PART VI: PROTEIN THERAPEUTICS Flashcards
What is the phenomenon of “the crowded cell” in terms of proteins?
- that proteins function dynamically within larger macromolecular assemblies or in cellular pathways –> for protein-protein and protein-nucleic acid complexes
- so in reality there are HEAPS of proteins in the cell
Is any single experimental technique sufficient to solve the molecular architecture of biologcially relevant complexes?
- NO
- This is due to its size and complexity
What did the T3SS evolve from?
- the bacterial flagellum
What is the injectosome role?
- Inject and maintain host cell pathogenesis
When will the needle be synthesised in terms of the injectosome?
- When the bacterium comes into contact with the eukaryotic cell
What does the flagella motor resemble?
- THe type 3 secretion system
What forms the basal body in a bacterium?
-The OM and IM
How is the T3SS visualised?
- Using single particle microscopy
Does the basal body in bacteria remain intact after cell lysis?
- YES
How is the bacterial falgellum like the T3SS and how is it not?
- Only has proteins for rod hook and filament secreted instead of others
What is assembled first in the T3SS?
- The basal body
In the T3SS, what are the translocator proteins delivered thorugh and what do they form?
- The needle
- Form translocation pore
In the T3SS, where are the effector proteins delvered into?
- The target cell
In the T3SS are effector proteins folded or unfolded when threaded from the cytoplasm to needle complex?
- UNFOLDED
In the T3SS, what mediates the unfolding of effector proteins?
- T3S chaperones prior to export
Which factor is requred for chaperone mediated unfolding?
- ATPase
Do the core proteins from T3SS share SIGNIFICANT similarities with the components of the flagellum?
- YES
What does the bacterial flagellum have built into it ansd what does this allow for?
- A t3SS
- Allows for sequential export of rod hook and filament components
Did the injectosome (T3SS) evolve from the flagellum?
- YES
Which 2 structures are similar between T3SS and the bacterial flagellum?
- Basal bodies (part that spans the membrane)
- C-ring at the cytoplasmic side which serves as a docking platform for ATPase
In the bacteiral flagella motor, what is the secretion apparatus used for?
- Only used for self assembly
In the T3SS, what happens to form the T3SS?
- It secretes polymers to form the needle (doesn’t have spinning)
- Then once the T3SS is formed, it is used to secrete bacterial portions into the cell
Does the T3SS spin like the BFM (bacterial flagellar motor)?
- NO
In the BFM, which component is the one that can spin?
- the rod component –> power generating stator ring
Which part of the bacterial flagellar motor (BFM) functions as a T3SS?
- the basal body
- It secretes proteins that will poymerise into a hook and filament of the flagellum
- Thus secretion apparatus only used for SELF ASSEMBLY
What is an example of a hybrid structural biology approach in terms of needle in T3SS and what did this show?
- docking the structure of the monomer (determined by NMR) into the EM density of the entire T3SS needle
- UNAMBIGUOUS PROOF THAT THE NEEDLE IS HOLLOW
- ## Showed that you can’t squeeze a folded protein through therefor must be unfolded
What are the main similarities between the T3SS and the bacterial flagellum?
- Helical tubular structure –> helical polymer of one protein; FLAGELLIN
- Flagellin units are produced in cytosol and UNFOLDED BEFORE TRANSLOCATION and transported in the unfolded form through narrow CENTRAL CHANNEL in filament
- Flagellum grows from the tip
In the T3SS and the flagellum which direction do these grow from?
- Both grow from the tip
What would happen if there were no chaperones in the cytoplasm?
- The filaments would accumulate in the cytoplasm and this would be toxic to the cell thus causing cell lysis
Is ATPase essential for the function of T3SS and flagellum?
- YES
What is the energy of ATP hydrolysis used to unfol?
- Used to unfold the proteins prior to translation
- T3SS: Unfold the effector proteins
- In flagellum: to unfold the components of the rod, hook, and filament
What force does the translocation of the effector proteins through the T3SS require?
- The proton motive force
What are two things the proton motive force does in terms of the T3SS and BFM?
- Energizes the translocation of unfolded proteins through the channel in the flagellum during its self assembly
- Provides the energy for the rotation of the flagellum
Is rotation driven by proton motive force or ATP?
PROTON MOTIVE FORCE (not ATP!!)
Roughly how many proteins were approved for treatment by the US Food and Drug administration by 2016?
- > 206 proteins
Of the proteins approved by the US Food and Drug Administration, what form were most of them and where were their sources?
- Most were recombinant
- Sources include Bacteria, yeast, insect cells, mammalian cells
- Also sourced from transgenic animals or plants
What is the advantage of having transgenic plants or animals?
- They are in a MORE CONTROLLED ENVIRONMENT
- Increased yield as well but mainly the controlled environment
Which protein pharmaceuticals are dominating the market? (5 things with one huge thing)
- Monoclonal antibodies (HUGE)
- Insulin
- Interferon beta (used for MS)
- G-CSF (granulocyte colony stimulating factor
- Coagulation factors
What are the disadvantages of protein therapeutics compared to to small molecule therapeutics? (7 things!)
- Difficulty/cost of large scale production
- Difficulty of purification
- Heterogeneity (including PTMS)
- Immunogenicity (if not natural human protein)
- Oral delivery not usually possible bc proteases in stomach and mouth
- May degrade in plasma
- REDUCED bioavailability (generally limitied to extracellular targets
Why is purification difficult (especially recombinant) in disadvantages of protein therapeutics?
- Protein drugs may not be stable or soluble outside cell so must be engineered to be stable
Why is heterogeneity hard in protein therapeutics?
- bc. recombinant proteins must be produced that contain the same PTMs as eukaryotic cells
What does bioavailability mean?
- The fraction of the drug that gets to the site of action
What are 6 advantages of protein therapeutics over small molecule therapeutics?
- High specificity and hence reduced side effect
- LOW toxicity
- Can replace deficient or dysfunctional natural proteins
- Faster development and approval times (by about 1 yr)
- Patient production relatively straightforward
- Proteins can be READILY ENGINEERED to IMPROVE PROPERTIES
What are two main examples of protein engineering to improve therpeutic utility?
- Insulin –> enhancemet of bioavailability profile
- Monoclonal antibodies –> reduction of immunogenicity
Can insulin form a hexamer?
- YES (R6 hexamer)
What kind of PTMs does insulin contain?
- Disulfide bonds
Is insulin similar to the pig and cow?
YES
- 3 aa difference from bovine insulin
- 1 aa difference from pig
- Very effective in humans as long as they are pure
What do the genetic engineering techniques do for insulin?
- Reduce reaction impurity problems
What are 3 things that a diabetic patient needs from the insulin?
- Insulin that is stable on the shelf
- Steady baseline level of insulin (long acting insulin analogues)
- Rapid source of insulin at meal time (need fast acting insulin analogues)
What are two long acting insulin analogues?
- Insulin glargine
- Insulin determir
How has insulin glargine been engineered to make it good (longer duration) for the basal insulin requirements?
- Increased isoelectric point (more positively charged)
- this means it is LESS soluble at physiological pH
- This SLOWS the absorption into tissue
How has insulin determir been engineered to make it a good long lasting insulin for the basal insulin requirements?
- It is acetylated
- This means there is reversible binding to albumin and SLOWER absorption into the tissue
What structure formation is insulin ideally in for a fast acting analogue?
- Monomeric form
What does the absroption of insulin depend on?
- Its association state e.g. monomeric, dimeric, tetrameric etc.
What are the 4 different association states that insulin can be in?
- Monomer
- Dimer
- Tetramer
- Zn-Hexamer
Which association state of insulin predominates and what is the absorption rate like?
- Zn-hexamer
- Slow absorption
Do the insulin complexes need to dissociate to be absorbed?
- YES
Why is insulin injected into the fat?
- Because this will encourage the uptake of glucose and prevent glucose release from the liver
What is a way that insulin can be engineered to not be the hexamer form, thus allowing for fast absroption?
- By modifying the intermolecular interface
- This will INHIBIT association of monomers and allow them to be quickly absorbed
Which two residues are very important for dimerisation in insulin?
- Proline 28 and Lysine 29
What is done in terms of engineering to the insulin structure to allow it not to self associate (dimerise) to allow the fast form (monomeric) to dominate?
- Proline 28 and Lysine 29 are mutated
- e.g. Insulin luspro and insulin aspart
What does insulin lispro have that is different (fast acting)?
- Lysine instead of the proline
- Proline instead of the lysine
What does insulin aspart have that is different (fast acting) ?
- Aspartic acid instead of proline
Does Lys-Pro Insulin act faster than the WT form of insulin?
- YES
What can the killing of tumour cells using monoclonal antibodies result from?
- Receptor blockade
- Payload delivery (cytotoxic T cells)
- Other mechanisms
What are examples of tumour antigens that have been successfully targeted by monoclonal Igs?
- EGFR, ERBB2, VEGF, CTLA4, CD20, CD30, CD52
Roughly, how are monoclonal antibodies (mAbs) made?
- From cell culture, grown into myeloma cells, then at same time a mouse is immunized with antigen e.g. EGFR and spleen cells are extracted from mouse
- Spleen B cells and myeloma cells fuse–> proliferate
- Screened
- mAbs formed
What is a diadvantage of mAbs?
- Undesirable effects bc. it’s a mouse antibody
- Immunogenicity
what is a way to try and reduce the immunigenicty of the mouse antibodies?
- Huminisation of mouse antibodies
- done by combining mouse CDR sequences with human sequences in the rest of variable regions
What is an even better way of trying to reduce immunogenictiy than the huminisation of mouse antibodies?
- huminising the mouse BEFORE immnization
What , in theory is the best way to generate human monoclonal antibodies (mAbs)?
- Phage display
What does phage display involve in terms of generating a phage display library?
- B cells are isolated, after which RNA is isolated and reverse-transcribed into cDNA.
-the cDNA is then used as template for PCR amplification of the VH chain (variable heavy chain) and VL chain (variable light chain) of the encoded antibodies to generate a phage display library that can theoretically represent all antibody specificities in a particular individual.
The PCR products are then cloned into the phage display DNA vector known as phagemid in a manner that they are fused to the gene Gene III in the phagemid.
Gene III encodes the phage capsid protein, pIII, which is a surface protein.
Thus, the phage particles express the different recombinant IgG-pIII fusion proteins on their surface, allowing the recombinant IgG molecules to be expressed (displayed) on the surface of the phage.
What can a phage library theoretically represent?
- all antibody specificities in a particular individual.
What is the process of biopanning using phage display library to select monoclonal antibodies of desired antigen specificity?
- An antibody phage display library is screened for phage binding to an antigen through its expressed
surface mAb by a technique called (bio-)panning. - After several rounds of “panning”, the pool is enriched for tight and specific binders
- Specific binders are then selected out from the pool by washing away non-binders and selectively eluting binding phage clones with low pH or excess ligand.
- The corresponding DNA is isolated and sequenced to identify the binders.
- The selected antibody genes are then cloned into whole human IgG expression vectors, which will then be introduced into eukaryotic cell lines by transfection to produce fully human monoclonal antibodies.
What are the antibody engineering approaches?
- Conventonal immunization via a mouse
- huminisation of mouse antibodies
- Phage display (no mouse)
What are two examples of therapeutic antibodies?
- Bevacizumab (Avastin)
- Infliximab (remicade)
What is bevacizumab?
- recombinant humanised monoclonal antibody to inhibit angiogenesis by binding to VEGF
- Implications for colorectal cancer and lung cancer
How was bavacizumab humanised?
- retaining the binding region and replacing the rest with a human full light chain and a huamnsied truncated IgG1 heavy chain (also other substitutions)
What is infliximab?
- Artificial antibody
- Originally developed in mice but bc. of reaction the mouse common domains were replaced with similar human antibody domains
- mAbs have IDENTICAL structures and affinities to target
how does infliximab work?
- Binds and neutralises TNF-alpha
- ANti-inflammatory
- Implications for chron’s disease and rheumatoid arthritis
What does SAR stand for?
- Structure Activity Relationship
What is the pathway for traditional drug discovery programs?
- Identify the target, develop assay
- Assay compounds
- Characterise active compounds
- The clinic
What is phase II of the drug development process comparing the drug to?
- The gold standard treatment at the time
What are the 7 steps to drug discovery?
- Discovery–> target identification and validation, assay development, hit generation and optimisation, lead selection and optimisation
- Non clinical
- Phase I (20-30 healthy volunteers)
- Phase II (100-300 patient volunteers)
- Phase III ( 1 000- 5 000 patient volunteers)
- Regulatory review (market approval)
- Launch
How many years in total can development of a drug take?
- about 14 years
What does a hit mean?
- That some activity is shown
What does a lead mean in terms of drug development?
- Family of compounds with similar structures that ALL show activity
What does SAR (Structure Activity Relationship) tell us?
- There is a correlation between the structure in compounds and activity in the assay
What does a classic SAR analysis involve?
- Different subsitutions all at one site with correpsonding activities for each to tell which one is the best
Is there a size limit in SAR analysis?
- YES
Does SAR analysis provide qualitative AND quantitative analysis?
- NO
- Only qualitative analysis
How do you get quantitative information with drug design analysis?
- Rational drug design–> QSAR –> Quantitative Structure Activity Relationship
What 4 questions do we ask when using rational drug designing?
- Do we have a target structure?
- Do we have existing compounds?
- Do we have both?
- Do have have nothing but an assay?
For a target based drug design, which two techniques could be used to get new hits is there was a target structure but no known ligands?
- In silico screening
- De novo design
For a ligand based drug design, which technique can be used to get accurate hit developments when there is no target structure but ligand is known?
- QSAR
For a ligand based drug design, which technique can be used to get an altered hit series when there is no target structure but ligand is known?
Scaffold hopping
When there is no target structure or ligand, which technique can be used for drug design?
- HTS–> High throughput screening
if there is both a target and a ligand available, can all the techniques be used?
- YES
What can help HTS? (High Throughput Screening)
Library filtering:
- Only selecting molecules for screening purposes that are drug like
- Using the lipinski rule of 5 (MW<500, logP<5, Hbond donors <=5, Hbond acceptors <=10) (SAVES TIME)
- Removing promiscuous inhibitors
What are promiscuous inhibitors?
- Compounds that ALWAYS have activity no matter what the assay is
What are PAINS?
- Pan-Assay INterference compoundS
- They are the promiscuous inhibitors
- They ALL retain a reactive centre
What can you do to knock out the promiscuous inhibitors?
- Add detergent to detect the REAL compounds
- the promiscuous inhibitor will become inactive because it sticks to EVERYTHING
What is de novo drug design?
- Contructing novel ligand in the active site on the basis of very local preferences for particular functional groups
- Assemble piecewise a potential drug into the binding site of the target
- Calculate the interactions as each piece of the new compound is added
Is de novo desgin automated? (like when they take it to the experimental chemist)
- YES
What are the 4 steps of de novo desgin?
- Library of drug like fragments
- Collection of linking chemistries
- Rules of binding (‘forcefields’)
- Scoring functions
What is the proper definition for the ‘hit rate’?
- The number of active compounds per screen –> usually around 0.1%
- For every 1000 molecules, ONE shows some activity
What is the rate of useful activity in terms of hit rate?
- (sub microM concentration) 1/10 of the hit rate
- So about 0.01 % –> so 1/10 000 molecules should show some activity
What is in silico screening used for?
- To improve the hit rates
What are the 5 steps to in silico screening?
- Take structure of a target biomolecule
- Take a library of commercially available small molecule structures
- Fit every small molecule into the protein target, scoring how well it fits
- Rank the scored small molecules
- Buy a selection of the top of the ranked list and test in your in vitro assay
What is the advantage for in silico screening?
- Enrichment
- Top 10 ranked compounds of the 10^6 compound database will probably NOT be active
- BUT the 100 active compounds in library are probs all in the top 2000 ranked compounds
- You must assay 0.2% of the compounds to find the same number of active molecules
What is the problem with in silico screening?
- The large numbers
- there will be 9990 false actives found —> so need a second test/assay to ensure only the real actives get through
What is docking?
- Computational creation of a protein-ligand complex by simulation
What does docking rely on in terms of structure?
- Relies on detailed structure of the protein and particularly its active site
What is docking used to do?
- Derive theroetical structures of unsolved complexes
- Investigate details of ligand-protein interaction
What three things is QSAR used to derive?
- Details of the role of various functional groups
- Relative binding affinities of similar compounds
- Models of pharmacophores for screning databases
For QSAR does there have to be knowledge of the target?
- NO
What is the rough process in general of QSAR?
- Detailed computer modelling of properties of ligand or lead compound and the correlaton of this information with activity data
What is CoMFA and what does it do?
- A QSAR technique
- Superimposes all molecules
- Measures a grid based electrostatic and steric energies around each of the molecules
- Correlates it with activity
- Develops a predictive model from statistical analysis of CoMFA data and activity
- 95% accurate activity
What are the specific steps in CoMFA?
- Align molecules
- Define surrounding strucutres
- Calculate properties for each molecule in smaller volumes
- Use these parameters – with a list of activities– to generate statistical models of SAR
What is an example of using CoMFA?
- Steroids interacting with human testosterone binding protein
- Superimpose and build CoMFA based model
- Correlation b/w predicted activity and experimental r=0.98
What is scaffold hopping?
- If you have a lead series of active compounds and they are bad–> poor ADME, toxicity, off-target activities, synthetically intractable etc.
- So…you RETAIN the activity while moving to a new chemistry –> which hopefully doesn’t suck
What are pharmacophores?
- A map of the physicochemical properties that give your compounds activity
- e.g. Hbond donors/acceptors (position and orientation)
- Charged groups (position)
- Aromatic systems (Centroids, normal)
- Hydrophobic sites
What are examples of pharmacophores?
- Tagamet and Zantac
- Look at the features in 3D
- Both have a H-bond donor, Hydrophobe, H-bond acceptor
What is the rough pathway for finding pharmacophores/-
- Molecular strucutres
- Low E conformations
- Pharmacophore model
What are shape based analogues?
- Structure of a known ligand defines the binding site
- Find other compounds that can present the same ‘shape’ –> and ideally similar atomic properties
What are the main issues with Drug design?
- biologcial systems are notoriously finicky (protein movements, adaptation to ligand, weak bonds are very important –> 1 H bond = 10x ligand affinity
- Little mistakes in the structures= big mistakes in the models
