Unit 3 - eukaryotic and prokaryotic drug systems Flashcards
Give some examples of recombinant proteins produced from E.coli?
Human Growth Hormone Interferon alpha Interferon gamma Tissue plasminogen activator Relaxin Alpha-antitrypsin Human insulin
What limitations are there of bacterial expression systems?
Some eukaryotic proteins are unstable when expressed in bacteria, or lack biological activity
Prokaryotes lack of the ability to undertake the following post-translational modifications of cloned proteins
- glycosylation
- disulphide bond formation
Recombinant proteins expressed at high levels are stored as inclusion bodies
- need to be refolded
Prokaryotes harbour pyrogens such as LPS
- stimulate the immune system
- need to be removed from the final product
Why is post-translational modifications important in drug production?
It affects the shape of the therapeutic protein
Influences biological activity
- antibodies
- enzymes
Affects protein half life in the body
- misfolded proteins are degraded by host proteases
- proteins with the wrong sugar groups such as antibodies are removed from the circulation
What is eukaryotic post-translational modification protein phosphorylation?
Protein phosphorylation covalently attaches a phosphate group to an amino acid side chain or at the protein’s C or N termini
It alters the structural conformation of a protein which in the case of enzymes can result in them being activated or deactivated
The most common form of post-translational modification in eukaryotic cells with approximately 13,000 human proteins have phosphorylation sites
It plays an important role in cell signalling, gene expression and differentiation
Minor role in recombinant protein expression
Why does post-translational modification of therapeutic proteins make disulphide bonds?
Disulphide bonds in proteins are formed between the thiol groups of cysteine residues
Stabilises protein structure in a number of ways
- links protein strands and helps the protein form in final structure
- forms hydrophobic regions which influence the final structure of the protein
In eukaryotic cells, disulphide bonds are generally formed in the lumen of the rough endoplasmic reticulum but not the cytosol. This is due to the oxidative environment of the endoplasmic reticulum and the reducing environment of the cytosol
Disulphide bonds are mostly found in
- secretory proteins
- lysosomal protein
- exoplasmic domains of membrane proteins
Why are post-translational modification of therapeutic proteins glycosylated?
A principle co-translational and post-translation modification step in the synthesis of membrane and secreted proteins
~ 50% of all eukaryotic proteins synthesised in the rough ER are glycosylated
- enzyme directed site specific process
Two types of glycosylation exist
- O-linked oligosaccharides
- Ser, Thr linked
- linked to hydroxyl groups of the amino acids serine and threonine
- N-linked oligaccharides
- Asn linked
- linked to amide group
Why is glycosylation important?
70% of protein products approved for human use as glycoproteins
They include
- antibodies
- hormones
- growth factors
- immune regulators
- interferons
Failure to glycosylate a protein with the correct combination of sugars will alter
- molecular size and charge and will result in its removal by receptors in the liver and the recticuloendothelial system
- immunogenicity
- xylose
- an oligosaccharide from plants
- pharmacological activity
- IgG heavy chain contains a single N-linked glycosylation site
- 2 sites for each IgG molecule
- aglycosylated forms display significant loss of antibody associated cell mediated cytotoxicity cannot adhere to cell surface receptors
How does preproinsulin become proinsulin?
Disulphide bond formation
How does proinsulin become insulin?
Trimming by carboxypeptidase B-like enzyme removes two basic residues from each of the new ends
What is Atryn?
Anticoagulant antithrombin
Extracted from the milk of goats
- genetically modified to produce human antithrombin
One genetically modified goat can produce the same amount of antithrombin in a year as 90,000 blood donations
What type of insulin was used initially?
Purified animal-sourced insulin was the only type of insulin available to diabetics until genetic advances occurred later with medical research
- pigs
- cows
What are the advantages of using genetically engineered insulin?
Synthetic human insulin was produced in 1978 from E.coli
- the protein does not require glycosylation to be biologically active
What are the disadvantages of using genetically engineered insulin?
Currently human insulin made from bacteria are almost twice as expensive as porcine insulin
How is insulin produced in E.coli?
cDNA LacZ Beta-galactosidase
Clone in E.coli
Isolate product
Treat with cyanogen bromide to cleave at methionine amino acid link
How is the Covid-19 vaccine produced?
Genes coding for the spike protein are identified and isolated
Genes inserted into ChAdOx1 viral vector
Injected into body
- cells express spike protein
- body produces antibodies against spoke proteins
If infected, immune system attacks SARS-CoV-2
Why are post-translational modifications needed?
Affects the shape of the final protein
Influences biological activity of
- antibodies
- enzymes
Affects drug half life in the body/drug stability
- protein with the wrong sugar groups such as antibodies are removed from the circulation
What is the function of interferon?
Hinder the replication of viruses Affect the immune response Three types - alpha - beta - gamma Used to treat multiple sclerosis Relatively quickly broken down - need for repeated injection
How much less active is E.coli produced interferon?
10 times less
- non-glycosylated
- reduced solubility
- formation of insoluble disulphide links
- removal of sugar group
Where are monoclonal antibodies glycosylated?
All antibodies are glycosylated at conserved positions in their constant regions
Why are monoclonal antibodies glycosylated?
Affect antibody activity
- complement binding
- attachment to immune cells
- antibody dependent cell cytotoxicity
What does chimerisation of monoclonal antibodies increase?
Persistence of the monoclonal antibody in the human circulation from 2 days (murine monoclonal antibody) to over 2 weeks (chimeric monoclonal antibody)
What are the four types of monoclonal antibodies?
Animal
Chimeric
Humanized
Human
What are the advantages of plant produced antibodies?
Relatively low cost to make
Potential to produce high biomass
- kg per acre
Disease free
What plant produced antibody residues attached to the asparagine in the ER?
In plants and mammals core N-acetylglucosamine and mannose residues are attached to the asparagine in the ER
What plant produced antibodies are further modified in mammals?
Further modification in the Golgi
- N-linked glycans
- alpha 1,6 fucose
- terminal sialic acid residues
What plant produced antibodies are further modified in plants?
Modification in the Golgi
- N-linked glycans
- alpha 1,3-linked fucose
- B-1,2-linked xylose
What is an immunoconjugate?
Development of the biologic class
- highly sophisticated drug delivery systems
Take advantage of the ability of the immune system’s ability to recognise very specific types of cell
Monoclonal antibodies have very high affinity and good specificity for certain cell types and are very well tolerated, especially when partially (chimeric) or fully humanised
After cell surface binding monoclonal antibodies are internalised into the cell and subject to degradation
- endocytosis
What is an immunoconjugate also known as?
Antibody-Drug conjugate
How does an antibody-drug conjugate work?
If the conjugate possesses suitably labile chemical bond(s), enzyme mediated bond cleavage can occur
- releases the therapeutic component within a target cell
Why are marine organisms a surprisingly good source of anti-cancer and anti-bacterial drugs?
The sea is a very competitive place to live
What are microtubules essential for?
Cell division and mitosis
- pull chromosomes apart in mitosis
How can inhibiting microtubule formation prevent cancer?
Blocking microtubule action prevents mitosis
What is the function of Dolastatins?
Bind to tubulin to prevent formation of microtubules
What is a monoclonal antibody ? and its main structural components.
Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are made by identical immune cells that are all clones of a unique parent cell and as a consequence all recognise the same target
Fab region: allows binding to the antigen
Fc region binds to cellular receptors and compliment
Hinge region: allows flexibility
In the shape of Y
heavy chain for bottom portion and start of Y
What is the differences between murine, chimeric,
humanized and fully human monoclonal antibodies and
why are these differences important
Mouse proudced Mab Recognised as foreign by human immune system • Have murine glycosylation patterns • Do not bind efficiently to immune effector cells via FC receptors • Removed by the liver
Mouse DNA encoding the binding portion of a monoclonal antibody is merged with human antibodyproducing DNA in cell culture to yield partially mouse, partially human monoclonal antibodies. The cell and compliment binding (Fc) region of the antibody has a human glycosylation pattern
Humanised Antibodies which have been genetically engineered to remove murine regions (T cell epitopes) that lead to an adverse immune response.
Fully Human Made from Transgenic mice which have been given human immune system Made using phage display
What are main therapeutic uses of monoclonal
antibodies and in each case give examples
Cancer (2 marks)
• Herceptin® (trastuzumab) – inhibits cell replication
• ozogamicin (gemtuzumab) - cancer cell targeting- Monoclonal antibodies
for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC,
antibody dependent cell-mediated cytotoxicity; CDC, complement
dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv
fragment.
Auto immune disease (2 marks)
• Omalizumab inhibits human immunoglobulin E (IgE) and is useful in
moderate-to-severe allergic asthma.
• Infliximab which are effective in rheumatoid arthritis
Infection (2 marks)
• Palivizumab (brand name Synagis which is manufactured by MedImmune)
is a humanized monoclonal antibody (IgG) to treat RSV
• Raxibacumab (GSK) – is a human monoclonal antibody (IgG) directed
against the toxin of B.anthracis.
Why is the glycosylation status of an
antibody important.
Affects immunogenicity
• Causes a significant loss of antibody associated cell mediated
cytotoxicity-cannot adhere to cell surface receptors
• Affects half life in the body
• Give an example where glycosylation is important
