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

BIND: repressors  - turn - 

DNA & RNA BINDING Zn-fingers Leu-zipper

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

BIND: Immunoglobulins

Immunoglobulin

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

The Catalytic Triad

chymotrypsin

CHAIN CUT-INDUCED DEFORMATION MAKES ENZYME ACTIVE Chymotripsin Chymotripsino gen non- active cat. site  active cat. site

SER-protease:catalysis

Chymotrypsin Protein Hydrolysis Stage #1

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 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

Monoclonal antibody production Why monoclonal? Don’t tell PETA Two forms of hybridoma preps: –Mouse Ascites –In vitro tissue culture.

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

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 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

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.