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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