PROTEIN PHYSICS LECTURE 24 PROTEINS AT ACTION: BIND TRANSFORM RELEASE BIND: repressors - turn - Znfingers DNA & RNA BINDING Leu-zipper BIND TRANSFORM BIND: Repressors -BINDING-INDUCED DEFORMATION MAKES REPRESSOR ACTIVE, and IT BINDS TO DNA BIND: Immunoglobulins Immunoglobulin BIND TRANSFORM RELEASE: ENZYMES chymotrypsin Note small active site Chymotrypsin catalyses hydrolysis of a peptide Spontaneous hydrolysis: very slow 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. 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 The Catalytic Triad chymotrypsin CHAIN CUT-INDUCED DEFORMATION MAKES ENZYME ACTIVE active cat. site Chymotripsin nonactive cat. site Chymotripsinogen SER-protease: catalysis Stage #1 Chymotrypsin Protein Hydrolysis Chymotrypsin Protein Hydrolysis Stage #2 Chymotrypsin Protein Hydrolysis Stage #3 Chymotrypsin Protein Hydrolysis Stage #4 Chymotrypsin Protein Hydrolysis Stage #5 Chymotrypsin Protein Hydrolysis Stage #6 Transition State Stabilization Chymotrypsin Kinetics The initial "burst" in chymotrypsin-catalysed hydrolysis of the p-nitrophenyl acetate CHYMOTRYPSIN ACTIVE SITE with INHIBITOR Catalytic antibodies ABZYM = AntyBody enZYM Transition state (TS) Antibodies are selected to TS-like molecule Preferential binding of TS: RIGID enzyme A novel approach to drug delivery: abzyme-mediated drug activation Levi Blazer 11/19/04 Immunology Review Immunoglobulin G Monoclonal vs. polyclonal http://www.path.cam.ac.uk/~mrc7/igs/img09.jpeg 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 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. Energy Abzyme stabilization of transition state ΔG Progress Abzymes – nearly endless possibilities Acyl-transfer Cationic cyclization Disfavored ring closure Aldol/Michaels Hydride transfer Oxy-cope rearrangements 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 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. Reactive Immunization Enaminone absorbs at 316 nm 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. Tumor Suppression Chemotherapy agent Inactivator: Removable through enzyme/abzyme catalysis Benefits of ADEPT Minimized toxicity = better! Localized activation Potentially lower required doses 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 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! -- Problems? Antibodies bind molecules. How can you use this in humans? – Immune response – Diffusion – Protein stability – Side reactions? – Natural activation? Cost! Ethical concerns. Methodology: synthesize prodrug with standard inactivator Y Administer prodrug and catalytic antibody conjugate separately. Administer catalytic antibody directly into Tumor. Normal Tissue Localized activation reduces unwanted toxicity Tumor Doxorubicin activation Topoisomerase I & II inhibitor By abzyme 38C2 Prodox synthesis Conclusions Abzyme conjugated ADEPT: – Potentially more effective – Less toxic for non-cancerous cells – Sustainable for long periods of time due to antibody half-life.