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Chemical Proteomics Jürgen Eirich, 2016-10-12 Outline Definitions and general considerations The three parts of a chemical proteomics project: 1: The small molecule 2: The protein sample 3: Identification and quantification Your lab project Literature / Reviews 2 Chemical Proteomics... … is a multidisciplinary science spanning molecular and cell biology, biochemistry, pharmacology, analytical and organic chemistry, physics, informatics,… … aims to study how small molecules (“chemicals”) of synthetic or natural origin bind to proteins and modulate their function. … can be applied in drug target discovery or to identify small-molecule probes as research tools to study protein function. … often relies on current state-of-the-art in protein mass spectrometry (MS) as analytical strategy. DOI: 10.1007/978-1-61779-364-6 3 Definitions A small molecule is a low molecular weight organic compound (< 900 Daltons) that may help regulate a biological process. Most drugs are small molecules. A drug is any substance other than food, that when consumed causes a physiological change in the body. A chemical compound (or just compound if used in the context of chemistry) is an entity consisting of two or more atoms, at least two from different elements, which associate via chemical bonds. wikipedia.org – to encourage further reading... 4 A probe A chemical probe is a selective small-molecule modulator of a protein’s function that allows the user to ask mechanistic and phenotypic questions about its molecular target in biochemical, cell-based or animal studies. potency and selectivity target-dependent action (in cell-based or a cell-free context) structure and/or well-characterized derivative known utility as a probe to address the molecular target available to the academic community with no restrictions on use (in an ideal case…) DOIs: 10.1038/nchembio.1867, 10.1038/nchembio.296, 10.1038/nature09504 5 Different purposes and requirements for chemical probes and drugs. • may have undefined mechanism of action (MoA) • defined MoA is essential • selectivity • intelectual property (IP) restrictions; limited availability • compound itself and activity data (freely) available • must be bio-available • drug-like properties not necessarily required • high bar for physicochemical and pharmaceutic properties • structurally related inactive and structurally unrelated active compounds DOI:10.1038/nchembio.1867 6 Typical three parts of a project Typical three parts of a project (and this lecture): 1: selection, design and/or synthesis of the compound(s) 2: exposure to the protein samples 3: identification and quantification of target(s) DOI:10.1038/nchembio.216 7 Typical three parts of a project: summary 1: selection, design and/or synthesis of the compound(s) discovery of small molecule protein targets generation of specific chemical probes compound repurposing affinity capture (beads, micorarrays) tagged probes (biotin, bio-orthogonal “click” handles (alkyne, azide, …)) unmodified/label-free compounds enzyme classes vs. single proteins (drug target) protein kinases, dehydrogenase enzymes, adenine-binding proteins, lipid electrophiles (known) onco-protein/variant 8 Typical three parts of a project: summary 2: exposure to the protein samples (complex, physiological) environment: lysate, intact cells, model organism, clinical material (rather than on isolated individual proteins) enrichment of target proteins in the presence of competing free test compound Include cross-linking to study protein–drug interactions, or ligand modulation of protein–protein interactions 9 Typical three parts of a project: summary 3: identification and quantification of target(s) targeted vs. untargeted/unbiased antibodies vs. fluorescent dyes vs. MS quantitative MS with isobaric mass tags or metabolic labeling to determine the potency for a target find target protein(s) off-targets (as the cause of side effects) biomarkers for treatment research tool compound vs. drug candidate 10 Typical three parts of a project Examples from current literature to explain/highlight certain aspects in one of the typical three parts of a chemical proteomics project/paper. 11 1: The compound discovery of small molecule (off) targets generation of specific chemical probes compound repurposing DOI: 10.1146/annurev-biochem-060713-035708 12 Aspirin-Dependent Protein Modification in Living Cells Aspirin covalent inhibition of cyclooxygenase enzymes through non-enzymatic acetylation of key serine residues identification of 120 proteins, 112 of which had not been previously reported to be acetylated by aspirin DOI: 10.1021/ja408322b 13 1: The compound affinity capture (beads, micorarrays) tagged probes (biotin, click handles (alkyne, azide, …)) unmodified compounds DOI: 10.1146/annurev-biochem-060713-035708 14 Analysis of glycans using a bio-orthogonal chemical reporter strategy. attachment of a bio-orthogonal functional group (an azide -N3) to chemically modified precursors of monosaccharides (sugars) incorporated into glycans by living cells after uptake detection by bio-orthogonal ligation and adding a compound that carries a readily detectable tag DOIs: 10.1038/nprot.2007.422, 10.1038/nbt1004-1244 15 1: The compound enzyme classes vs. single proteins (drug target) protein kinases, dehydrogenase enzymes, adenine-binding proteins, lipid electrophiles (known) onco-protein/variant DOI: 10.1146/annurev-biochem-060713-035708 16 Kinobeads Strategy for assessing inhibitor selectivity in proteomes: kinase native Kinobeads use immobilized, broad-spectrum kinase ligands to enrich active kinases from complex proteomes. DOI: 10.1146/annurev-biochem-060713-035708 17 Kinobeads compound concentration protein displacement protein compound affinity DOIs: 10.1038/nbt1328 & 10.1021/pr5012608 18 Targets of Imatinib Western blot analysis of proteins captured on kinobeads: Imatinib treatment of lysate (top panel) and cells in culture (second panel) similarly reduces the amount of target protein DDR1 captured on kinobeads. phosphorylation-specific Y703P-KIT antibody (third panel) and a general KIT antibody (bottom panel) show that only the phosphorylated species are affected by Imatinib. Discoidin Domain Receptor family, member 1 receptor Tyrosine KInase DOIs: 10.1038/nbt1328 & 10.1021/pr5012608 19 The other way around A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity Engineered, immobilized enzymes, but unmodifyed compounds, Fluoresence read out... DOI: 10.1038/nchembio.1578 20 2: The protein sample environment: lysate, intact cells, model organism, clinical material (rather on isolated individual proteins) enrichment of target proteins in the presence of competing, free test compound Include cross-linking to study protein–drug interactions, or ligand modulation of protein–protein interactions 21 In vivo imaging C. elegans embryos are labeled with the sugar-derived probe Ac4GalNAz (OAcetylated N-AZidoGalactosamine) and reacted with DIFO-488 (green) for detection via fluorescent microscopy incubated again with Ac4GalNAz to metabolically label a second, newer population of glycans reacted with DIFO-568 (red) to distinguish the old from the new populations DOI: 10.1021/cb900254y 22 In vivo imaging DIC: differential interference contrast, 488 and 568 fluorescence channels Larval stages L1 (top panels), L2 (middle panels), or adult (bottom panels) C. Elegans metabolically labeled with azide-sugars were reacted with DIFO-488 and DIFO-568 in different developmental stages. Yellow indicates co-localization. DOI: 10.1021/cb900254y 23 Competition assay in theory… inhibitor administered to a biological system (cell, animal model) proteomes are isolated and treated with a probe suitable for the targets of interest selective labeling of (still) active (uninhibited) enzymes. DOI: 10.1146/annurev-biochem-060713-035708 24 … and practice By competition with a enzyme class specific probe the selectivity of inhibitors for a target protein is measured DOI: 10.1021/jm400898x 25 3: Identification and quantification targeted vs. unbiased antibody-based vs. fluorescence vs. MS quantitative MS with isobaric mass tags or metabolic labeling to determine the potencies for a large number of targets in a single analysis. Find target protein(s) off-targets (as the cause of side effects) biomarkers for treatment binding sites DOI: 10.1146/annurev-biochem-060713-035708 26 Isotope-coded taggs and linkers ICAT (Isotope Coded Affinity Tag) reagent: mass difference of 8 Da at a charge state of two three elements: an affinity tag (biotin), to isolate labeled peptides; a linker with stable isotopes (X); a reactive group specific towards thiol groups of cysteines. The reagent exists in two forms, heavy and light where X either represents either deuterium or hydrogen leading to a mass shift. DOI: 10.1038/13690 27 Binding site identification MS/MS sequencing shows the binding of the aspirin probe to Lys46 of histone H1.4 Locations of aspirinmodified residues in the protein GAPDH. Numbers of the aspirin-modified amino acid residues. DOI: 10.1038/srep07896 28 Protein stabilization by ligand binding When proteins in their native conformation are subjected to denaturing conditions, they lose higher-order structures, leading to the unfolded state. Protein unfolding, or denaturation, can be deterred by the presence of a bound ligand (e.g. buffer component, drug, …). These changes are the bases for different assays that identify ligands or other conditions that increase the stability of a protein. DOI: 10.1056/NEJMcibr1308868 29 Protein stabilization by ligand binding protein (+/- ligand) isolated or in a cell denaturing conditions isolation of (native) proteins identification DOI: 10.1056/NEJMcibr1308868 30 Denaturing conditions (a) Drug Affinity Responsive Target Stability (DARTS): Ligand bound proteins are more resistant to proteolysis in the presence of denaturant. Non-binding proteins are hydrolyzed. Proteolysis resistant proteins can be analyzed by MS. DOIs: 10.1016/j.cbpa.2012.12.022, 10.1039/C6MD00045B 31 Denaturing conditions DOIs: 10.1016/j.cbpa.2012.12.022, 10.1039/C6MD00045B (b) Stability of Proteins from Rates of OXidation (SPROX): Ligand bound proteins are more resistant to oxidant, thus requiring higher amounts of denaturant to generate the same degree of oxidation compared to non-binders. 32 CEllular Thermal Shift Assay Western blot quantification DOIs: 10.1038/nprot.2014.138, 10.4155/fmc.15.50, 10.1146/annurev-pharmtox-010715-103715 33 MS TPP Thermal Proteome Profiling (TPP): a) Temperature Range (TR) experiment b) IsoThermal-Dose-Response (ITDR) Encoding temperatures or compound concentrations as TMT channels DOIs: 10.1038/nprot.2015.101, 10.1038/nmeth.3590 34 Hands on - Lab course The three parts of your lab project: 1: selection, design and/or synthesis and purchase of the probe Azide-tagged probe for click mediated pull down 2: exposure to the protein samples In cell lysate 3: identification and quantification of target(s) via MS 35 Literature: Review Articles Chemical Proteomics - Methods and Protocols, Editors: G. Drewes, M. Bantscheff DOI: 10.1007/978-1-61779-364-6 Target profiling of small molecules by chemical proteomics, U. Rix, G. Superti-Furga DOI:10.1038/nchembio.216 Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack, S. Ziegler, et al., DOI: 10.1002/anie.201208749 Enzyme Inhibitor Discovery by Activity-Based Protein Profiling, M. Niphakis, B. Cravatt DOI: 10.1146/annurev-biochem-060713-035708 36 ABPP: Activity-Based Protein Profiling probe reporter Curr Opin Chem Biol. 2012 (1-2): p 227-233. Probe design Reactive group: mechanism-based inhibitors, (protein-reactive) natural products general electrophilic moieties photoreactive chemotypes Binding group many vary in non-covalent interactions size hydrophobicity Tags: Dye, Biotin, Azide, Alkyne, … Annu Rev Biochem (2008) 77: 383 - 414 Photo cross linkers Aryl azides Alkyl/Aryl diazirines Benzophenones Affinity-Based Probe (AfBP) Top Curr Chem (2012) 324: 85-113