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PROTEIN PHYSICS LECTURE 24

PROTEIN PHYSICS LECTURE 24. PROTEINS AT ACTION: BIND  TRANSFORM  RELEASE . BIND: repressors.  - turn - . Zn- fingers. DNA & RNA BINDING. Leu-zipper. BIND  TRANSFORM  BIND : Repressors. -BINDING-INDUCED DEFORMATION MAKES REPRESSOR ACTIVE, and IT BINDS TO DNA.

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PROTEIN PHYSICS LECTURE 24

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  1. PROTEIN PHYSICS LECTURE 24 PROTEINS AT ACTION: BIND  TRANSFORM  RELEASE

  2. BIND: repressors -turn-

  3. Zn- fingers DNA & RNA BINDING Leu-zipper

  4. BIND TRANSFORM BIND: Repressors -BINDING-INDUCED DEFORMATION MAKES REPRESSOR ACTIVE, and IT BINDS TO DNA

  5. BIND: Immunoglobulins

  6. Immunoglobulin

  7. BIND TRANSFORM  RELEASE: ENZYMES chymotrypsin Note small active site

  8. Chymotrypsin catalyses hydrolysis of a peptide Spontaneous hydrolysis: very slow

  9. Chymotrypsin • Chymotrypsin is one of the serine proteases. • Chymotrypsin is selective for peptide bonds with aromatic or large hydrophobic side chains, such as Tyr, Trp, Phe and Met, which are on the carboxyl side of this bond. It can also catalyze the hydrolysis of easter bond. • The main catalytic driving force for Chymotrypsin is the set of three amino acid known as catalytic triad. This catalytic pocket is found in the whole serine protease family.

  10. Properties of an Active Site • A shape that fits a specific substrate or substrates only • Side chains that attract the enzyme particular substrate • Side chains specifically positioned to speed the reaction

  11. The Catalytic Triad

  12. chymotrypsin

  13. CHAIN CUT-INDUCED DEFORMATIONMAKES ENZYME ACTIVE non-active cat. site  active cat. site ChymotripsinChymotripsinogen

  14. SER-protease: catalysis

  15. Chymotrypsin Protein Hydrolysis Stage #1

  16. Chymotrypsin Protein Hydrolysis Stage #2

  17. Chymotrypsin Protein Hydrolysis Stage #3

  18. Chymotrypsin Protein Hydrolysis Stage #4

  19. Chymotrypsin Protein Hydrolysis Stage #5

  20. Chymotrypsin Protein Hydrolysis Stage #6

  21. Transition State Stabilization

  22. Chymotrypsin Kinetics The initial "burst" in chymotrypsin-catalysed hydrolysis of the p-nitrophenyl acetate

  23. CHYMOTRYPSIN ACTIVE SITE with INHIBITOR

  24. Catalytic antibodies ABZYM = AntyBodyenZYM Transition state (TS) Preferential binding of TS: RIGID enzyme Antibodies are selected to TS-like molecule

  25. A novel approach to drug delivery:abzyme-mediated drug activation Levi Blazer 11/19/04

  26. Immunology Review • Immunoglobulin G • Monoclonal vs. polyclonal http://www.path.cam.ac.uk/~mrc7/igs/img09.jpeg

  27. Monoclonal antibody production • Why monoclonal? • Don’t tell PETA • Two forms of hybridoma preps: • Mouse Ascites • In vitro tissue culture. http://ntri.tamuk.edu/monoclonal/mabcartoon.gif

  28. Abzyme stabilization of transition state Energy ΔG Progress Abzymes • Catalytic monoclonal antibodies: usually IgG, although in theory all Ig subclasses could be created. • Created by immunizing an animal against a transition state analog (TSA) of the desired reaction. • Any non-lethal TSA antigen that can be coupled to a carrier protein can potentially create a useful abzyme.

  29. Abzymes – nearly endless possibilities

  30. Abzymes • Specific for a particular reaction • But - varied enough to accept a variety of substrates • Can be produced for any non-lethal antigen. • Easier to humanize

  31. Reactive immunization • A novel method to select and create the most catalytically active abzymes. • Use an immunogen that will react a physiological pH or will bind covalently to a B-cell receptor.

  32. Reactive Immunization Enaminone absorbs at 316 nm

  33. TumorSuppression Chemotherapy agent Inactivator: Removable throughenzyme/abzyme catalysis Overview of the old system • ADEPT – Antibody directed enzyme prodrug therapy • Chemically modify a chemotherapy agent to make it minimally toxic. • Prepare an antibody-enzyme conjugate that catalyzes the activation reaction • Use a localized injection of conjugate to selectively activate drug in tumor tissue.

  34. Benefits of ADEPT • Minimized toxicity = better! • Localized activation • Potentially lower required doses

  35. Problems with ADEPT • Immunological response to non-host enzyme (the antibody section can be humanized) • Conservation of active sites across species • Selectivity of enzyme • Hard to engineer

  36. Mother Nature: Better than Reingold • Enzymes catalyze many reactions faster and with more specificity than synthetic catalysts. • Problem: difficult to engineer an enzyme if there is no natural analog. • Why not let Mother Nature do the design work for us? -- ABZYME! --

  37. Problems? • Antibodies bind molecules. • How can you use this in humans? • Immune response • Diffusion • Protein stability • Side reactions? – Natural activation? • Cost! • Ethical concerns.

  38. Methodology: synthesize prodrug with standard inactivator Y Administer prodrug and catalytic antibody conjugate separately. Administer catalytic antibody directly into Tumor. Tumor Normal Tissue Localized activation reduces unwanted toxicity

  39. Doxorubicin activation Topoisomerase I & II inhibitor By abzyme 38C2

  40. Prodox synthesis

  41. Conclusions • Abzyme conjugated ADEPT: • Potentially more effective • Less toxic for non-cancerous cells • Sustainable for long periods of time due to antibody half-life.

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