Forward Chemical Genomics Identifying the Target Protein through High-through-put Assays.
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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
- 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
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 - 5mM stock solutions after compound cleavage and resuspension in multiple well plates
De-coding using LC-MS for example
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
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
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
- 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 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
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
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)
Protein
Protein Microarrays
- Proteins are immobilized on a surface- Labelled compound incubated with the surface- Identification of compound-protein interaction through label
- 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
- 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?
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
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
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
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