libraries
Libraries of
of Compound
Compound Mixtures
Mixtures
„split and combine“ strategy
≡
One bead - one compound
by Furka
History
History or
or position
position of
of aa single
single bead
bead
(or
of
an
ensemble)
can
be
deduced
(or of an ensemble) can be deduced
mix
split
couple
Parallel
Parallel synthesis
synthesis of
of single
single compounds
compounds
mix
split
couple
History
History of
of the
the beads
beads is
is not
not deducible
deducible
Mixture
Mixture
Deconvolution
split-method. balls = polymeric carrier. R = number of parallel reactions
P = number of different compounds
Libraries of Compound Mixtures
Identification
Identification of
of active
active compounds
compounds in
in mixtures
mixtures
1.
Deconvolution
1a)
Iterative deconvolution
1b)
Recursive deconvolution
1c)
Positional Scanning
2.
Indexed (orthogonal) libraries
3.
Tagging (and Decoding)
Tag:
test compound
test compound
resin
3a)
Chloro-phenol-ethers
3b)
aromatic amines
3c)
peptides
3d)
DNA
tag
4.
Spectroscopic methods
affinity selection
SAR by NMR
1.
1.
!
a)
Deconvolution
Deconvolution
Iterative method for the identification of active compounds (particularly for solution-libraries)
Prerequisite: High precision and reproducibility of the bio-test
R1X
R2X
Iterative Deconvolution
Synthesis and Screening of sub-libraries
1. step:
Screening of the complete library
(44 = 256 substances)
2. step:
synthesis of 4 sub-libraries
with position 1 defined in each sub-library
(43 = 64 substances per library)
3. step:
Screening of the first set of sub-libraries
4. step:
Synthesis of 4 sub-libraries
with fixed position 1 and position 2
defined in each sublibrary
(42 = 16 substances per library)
5. step:
Screening of the second set of sub-libraries
6. step:
Synthesis of 4 sublibraries
with fixed positions 1 and 2 and position 3
defined (41 = 4 Substanzen je Bibliothek)
7. step:
Screening
8. step:
Synthesis of 1 final library
with fixed positions 1, 2, 3 and
defined position 4
9. step:
Screening
R4X
R1 - R4: 4 substituents
X: possibilities, to vary
the substituents
R3X
Possible sources of error during deconvolution:
S.M. Freier et al., J. Med. Chem. 1995, 38, 344
S.M. Freier et al., J. Med. Chem. 1996, 39, 2720
Chem. Rev. 1997, 97, 449
a)
a)
Iterative
Iterative Deconvolution
Deconvolution
Example: Combinatorial synthesis of pyrrolidines via 1,3-dipolar cycloaddition
Aim: Novel antihypertensive ACE-inhibitors structurally related to captopril
Typical example for a „focused library“
R2
, NEt3
O
O
NH2
O
ArCHO
CH(OMe)3
R1
N
O
R3
Ar
library >500 sulfanylacylprolines
AgNO3, CH3CN
R1
4 iterative screening-cycles with
corresponding sub-libraries
O
1.
O
O
R1
R4
H
N
Ar
R3
NEt3, THF
O
N
HO
2. TFA, CH2Cl2
R2
CO2H
O
R3
MeO2C
N
CO2H
SH
N
CO2H
O
O
Captopril
Ki = 450 pM
R1
Ar
R2
MeO2C
N
R4
O
Cl
SH
SH
Ki = 160 pM
M.A. Gallop et al., Acc. Chem. Res. 1996, 29, 144
M.A. Gallop et al., J. Am. Chem. Soc. 1995, 117, 7029
Ki = 5 µM
a)
a)
Iterative
Iterative Deconvolution
Deconvolution
Example: Combinatorial synthesis of pyrrolidines via 1,3-dipolar cycloaddition
Gly
Ala
CHO
Leu
CHO
CHO
Me
NC
MeO2C
Cl
AcS
O
Phe
CHO
OSiMe2tBu
OMe
MeO2C
tBuO2C
AcS
O
Cl
O
M.A. Gallop et al.
J. Am. Chem. Soc. 1995, 117, 7029
AcS
Cl
O
c)
c)
Positional
Positional Scanning
Scanning
Non-iterative deconvolution-method
Procedure:
1. step:
The complete library is prepared 4x by
synthesizing of 4x4 sub-libraries
2. Schritt
Screening
The optimal combination of substituents can be deduced directly
as there are definite preferences for the respective substituents
A1
+
A2
B1
+
B2
C1
C2
A1-B1-C1
A1-B1-C1
A2-B1-C1
A2-B1-C1
A1-B2-C1
A1-B2-C1
A2-B2-C1
A2-B2-C1
A2-B2-C1
A2-B2-C1
A1-B1-C2
A1-B1-C2
A2-B1-C2
A2-B1-C2
A2-B2-C2
A2-B2-C2
A1-X-X
A2-X-X
X-B1-X
X-B2-X
X-X-C1
X-X-C2
A1
B2
C1
c)
c)
Positional
Positional Scanning
Scanning
A 1-5
B 1-5
D 1-5
C 1-5
Substituent A
A1
B 1-5
D 1-5
Substituent B
A 1-5
B1
D 1-5
Substituent C
A 1-5
B 1-5
D 1-5
Substituent D
A 1-5
B 1-5
D1
C 1-5
C 1-5
C1
C 1-5
A2
A 1-5
A 1-5
A 1-5
B 1-5
D 1-5
B2
C 1-5
D 1-5
B 1-5
C 1-5
D 1-5
B 1-5
C2
Identification of the most active
Identification of the most active
library member:
library member:
1. The complete library is synthesized
1. The complete library is synthesized
4x in a format of 5x4 sublibraries
4x in a format of 5x4 sublibraries
2. The 20 sublibraries are screened
2. The 20 sublibraries are screened
D2
3. The compounds in frames are the
3. The compounds in frames are the
most active in each set of sublibraries
most active in each set of sublibraries
C 1-5
A2
A3
B 1-5
A 1-5
D 1-5
B3
A 1-5
D 1-5
B 1-5
A 1-5
D 1-5
B 1-5
D3
C 1-5
C 1-5
C3
C 1-5
A4
A 1-5
A 1-5
A 1-5
B 1-5
D 1-5
B4
D 1-5
B 1-5
D 1-5
B 1-5
D4
C 1-5
C 1-5
C4
C 1-5
A4
A 1-5
A 1-5
A 1-5
B 1-5
D 1-5
C 1-5
B5
D 1-5
C 1-5
B 1-5
D 1-5
C5
B1
B 1-5
C3
Basic presumption:
the activities are additive
and not synergistic
A synergistic behaviour
of the substituents affords
wrong results
D5
C 1-5
D5
Combinatorial Chemistry
Combinatorial
Combinatorial Synthesis
Synthesis in
in Solution
Solution
Synthesis in Solution
Solid Phase Synthesis
+ reaction conditions are usually more easily adapted to a
large variety of substituents
+ purification procedures achieved by simple filtrations
which can be easily automated
+ no limitations in synthesis-scale
+ excess of reagents can be used to drive reactions
to completion
+ no attachment / cleavage steps
+ synthesis of mixtures (split and combine)
+ easy to automate
- strict limitations in reagent excesses
- reaction conditions have to be established for each case
- extensive and time consuming, chromatographic purifications
are often necessary
- difficult on-bead characterization of the products
- parallelisation and automation usually requires
more initial effort
- chemistry on polymers is relatively expensive:
polymers, solvents
- resins and linkers limit the number of possible
reactions (orthogonality)
Synthesis in solution is especially suited for short reaction-sequences:
"One-pot" syntheses, multi-component reactions
Parallel Synthesis in Solution
Work-up
Work-up Procedures:
Procedures:
Liquid-solid phase extractions using polymer-bound scavengers
Auxiliary bases and acids are covalently linked to insoluble supports
Advantage: Excesses of reagents and by-products can be removed by simple filtration
N
O
O
R1 COOH
O
(1 eq)
Cl
R1
O
O
O
R2-NH2
O
R1
SO3H
O
N
H
R2
(1.1 eq)
N
SO3R2-NH +
O
3
x HCl
nucleophilic
basic
electrophilic
acidic
N
N
NH2
NH2
NH2
O
Me
N
Me
N
NCO
CHO
COCl
COOH
SO3H
Parallel Synthesis in Solution
Special
Special Methods:
Methods:
polymer-bound reagents or catalysts
“Inverse” solid phase synthesis:
N
O
O
R1 COOH
O
(1 eq)
Cl
R1
O
O
O
R1
bromination
SO3R2-NH +
+
R
N R - BH4
R
R
R
+
N R - BH4 x Ni(OAc)2
R
oxidants
Cr2O72-
PDC
+
SO4H
polyvinylpyridine x Zn(BH4)2
SiO2
CrO3
N Me
Me Me
Me
+
miscellaneous
SiO2
N
Me
Me
KMnO4
IO4-
N OsO4
O
N x HCl
PPh2
ionic exchange resins
D.H. Drewry, D.M. Coe, S. Poon
Med. Res. Rev. 19, 97-148 (1999)
OH
R
+
reductants
SnH
+
R2
3
ketone epoxidation
S
+
N
H
SO3H
O
N Me Br 3
Me
Me
O
R2-NH2
O O
O
O
S
Parallel Synthesis in Solution
Synthesizers
Synthesizers for
for Automated
Automated Synthesis
Synthesis in
in Solution
Solution
1.
ISRA-system
Requirements: All procedures of a synthesis-laboratory have to be automated:
weigh in, synthesis, work-up, HPLC-analysis, lab journal
Automation of this complete process is extreme difficult. No commercial solutions.
Advantage: fully-automatic, parallel synthesis laboratory, operating 24 h a day
2
3
4
5
6
23
8
9
10
11
12
7
2m
22
1
21
18
18
16
18
14 13
15
17
20
17
19
5m
1 robot
2 HPLC
3 starting rack
4 filter rack
5 filtration station
6 capping station
7 lid transfer port
8 lid magazin
9 drying station
10 drying columns
11 coolable rack
12 balance
synthesis
13 phase separation
14 phase detection
15 centrifuge
16 hand over station
17 evaporator
18 reaction blocks
work up
analysis
19 pipetting station
20 reagent rack
21 reactant rack
22 HPLC-transfer
23 vortexer station
1.
Robots for solution-synthesis: ISRA-system
High Throughput Screening
…
… an
an integral
integral part
part of
of the
the drug
drug research
research and
and development
development process
process
Identification of a
biological target
Development of an assay
High-throughput screening
Hit
Lead-Structure
Lead-Optimization
Development
Random Libraries
Combinatorial Chemistry
Focused Libraries
High Throughput Screening
Screening
Screening Systems
Systems
Molecular screening systems
Measuring parameter: Binding
receptor
antibody
ligand
antigen
DNA
DNA binding protein
Reductionism:
exploring a complex problem
by reducing it to simple parts
Animals
Organs
Measuring parameter: Enzymatic function
Tissues
Substrate turnover
Modification of a target molecule
Cells
Organelles
Subcellular screening systems
Molecules
Binding or enzyme assays using subcellular components such as
membrane preparations or cellular fractions
Cellular screening systems
Measuring parameters: Cellular Events
Secretion
Triggering of intracellular ion fluxes
Generation of signaling molecules
Stimulation of gene expression
Significantly more complex
High Throughput Screening
Screening
Screening Systems:
Systems:
Receptors
Enzymes
Recognition domains
Target Classes
Class of Target
Therapeutic Area
G-protein-coupled receptors
Intracellular receptors
Cytokine receptors
Tyrosine kinase receptors
Transcription factors
Ion channels
Kinases
Proteases
SH2 and SH3 domains
Central nervous system, inflammation, metabolic disease
Cancer, metabolic disease
Cancer, cardiovascular, inflammation
Cancer, cardiovascular, inflammation
Various areas, including osteoporosis and inflammation
Cardiovascular, central nervous system
Various areas including cancer and metabolic disease
Cardiovascular, arthritis, osteoporosis
Various areas including diabetes and cancer
Within Bayer >100 HTS assays and a comparable number of MTS assays are available
Only a limited number of diseases are covered
All large pharmaceutical companies are working in the same therapeutic areas
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Radiolabelled ligands
The challenge: To generate an easy-to-detect read-out signal
Incubation
Usually the binding of ligand to a protein can not be visualized directly.
A reporter group needs to be coupled to the target.
- Enzymes
- radionuclides
- fluorescent probe
- color dye covalently attached to the target
+
Filtration
The problem:
The ligands must be radiolabelled: expensive, dangerous, ….
Determination of the bound ligand
on the filter by liquid scintillation counting
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Enzyme-linked colorimetric screening
Alkaline phosphatase
5-bromo-4-chloro-3-indolyl phosphate (BCIP)
Horseradish peroxidase
tetramethylbenzidine
ß-galactosidase, glucose-oxidase
+
Biotin
Incubation,
Filtration
Biotin
Filtration,
Incubation with
streptavidin - enzyme
Biotin
Enzyme
Streptavidin
Addition of reagents for
color development
Color producing reaction
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Enzyme-linked colorimetric screening
O
HN
Vitamin H in humans
Essential cofactor for almost a dozen of enzymes that
have diverse metabolic functions
NH
H
H
COOH
S
Acetyl-CoA carboxylase, pyruvate carboxylase, carboxylase,
urea carboxylase, ….
Biotin serves as a covalent bound CO2-carrier for reactions in
which CO2 is fixed into an acceptor by carboxylases
Biotin
Alkaline phosphatase
5-bromo-4-chloro-3-indolyl phosphate (BCIP) or
OH
HO P O
O
O2N
Cl
Br
OPO3Na2
Cl
Alkaline phosphatase Br
N
H
Cl
OH
oxidation
N
H
O
Br
N
H
H
N
Br
O
Cl
Indigo type dye
insoluble
NO2
Examples of various screening methods for one-bead-one-compound combinatorial libraries
A) a positive bead in an enzyme-linked colorimetric assay using 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the substrate
B) a positive bead can be easily retrieved by a hand-held micropipette
C) a dual color enzyme-linked colorimetric assay using two different substrates
D) a whole-cell on-bead binding assay with a monolayer of intact cells on the surface of a positive bead
E) an autoradiogram showing positive (32P-labeled peptide beads in an on-bead functional assay for protein kinase peptide substrate
F) an in situ solution-phase releasable assay for detecting anticancer agent, showing a clear zone of inhibition surrounding a positive bead
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Antibody-linked colorimetric screening
receptor
+
Incubation,
Filtration
antibody
Enzyme
antibody
Enzyme
Addition of reagents for
color development
Color producing reaction
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Reporter gene assay (Whole cell assays)
With the cell based screening approach, a recombinant cell or microorganism is engineered
to respond in a specific manner to an effector of the molecular target of interest
The reporter gene assay is the most common cell-based assay used for HTS
Hormone,
Ligand
Receptor
Signal transduction
Luciferase
Luciferase Gene
DNA
Response Element
Reporter Gene
Luciferase reaction:
ATP + luciferin
adenyl-luciferin-O2
luciferase
adenyl-luciferin + PPi
adenyloxyluciferin + H2O + light
Other reporters:
ß-galactosidase, chloramphenicol acetyl transferase, alkaline phosphatase
High Throughput Screening
Screening
Screening Systems:
Systems:
Assay set-up
Functional assay (Whole cell based): kinase receptor activation
Ligand-binding
Receptor-phosphorylation
ligand
Stimulation
phosphorylated
tyrosines
cell
cell surface
receptor
cell
Immuno-assay
solubilized receptor
+
biotin
capture
antibody
Streptavidin-HRP
color reaction
biotinylated
anti-antibody
High Throughput Screening
HTS
HTS -- technical
technical implemantation
implemantation
96-MTP format
dilution
synthesis
tared tubes
sample processor
master plate
assay plates
assay plate file
synthesis file
master plate file
biological result file
dilution
structure
purity
amount
compound ID
concentration
well position
IC50 values
compound ID
concentration
well position
High Throughput Screening
HTS
HTS -- technical
technical implemantation
implemantation
Data - Handling
Screening - Robot
High Throughput Screening
HTS
HTS -- technical
technical implemantation
implemantation