Forward Chemical Genomics

Identifying the Target Protein through High-through-put Assays

Forward Chemical Genomics

Forward

(phenotype based screen)

Phenotype

Small MoleculesCausing

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)

Solid supports are combined, mixed and redistributed between synthetic steps

A single bead serves as individual reaction vessels during split-pool library synthesis

Diversity Oriented Synthesis

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 glass-slides (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 Specificity- A 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

- 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

Using Dos and SMMs for Specific Modulation of Proteins

- 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

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 lysate2. 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

FKBP12RECEPTOR

Detection of Binding to Ligands with Varying Affinity

- Derivatives of AP1497 (a known binder of FKBP12) synthesized and tested

- Specific Tag-antibodies, 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 2.24- positive (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 Cy5-labeled 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

MMiicrocroMMaacrocro

SurfaceSurfaceglass glass slidesslides

flat cellulose, filter flat cellulose, filter paperpaper

FabricatioFabricationn

spotted spotted synthesized directly synthesized directly on array via solid-on array via solid-phase synthesisphase synthesis

SizeSize0.1 mm0.1 mm6 mm6 mm

Small Molecule MACROarays vs. MICROarrays

MMiicrocroMMaacrocroAmount of Amount of compoundcompoundpicomolespicomolesnanomolesnanomoles

Cleavage Cleavage and and isolation isolation of of compoundcompound

difficult, difficult, low low quantitiesquantities

Relatively easy, large Relatively easy, large quantitiesquantities

Feature Feature densitydensityhighhighlowerlower

Small Molecule MACROarays vs. MICROarrays

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

On Support

Off Support

Protein

Protein Microarrays

- Proteins are immobilized on a surface- Labelled compound incubated with the surface- Identification of compound-protein interaction through label

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 & Small Molecules

Protein Microarrays

A 10,800 features protein array

- 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

Protein Microarrays & Small MoleculesProof 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 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

Test protein

Interaction?

Introduction to Secondary Metabolism

מטבוליטים מטבוליטים ראשונייםראשוניים

מופיעים בד"כ בכל מופיעים בד"כ בכלהאורגניזמיםהאורגניזמים

חיוניים לחיי התא וריבויו חיוניים לחיי התא וריבויו

מטבוליטים משנייםמטבוליטים משניים)חומרי טבע()חומרי טבע(

חיוניים להישרדות האורגניזם חיוניים להישרדות האורגניזם

,תפקיד בהתמודדות עם תנאי הסביבה, תפקיד בהתמודדות עם תנאי הסביבה עקות ביוטיות וא-ביוטיות עקות ביוטיות וא-ביוטיות

בצמחים חשיבות רבה מכיוון ואינם זזים בצמחים חשיבות רבה מכיוון ואינם זזים

חומרים משניים )חומרי טבע( חומרים משניים )חומרי טבע( בצמחיםבצמחים

חומרים משניים ידועים מצמחים50,000כמעט

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 Main Groups of Secondary Metabolites in PlantsMetabolites in Plants

29,000 terpenes29,000 terpenes

12,000 alkaloids12,000 alkaloids

8,000 phenolics8,000 phenolicsCroteau et al., 2000

Secondary Metabolites are Derived from Primary Metabolites

The Terpenoids or The Terpenoids or IsoprenoidsIsoprenoids

TerpenoidsTerpenoids

More than 29.000 different structures More than 29.000 different structures

known in natureknown in nature

Built out of C5 isoprene unitsBuilt out of C5 isoprene units

Monoterpene (C10), Sesquiterpene (C15) Monoterpene (C10), Sesquiterpene (C15)

and largerand larger

OPP

Terpenoids - Important in PlantsTerpenoids - 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

Polyterpenes

Farnesylproteins

Sesquiterpenes

FPP

Geranylgeranyl proteins

DMAPPIPP

GPP

GGPP

DMAPP IPP

Phytoene

Triterpenes

ChlorophyllsTocotrienols

Monoterpenes

Plastoquinones

GPP

GGPP

Phytol

IrregularTerpenes:

Anistomene

Isoprene

Methylbutenol

IrregularTerpenes:

Chresantemyl-PPLavandulyl

Cytokinins

Bioactive Diterpenes

HMBPP

Giberellines

SPPChlorophyllsTocopherolls

Phylloquinones

PrenilatedFlavonoids

Cytosol Plastid

Mitochondria

IPP

Ubiquinone

MVA pathway

MEPpathway

Phytofluene

Zeta-Carotene

Neurosporene

Pro-Lycopene Lycopene

Me-CPP

CDP-MEP

CDP-ME

MEP

DXP

G3PPyruvate

Mevalonatediphosphate

Mevalonate

HMG-CoA

Aceto-acetyl-CoA

2x Acetyl-CoA

The Isoprenoid Pathway in Plants and its Branches Other carotenoids

Sterols

Brassinosteroids

Monoterpenes Monoterpenes (C(C1010))

TriterpenoidsTriterpenoids(C(C3030))

Tetra-terpene / Carotenoids Tetra-terpene / Carotenoids (C(C4040))

The AlkaloidsThe Alkaloids

The AlkaloidsThe Alkaloids

Alkaloids are nitrogen containing substancesAlkaloids are nitrogen containing substances Mostly synthesized from amino acidsMostly synthesized from amino acids They are typically bitter in taste and in a lot of They are typically bitter in taste and in a lot of

cases toxiccases toxic They are most commonly found in vascular They are most commonly found in vascular

plants but many more are being found in fungi, plants but many more are being found in fungi, microbes, insect or animalsmicrobes, insect or animals

The AlkaloidsThe Alkaloids

""Interesting" Interesting" AlkaloidsAlkaloids

NCH3

O

COCH3

O

C

OH

Cocaine

N

NH

CH3

Nicotine

""Interesting" Interesting" AlkaloidsAlkaloids

PhenylpropanoidsPhenylpropanoids

PhenylpropanoidsPhenylpropanoids

Phenyl

Propane

Phenylpropanoids: Anthocyanin Pigments

"Interesting" Phenylpropanoids