Transcript No Slide Title

Forward Chemical Genomics
Identifying the Target Protein
through High-through-put Assays
Forward Chemical Genomics
Forward
(phenotype based screen)
Phenotype
Small
Molecules
Causing
Phenotype
Target (e.g. protein)
?
More recent
High-throughput-assay Formats for Detecting
Small Molecule-Protein Interactions
- Small molecules microarrays
- Protein microarrays
- DNA-Cell arrays
- Yeast three-hybrid system
Small-Molecule Microarray
- Small molecules
arrayed
- Labelled protein
brought in contact
- Protein binds to
metabolite
- Interaction
detected
Small-Molecule Microarray
Small-Molecule Microarray
- The yeast Ure2p is a central
repressor of genes involved in
nitrogen metabolism
- Part of the Tor protein signaling
cascade
- No compound binding it is known
Small-Molecule Microarray
- 3780-member small molecule
microarrays was used (800 spots
cm2)
- The collection of molecules was
prepared by diversity oriented
synthesis (DOS) approach
- Thus, molecules unbiased towards
a particular protein target
Preparation of a DOS Library
-
Preparation of a DOS Library
Generation of the Library
- "One-bead, one-stock solution" approach
- Small molecules are synthesized on
polystyrene macrobeads
Macrobead
Macrobead/linker
system
(a backbone might
also be attached
to the linker)
A single bead serves as individual
reaction vessels during split-pool library
synthesis
Diversity Oriented Synthesis
Solid supports are
combined, mixed
and redistributed
between synthetic
steps
Generation of the Library
- Using chloroaromatic
TAGS to encode the
Microbeads
- The tag could be
cleaved
Generation of the Library
- Bead arraying
Generation of the Library
- 5mM stock solutions after compound
cleavage and resuspension in multiple well
plates
De-coding using
LC-MS for example
Preparation of the Library
+ decoding
Generation of the Microarray
- Stock solutions in DMF spotted on glassslides (1nl) using a quill-pin
Molecules bound through
reactive functional groups
Hybridization and Identification of "hits"
- Array probed with fluorescently labelled
purified Ure2p and 8 "hits" identified
Testing an Individual "hit"
- Eight molecules re-synthesized
- Tested of modulation of Ure2p function
with PUT1-lacZ reporter system
- PUT1 is known to be repressed by URE2
- A compound named uretupamine A, gave
a concentration dependent doze response
Testing an Individual "hit"
Structure-Activity Relationship
Higher activity of Uretupamine B, removal of phenyl group
Uretupamine A & B SpecificityA Whole-Genome Gene Expression Profiling
- A subset of genes known to be repressed by
Ure2p where up regulated
- These subset of genes was not changed in
expression when a Ure2p deletion strain was
assayed with Uretupamine A & B
Using Dos and SMMs for Specific
Modulation of Proteins
- The approach described allows
modulating specific aspects of protein
function
- It could be used more specifically than
providing a physiological stimulus or a
genetic deletion
New type of SMMs
- A few DOS libraries spotted
- On the same slide, commercially available
natural products
- Using Isocyanate coated surface
- Isocyanates react with a number of nucleophilic
functional groups and allows to increase the
diversity of small molecules spotted
New type of SMMs
Genistein
Gibberellic
acid (GA)
Hybridization to Cell Lysates with
no Prior Purification
- Earlier studies with SMMs relied on
incubation with a purified protein of interest
- Limited by expression of large-proteins,
solubility, post translational modification
state, activity, and yield
- Described method used epitope-tagged
target proteins from cell-lysates without
purification
Hybridization to Cell Lysates with
no Prior Purification
- Transient transfection to mammalian cell line of
tagged protein of interest
- Arrays incubated serially with:
1. clarified lysate
2. primary (anti-epitope antibody)
3. fluorophore labelled secondary antibody
- Array washed and scanned
- Fluorescence intensity compared to an identical
array hybridized with a mock transfected identical
cell line
Detection of Binding to Ligands
with Varying Affinity
FKBP12
RECEPTOR
Detection of Binding to Ligands
with Varying Affinity
- Derivatives of AP1497
(a known binder of
FKBP12) synthesized
and tested
- Specific Tagantibodies, GFP fusion
and a FKBP12
polyclonal antibody
could be used for
detection
Detection of FKBP12 Binders
- An array of 10,800 features printed
- Contains rapamycin (known to bind FKBP12)
- Contains 27 features corresponding to FKBP12
synthetic ligands
- 10 arrays hybridized (5-FKBP12-Tagged (Flag)
and 5-control
Detection of FKBP12 Binders
-Detection by anti-Flag mono-clonal
antibody and subsequently a Cy5-labeled
anti-mouse antibody
- Scanned for fluorescence at 635 nm
- Signal to noise ratio greater than 2.24positive (compared to arrayed solvent)
Detection of FKBP12 Binders
- Contains rapamycin
(known to bind FKBP12)
- Contains 27 features
corresponding to FKBP12
synthetic ligands
- 10 arrays hybridized
(5-FKBP12-Tagged (Flag)
and 5-control
-Detection by anti-Flag
mono-clonal antibody
and subsequently a Cy5labeled anti-mouse
antibody
- 24 features positive
Significance of the Study
1. New method for SMMs
2. Significant number (approx. 11,000) and
diversity of natural products and synthetic
bioactives on glass microarrays
3. Using cellular lysates instead of purified
proteins
Small Molecules MACROarrays
Small Molecules MACROarrays
Small Molecule MACROarays vs. MICROarrays
Micro
Surface
Macro
glass slides
flat cellulose, filter paper
Fabrication
spotted
synthesized directly on
array via solid-phase
synthesis
Size
0.1 mm
6 mm
Small Molecule MACROarays vs. MICROarrays
Small Molecule MACROarays vs. MICROarrays
Micro
Amount of
compound
Cleavage
and
isolation of
compound
Feature
density
picomoles
Macro
nanomoles
difficult, low Relatively easy, large
quantities
quantities
high
lower
Other features of SMMAs
Fabrication and Use
- Plane support (filter) is derivatised by a
spacer (for on support screening)
- Subsequently, covalent attachment of a
linker unit (attachment of growing
molecules and for further cleavage)
Other features of SMMAs
Fabrication and Use
- Mainly manual pipetting (1-5 micro liters)
but also robotic for larger size arrays
- Reaction at room temperature, large
excesses of reagents, Microwave heating for
reactions
- Cut spot out with a hole-punch, cleavage
and examination with TLC, LC-MS
Other features of SMMAs
Fabrication and Use
On and Off Support Assays
Off-support assays
Compound Classes
Accessed with SMMAs
Use of SMMAs as a Platform for the
Discovery of Fluorescence Dyes
Use of SMMAs as a Platform for the
Discovery of Fluorescence Dyes
- Synthesized a library of chalcones using
a SMMAs platform
- 0.3 cm2; loading 100nmol
compound/spot; 30 chalcone derivatives
- By one-step condensation reaction the
chalcone could be transformed to a
variety of nitrogen containing hetrocycls
(some of them are fluorescent)
Use of SMMAs as a Platform for the
Discovery of Fluorescence Dyes
chalcone
On and Off Support Spectral
Properties Analysis
Off Support
On Support
Protein Microarrays
- Proteins are
immobilized on a surface
- Labelled compound
incubated with the
surface
- Identification of
compound-protein
interaction through label
Protein
Protein Microarrays & Small Molecules
Protein Microarrays & Small Molecules
- Proteins are purified, full-length correctly
folded
- Proteins covalently attached to glass
microscope slides
- Use of a printing robot (like DNA arrays
printing)
-150-200 micron diameter of each spot
(1600/cm2)
Protein Microarrays
A 10,800 features protein array
Protein Microarrays & Small Molecules
- Assays with :
1. DIG- steroid dioxygening-like- recognized by
a mouse monoclonal antibody
2. Biotin, a Vitamin recognized by Avidin
3. AP1497, a ketoamide recognized by the
FKBP12 receptor
Protein Microarrays & Small Molecules
Proof of concept:
- Proteins for the corresponding
molecules spotted
- Metabolites bound to BSA that
was previously labelled with a
different fluorophore
- Unique dyes allow
simultaneous analysis of all
three interactions
DNA-CELL ARRAYS
- DNA expression plasmid
arrayed on the surface
- Cells plated on top of them
- Cells take-up DNA and produce
the protein
- Labelled compound hybridized
Yeast Three - Hybrid system
- Done in yeast or E.Coli cells
- Test protein fused to an activation
domain
Test protein
- Test compound chemically linked to
an anchor compound that interacts
with an anchor protein that has a
DNA binding domain activating a
reporter gene
- Once the activation and binding
domain are in close proximity the
reporter is activated
Interaction?
Introduction to
Secondary Metabolism
‫מטבוליטים ראשוניים‬
‫‪ ‬מופיעים בד"כ בכל האורגניזמים‬
‫‪ ‬חיוניים לחיי התא וריבויו‬
‫מטבוליטים משניים‬
‫(חומרי טבע)‬
‫‪‬‬
‫חיוניים להישרדות האורגניזם‬
‫‪ ‬תפקיד בהתמודדות עם תנאי הסביבה‪,‬‬
‫עקות ביוטיות וא‪-‬ביוטיות‬
‫‪ ‬בצמחים חשיבות רבה מכיוון ואינם זזים‬
‫חומרים משניים (חומרי טבע) בצמחים‬
‫כמעט ‪ 50,000‬חומרים משניים ידועים מצמחים‬
‫שייכים לקבוצות מטבוליות שונות‪ ,‬לפעמים קשורות‬
‫אחת בשנייה‬
‫מבנים כימיים רבים ומסובכים עם התמרות שונות‬
‫תפקיד חשוב בקביעת ערך מזוננו (וויטמינים‪ ,‬צבע‪,‬‬
‫טעם)‬
‫מקור חשוב לתרופות‪ ,‬חומרי בריאות‪ ,‬חומרי בושם ועוד‬
‫מוצרים תעשייתיים‬
Main Groups of Secondary
Metabolites in Plants
29,000 terpenes
12,000 alkaloids
8,000 phenolics
Croteau et al., 2000
Secondary Metabolites are Derived from
Primary Metabolites
The Terpenoids or
Isoprenoids
Terpenoids

More than 29.000 different structures
known in nature

Built out of C5 isoprene units

Monoterpene (C10), Sesquiterpene (C15)
and larger
OPP
Terpenoids - Important in Plants !
C5 - hemiterpenes - e.g. isoprene
C10 - monoterpenes - e.g. limonene
C15 - sesquiterpene - e.g. abscisic acid (ABA)
C20 - diterpene - e.g. gibberellin
C30 - triterpne - e.g. brassinosteroids
C40 - tetraterpenes - e.g. carotenoids
> carbons - polyterpenes- e.g. ubiquinones, rubber
mixed biosynthetic origins - meroterpenes - e.g.
cytokinines
2x Acetyl-CoA
Cytosol
MVA
pathway
Pyruvate
Plastid
G3P
CDP-ME
Aceto-acetyl-CoA
MEP
pathway
CDP-MEP
HMG-CoA
Mitochondria
IPP
Ubiquinone
Mevalonate
Me-CPP
DXP
Mevalonate
diphosphate
HMBPP
MEP
DMAPP
IPP
Prenilated
Flavonoids Irregular
Terpenes:
Chresantemyl-PP
Lavandulyl
Cytokinins
IPP
DMAPP
Methylbutenol
GPP
Farnesyl
proteins
Sesquiterpenes
Monoterpenes
Chlorophylls
Tocotrienols
FPP
Phytol
SPP
GGPP
Polyterpenes
Phytoene
Bioactive
Phytofluene Diterpenes
Geranylgeranyl
proteins
Brassinosteroids
Irregular
Terpenes:
Anistomene
GGPP
Triterpenes
Sterols
Isoprene
GPP
Plastoquinones
Chlorophylls
Tocopherolls
Phylloquinones
Zeta-Carotene
Neurosporene
The Isoprenoid Pathway
in Plants and its Branches
Pro-Lycopene
Lycopene
Other carotenoids
Giberellines
Monoterpenes
(C10)
Triterpenoids
(C30)
Tetra-terpene / Carotenoids
(C40)
The Alkaloids
The Alkaloids




Alkaloids are nitrogen containing substances
Mostly synthesized from amino acids
They are typically bitter in taste and in a lot of
cases toxic
They are most commonly found in vascular
plants but many more are being found in fungi,
microbes, insect or animals
The Alkaloids
"Interesting"
Alkaloids
CH3
N
Cocaine
O
COCH3
O C
H
O
"Interesting"
Alkaloids
Nicotine
H
N
N
CH3
Phenylpropanoids
Phenylpropanoids
Propane
Phenyl
Phenylpropanoids: Anthocyanin Pigments
"Interesting" Phenylpropanoids