BIND TRANSFORM RELEASE : ENZYMES Note small active site chymotrypsin
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
Chymotrypsin Kinetics The initial "burst" in chymotrypsin-catalysed hydrolysis of the p-nitrophenyl acetate
CHYMOTRYPSIN ACTIVE SITE with INHIBITOR CHYMOTRYPSIN ACTIVE SITE with INHIBITOR
Catalytic antibodies ABZYM = AntyBody enZYM Antibodiesareselected to TS-like molecule Transition state (TS) Preferentialbinding of TS: RIGIDenzyme
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 ΔGΔG Progress Abzyme stabilization of transition state
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. Chemotherapy agent Inactivator: Removable through enzyme/abzyme catalysis Tumor Suppression
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 Administer prodrug and catalytic antibody conjugate separately. Administer catalytic antibody directly into Tumor. Localized activation reduces unwanted toxicity Normal Tissue Tumor Y
Doxorubicin activation By abzyme 38C2 Topoisomerase I & II inhibitor