PROTEIN PHYSICS LECTURE 24

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 SiteA shape that fits a specific substrate or substrates onlySide chains that attract the enzyme particular substrateSide 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 Blazer11/19/04

Immunology ReviewImmunoglobulin GMonoclonal 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.

Ene

rgy

ΔG

Progress

Abzyme stabilization of transition state

Abzymes – nearly endless possibilities

Acyl-transferCationic cyclizationDisfavored ring closureAldol/Michaels Hydride transferOxy-cope rearrangements

AbzymesSpecific for a particular reactionBut - varied enough to accept a variety of substratesCan 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 activationPotentially lower required doses

Problems with ADEPT

Immunological response to non-host enzyme (the antibody section can be humanized)

Conservation of active sites across speciesSelectivity of enzymeHard 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.