Notes 20101020 CHEM 731 Therapeutic Proteins
From SnOwy - Ed's Wiki Notebook
Continuing on ...
Contents |
Last week
- antibody protein therapeutics
- target diseases
- drug targets
- versus small molecules
Enzyme therapeutics
- PEGylated L-asparaginase to degrade extracellular asparagine to slow down leukemia -- starve a tumour of an essential amino acid
- PEG-arginine deaminase in trials for melanoma and carcinoma
- glycosylated human DNase I -- degrades mucus in cystic fibrosis
Editing existing proteins
- changing the C-terminus arginine to glycine changes the isoelectric point of the insulin -- precipitates
Title: Computational stabilization of human growth hormone (HGH)
- problem: human growth hormone degrades too quickly in the body and also on the shelf
- recommendation: increase lifespan of HGH
To be honest, at this point in the class, I was so worried about my biol614 project that I couldn't really concentrate. I'm sure I did a good job with the proposal; I even included a glossary since I was told I was too vague previously. I think I might go home at the break, I simply am too full of anxiety to take good notes.
Title: Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching"
- changed stability by altering the histidine composition of a protein (position specific)
Okay, here begins the second hour of the class -- I think I'm feeling a bit better -- we're now discussing non-antibody scaffolds.
Non-antibody scaffolds
- certain folds are very good at binding lots of different things
- most natural scaffolds come from binders which have multiple targets
- examples are of course antibodies
- other examples: tim barrels, ankyrin repeats (which are immunity proteins in fish)
- antibodies are large and expensive to make -- is a heterodimer, requires glycosylation, disulfide bonds
- we can avoid all of these challenges by using smaller scaffolds
Protein-protein binding determinants
- correct electrostatic distribution on exposed surface
- complementary shape
- both add to a well formed interface
Title: Engineered protein scaffolds as next-generation antibody therapeutics
- in the early 2000s, over 50 scaffolds have been proposed
- in recent years, we have been focusing on a select subset
- includes affibodies (small: 58 residues; triple alpha-helix) -- things bind to the helices
- monobodies; anticalins -- bind lipids, beta barrel; DARPins -- Designed Ankyrin Repeat Protein
- consolidation -- ?
- modeling an induced fit is currently difficult -- selecting for an induced fit is easier
- is not often the case that induced fits occur in antibodies
- an induced fit is the phenomenon of a binding interface changing conformation in order to match its ligand
Successful scaffolds
- immunoglobulin-like
- multi-domain -- multiple binding sites: recognition of interfaces between two binding sites
- momeric
- when designing interfaces -- particular residues are excellent at binding -- example is tyrosine.
Modulating protein structure
- core repacking -- given a scaffold, redesign the core sequence, many successes
- changing protein structure in a predictable way
- must be able to predict core sequence well
- computation used more than selection
- early core designs: fixed backbone
- rotamer library assists in modeling side chain conformation
- known structures favour particular rotamers
- a significant (~1/3) number of amino acids have no specific rotamer
- complete repacks, surface design
- core hydrogen bonds, polar residues
.
.
.
Entropy
Title: Conformational entropy of alanine versus glycine in protein denatured states
- replacing a glycine in a helix with alanine will cause that helix to stabilize
- glycine has been thought to increase the entropy of the folded state of proteins due to the number of conformations it may take
- it is now known that the entropy increased is counter balanced by the enthalpic term due to the interaction of alanine with water
- we now think that the alanine has a stabilizing effect due to its sidechain interaction with its neighbours
