protein physics lecture 24 proteins at action: bind transform release
TRANSCRIPT
PROTEIN PHYSICS
LECTURE 24
PROTEINS AT ACTION:
BIND TRANSFORM RELEASE
BIND: repressors
- turn -
DNA & RNABINDING
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 Chymotripsinogen
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
Catalytic antibodies ABZYM = AntyBody enZYM
Antibodiesare
selectedto TS-likemolecule
Transition state (TS)
Preferentialbinding of TS:RIGID
enzyme
A novel approach to drug delivery:
abzyme-mediated drug activation
Levi Blazer
11/19/04
Immunology ReviewImmunoglobulin 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
AbzymesCatalytic 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.
En
erg
yΔG
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 systemADEPT – Antibody directed enzyme prodrug therapyChemically 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 throughenzyme/abzyme catalysis
TumorSuppression
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 TissueTumor
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.