First lecture


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First lecture

  1. 1. English presentation skills: seminars in chemical biology
  2. 2. Tell me and I'll forget Show me and I may remember Involve me and I'll understand Chinese proverb
  3. 3. Overview of the course 1 st lecture: General information Introduction in Chemical Biology 2 nd lecture: How to search for papers How to prepare for a lecture 3 rd lecture: Technical aspects: Communication & Powerpoint use
  4. 4. Overview of the course <ul><li>Seminar 4-7: 1 short presentation (scientific paper) </li></ul><ul><li>Seminar 8-end: long presentation (topic/subfield in </li></ul><ul><li> chemical biology) </li></ul><ul><li>Topic is free to choose: </li></ul><ul><ul><li>Restricted to chemical biology </li></ul></ul><ul><ul><li>A list of sample topics + authors will be provided </li></ul></ul>
  5. 5. Overview of the course <ul><li>After every talk: </li></ul><ul><ul><li>Questions & Anwsers </li></ul></ul><ul><ul><li>Comments about presentation style </li></ul></ul><ul><ul><li>10 minutes (paper); 25-30 minutes (topic) </li></ul></ul><ul><ul><li>5 minutes Q&A </li></ul></ul><ul><ul><li>5 minutes input </li></ul></ul>
  6. 6. Overview of the course <ul><li>Presenter: gives presentation </li></ul><ul><li>Chair: introduces speaker + handle Q&A </li></ul><ul><li>Comments: </li></ul><ul><ul><li>At least: one positive comment </li></ul></ul><ul><ul><li>suggestions for improvement </li></ul></ul>
  7. 7. Evaluation <ul><li>Verbal communication </li></ul><ul><li>Non-verbal communication </li></ul><ul><li>Powerpoint slides </li></ul><ul><li>Explanation of presented topic </li></ul><ul><li>Participation in class </li></ul>
  8. 8. Attending the class <ul><li>“ Seventy percent of success in life is showing up” </li></ul>Woody Allen “ 95% of success in this class, is showing up!” (max 1 lecture not present)
  9. 9. E-mail <ul><li>[email_address] </li></ul>
  10. 10. What is Chemical Biology? English presentation skills: seminars in chemical biology
  11. 11. Chemical Biology is… <ul><li>Chemical Biology seeks to develop new tools based on small molecules that allow minimal perturbation of biological systems while providing detailed information about their function. </li></ul>
  12. 12. Why small molecules if you can make knock-out mice? <ul><li>Small molecules: </li></ul><ul><ul><li>Allow Temporal controll </li></ul></ul><ul><ul><li>No compensation effects </li></ul></ul><ul><ul><li>Allow study of lethal knock-outs </li></ul></ul><ul><li>Lead compound for drug development </li></ul>
  13. 13. Overview <ul><li>Small molecule screens: looking for targets </li></ul><ul><li>Proteomics: identifying targets </li></ul><ul><li>Probe design for enzymes: smart targeting </li></ul><ul><li>Imaging techniques: visualizing targets </li></ul>
  14. 14. Forward chemical genetics <ul><li>Libraries of small molecules </li></ul><ul><li>Looking for phenotypes </li></ul><ul><ul><li>Easy readout </li></ul></ul><ul><ul><li>High throughput (96-well, 384-well) </li></ul></ul><ul><li>Chemicals as ‘mutagens’ </li></ul>
  15. 15. Read-out <ul><li>Fluorescence widely used detection method </li></ul><ul><ul><li>Antibodies </li></ul></ul><ul><ul><li>GFP reporters </li></ul></ul><ul><li>More elaborate, but also possible: </li></ul><ul><ul><li>Automated image analysis </li></ul></ul>
  16. 16. Screening = sifting <ul><li>Be aware of: </li></ul><ul><ul><li>False positives </li></ul></ul><ul><ul><li>False negatives </li></ul></ul><ul><li>False positives: signal with inactive compound </li></ul><ul><li>False negative: no signal with active compound </li></ul>
  17. 17. Screening = sifting <ul><li>Often: several selectivity-filters </li></ul><ul><li>2 nd or 3 rd screen: lower throughput </li></ul><ul><ul><li>Gel-based analysis </li></ul></ul><ul><ul><li>Microscopy-based analysis </li></ul></ul>
  18. 18. Example
  19. 19. Goal <ul><li>Get to know more about mitosis </li></ul><ul><ul><li>DNA duplicates and condenses </li></ul></ul><ul><ul><li>Mitotic spindle forms, chromosomes align </li></ul></ul><ul><ul><li>Centrosomes divide </li></ul></ul><ul><ul><li>Nuclear envelope is formed </li></ul></ul><ul><ul><li>Production of two daughter cells </li></ul></ul>
  20. 20. Question <ul><li>What proteins are involved? </li></ul><ul><li>Only known small molecule inhibitors </li></ul><ul><li>target tubulin </li></ul>Green: tubulin Blue: chromatin Taxol Vinblastine
  21. 21. 1 st Screen: mitotic arrest <ul><li>Nucleolin -> phosphorylated during mitosis </li></ul><ul><li>‘ cytoblot’ assay </li></ul><ul><ul><li>Detects post-translational modification in whole cells </li></ul></ul><ul><ul><li>Low volumes ( μ l-nl) </li></ul></ul>
  22. 22. ‘ Cytoblot’ <ul><li>Culture cells in 384, 1536- or 6144-well plates </li></ul><ul><li>Incubate with library compounds/controls </li></ul>Stockwell, Schreiber, Chem. Biol. 1999 <ul><li>Fix & detect by antibody-HRP conjugate </li></ul>
  23. 23. Screening for mitotic arrest <ul><li>Use antibody against Phospho-Nucleolin </li></ul><ul><li>139 of 16320 compounds show increase </li></ul>
  24. 24. Second screen: in vitro tubulin polymerization <ul><li>Why? We don’t want tubulin-targeting molecules </li></ul><ul><li>86 show no effect </li></ul><ul><li>52 destabilization </li></ul>Q: which ones to continue with?
  25. 25. Third screen: fluorescent microscopy <ul><li>86 compounds: </li></ul><ul><ul><li>27: no effect -> false positives </li></ul></ul><ul><ul><li>42: effects in interphase & mitosis </li></ul></ul><ul><ul><li>5: only affect mitosis </li></ul></ul><ul><li>Stain: </li></ul><ul><ul><li>Tubulin (green) </li></ul></ul><ul><ul><li>Chromatin (blue) </li></ul></ul><ul><li>Visual inspection of microtubules </li></ul><ul><li>and chromosomes </li></ul>
  26. 26. One interesting compound: “Monastrol” <ul><li>Result: no spindle bipolarity </li></ul><ul><li>Eg5 (a kinesin – motor protein): </li></ul><ul><li>involved in separation of </li></ul><ul><li>centrosomes </li></ul><ul><li>Not a general kinesin inhibitor: </li></ul><ul><li>organel localization normal </li></ul>
  27. 27. Overview <ul><li>Small molecule screens: looking for targets </li></ul><ul><li>Proteomics: identifying targets </li></ul><ul><li>Probe design for enzymes: smart targeting </li></ul><ul><li>Imaging techniques: visualizing targets </li></ul>
  28. 28. Proteomics <ul><li>Proteomics is the study of the proteome , which is the complete set of proteins expressed by the genome of an organism, tissue or cell under a given set of conditions. </li></ul><ul><li>Central techniques: </li></ul><ul><ul><li>Protein separation </li></ul></ul><ul><ul><li>Protein identification </li></ul></ul>
  29. 29. Proteomics by gel electrophoresis <ul><li>2D gels: </li></ul><ul><ul><li>Spot intensity ~ protein abundance </li></ul></ul>pI MW Normal state Disease state <ul><li>Comparison of different cell states: </li></ul><ul><ul><li>Look for up- or downregulated proteins </li></ul></ul>
  30. 30. Proteomics by chromatography <ul><li>1 or 2 liquid chromatography columns: </li></ul><ul><ul><li>Reverse phase column </li></ul></ul><ul><ul><li>Sometimes in series after SCX column </li></ul></ul>SCX cation exchange reverse phase Mass spectrometer
  31. 31. Use small molecule as fishing hook Leslie, Hergenrother, Chem. Soc. Rev. 2008 , 1347-60 <ul><li>Make affinity-resin </li></ul>
  32. 32. Enriching for your target <ul><li>Incubate with cell lysate/tissue homogenate </li></ul><ul><li>Wash to eliminate non-binding proteins </li></ul><ul><li>Elute: </li></ul><ul><ul><li>Denature </li></ul></ul><ul><ul><li>Add free ligand </li></ul></ul>
  33. 33. Identify targets by Mass Spectrometry <ul><li>After elution: purification and digestion </li></ul>
  34. 34. Identify your target: mass fingerprint <ul><li>Ionize tryptic peptides </li></ul><ul><li>Search database: </li></ul><ul><ul><li>Which proteins give rise to these tryptic peptides? </li></ul></ul><ul><ul><ul><li>748.404 </li></ul></ul></ul><ul><ul><ul><li>920.415 </li></ul></ul></ul><ul><ul><ul><li>1271.61 </li></ul></ul></ul><ul><ul><ul><li>1378.78 </li></ul></ul></ul><ul><ul><ul><li>1484.97 </li></ul></ul></ul><ul><ul><ul><li>1502.62 </li></ul></ul></ul><ul><ul><ul><li>1562.8 </li></ul></ul></ul><ul><ul><ul><li>1606.82 </li></ul></ul></ul><ul><ul><ul><li>1815.83 </li></ul></ul></ul><ul><ul><ul><li>1853.89 </li></ul></ul></ul><ul><ul><ul><li>1884.95 </li></ul></ul></ul><ul><ul><ul><li>1981.97 </li></ul></ul></ul><ul><ul><ul><li>Example: </li></ul></ul></ul>
  35. 35. Enter tryptic peptide values <ul><li>Online engine at </li></ul><ul><li> </li></ul>
  36. 36. And there are your hits…
  37. 37. Identify your target: tandem MS <ul><li>Ionize tryptic peptides </li></ul><ul><li>Fragment peptides </li></ul><ul><li>Algorithm can assign </li></ul><ul><li>peptide sequence & search </li></ul><ul><li>database </li></ul>
  38. 38. For more info <ul><li>See: for </li></ul><ul><li>proteomic tutorials and resources </li></ul>
  39. 39. Overview <ul><li>Small molecule screens: looking for targets </li></ul><ul><li>Proteomics: identifying targets </li></ul><ul><li>Probe design for enzymes: smart targeting </li></ul><ul><li>Imaging techniques: visualizing targets </li></ul>
  40. 40. ‘ Activity-based Proteomics’ <ul><li>Use enzyme mechanism => covalent tagging </li></ul><ul><li>Why important? </li></ul><ul><ul><li>Enzymes translated as zymogens </li></ul></ul><ul><ul><li>Activity tightly regulated </li></ul></ul>
  41. 41. Regulation of protease activity <ul><li>Activation of proteases: </li></ul><ul><ul><li>pH </li></ul></ul><ul><ul><li>[Ca 2+ ] </li></ul></ul><ul><ul><li>dimerization </li></ul></ul><ul><ul><li>post-translational modifications… </li></ul></ul><ul><li>Endogenous inhibition of proteases </li></ul>Riedl et al ., Cell 2001
  42. 42. Proteases: mechanism of action
  43. 43. ‘ Activity-based Probes’ <ul><li>Small molecule reporters of active enzymes </li></ul>Tag Spacer Warhead Activity-based Probe (ABP) Biotin Fluorophore Radioisotope Tag - Detection … Peptide Alkyl Spacer - Selectivity Electrophile Warhead
  44. 44. Activity-based probes <ul><li>Covalent inhibitors with a tag </li></ul>Cravatt et al ., PNAS 1999 , Bogyo et al. , Chem Biol. 2000
  45. 45. Covalent modification of active site <ul><li>Only with active enzyme </li></ul>
  46. 46. Profiling of protease activity <ul><li>Profiling of Protease Activity </li></ul>Mouse Pancreatic Cancer Model: Cathepsin activities upregulated Joyce et al ., Cancer Cell 2004
  47. 47. When it’s hard to get selectivity… <ul><li>Kinases: 491 kinase domains in human genome </li></ul><ul><li>most drugs target ATP binding site </li></ul><ul><li>~ 20% of kinases have small Thr- “gatekeeper” </li></ul>Bishop, A.C.; Shokat, K. M. et al ., Nature 2000 , 407 , 395
  48. 48. Look for a second selectivity filter <ul><li>Cysteine in flexible loop in 11 kinases </li></ul>Cohen, M. S, Taunton, J et al ., Science 2005 , 308 , 1318
  49. 49. Inhibition of specific kinases <ul><li>RSK2 kinase: </li></ul><ul><ul><li>Has got both Thr and Cys </li></ul></ul><ul><ul><li>Can autophosphorylate itself </li></ul></ul>Detection: autophosphorylation of RSK2 Cohen, M. S, Taunton, J et al ., Science 2005 , 308 , 1318
  50. 50. Inhibition of specific kinases <ul><li>Mutating MSK1 gatekeeper: sensitization </li></ul><ul><li>Does it work the other way around? </li></ul><ul><li>MSK1 kinase: </li></ul><ul><ul><li>Has Cys but NO Thr </li></ul></ul><ul><ul><li>Can autophosphorylate itself </li></ul></ul>Cohen, M. S, Taunton, J et al ., Science 2005 , 308 , 1318
  51. 51. What about specificity? <ul><li>Autophosphorylation: </li></ul><ul><ul><li>indirect measurement of inhibitor binding </li></ul></ul><ul><ul><li>does not confirm selectivity </li></ul></ul><ul><li>Direct labeling experiment: </li></ul>Cohen, M. S, Taunton, J et al ., Nature Chem. Biol. 2007 , 3 , 156
  52. 52. Implications of this study <ul><li>Inhibition of specific kinases by mutation & small molecules </li></ul><ul><li>Useful tool to study functional role of a single kinase </li></ul>
  53. 53. Overview <ul><li>Small molecule screens: looking for targets </li></ul><ul><li>Proteomics: identifying targets </li></ul><ul><li>Probe design for enzymes: smart targeting </li></ul><ul><li>Imaging techniques: visualizing targets </li></ul>
  54. 54. Why imaging? <ul><li>Protein localization </li></ul><ul><li>Detection of pathologies in vivo </li></ul><ul><li>Most common: GFP and derivatives: </li></ul><ul><ul><li>Different colors available </li></ul></ul><ul><ul><li>Large protein </li></ul></ul><ul><li>Alternatives: </li></ul><ul><ul><li>Target small peptide sequences (modified protein) </li></ul></ul><ul><ul><li>Target certain activity </li></ul></ul>
  55. 55. Targeting modified proteins Chen et al. 2005 Curr. Opin. Biotech. 16 , 35-40 Protein Tag Peptide Tag Enzyme mediated Tag Unnatural amino acid
  56. 56. Peptide tags <ul><li>‘ Chemical fluorophores’ </li></ul><ul><ul><li>FlAsH </li></ul></ul><ul><ul><li>… (a ton more) </li></ul></ul>Tsien et al. 2000 Methods Enzymol. <ul><li>FlAsH: </li></ul><ul><ul><li>Tight binding </li></ul></ul><ul><ul><li>Multiple colors </li></ul></ul>
  57. 57. FlAsH application <ul><li>Study dynamic structures: </li></ul><ul><li>Gap junction plaques </li></ul><ul><ul><li>Small molecule signaling between cells </li></ul></ul><ul><ul><li>Half life <5 hours </li></ul></ul><ul><li>Tetracysteine tag on Connexin43 </li></ul>Tsien, Ellisman et al ., Science 2002
  58. 58. Target certain activity <ul><li>Activity-based probe mediated imaging </li></ul><ul><ul><li>Clan CA proteases in Pancreatic cancer </li></ul></ul>Normal islets Angiogenic islets Tumors
  59. 59. Is this all? <ul><li>Carbohydrate detection </li></ul><ul><li>Detection of other post-translational modifications </li></ul><ul><li>Probes to monitor [Ca 2+ ] or other metals </li></ul><ul><li>… </li></ul>