Tut 3: How to improve protein drugs (Recombinant proteins) Flashcards
What is insulin?
Insulin is one of the smallest proteins, consisting of two polypeptide chains linked together by disulphide bonds. Insulin promotes nutrient storage via anabolic action.
After a meal, blood glucose rises causing insulin to be released by pancreatic Beta-cells. It stimulated the uptake of glucose, amino acids and fatty acids into the cells to decrease the concentration in blood to restore homeostasis. It promotes the synthesis and storage of carbohydrates, proteins and lipids while inhibiting their degradation and release into blood.
What are the benefits of recombinant proteins?
1) Transcription and translation of an exact human gene can lead to a higher specific activity of the proteins and a decreased chance of immunological rejection.
2) Recombinant proteins are often produced more efficiently and inexpensively, and produced a potentially limitless quantity.
3) Reduction of exposure to animal/human diseases
4) Allows modification of a protein or selection of a particular gene variant to improve function, activity or specificity, or even create a new function. Eg Increase half-life, stability, penetration etc.
Explain insulin as an example for use in protein therapeutics and what led to it becoming a recombinant protein? I.e. how did the use of animal pancreas to the use of bacteria come about.
In 1922, insulin was first purified from bovine/porcine pancreas and injected into diabetic patients.
There were a number of problems that hindered its continued use including;
- Availability of animal pancreas for purification
- Cost of insulin purification from animal pancreas
- immunological reaction that some patients expressed from the animal pancreas
These problems were addressed by isolating the human insulin gene and engineering E.coli to express it using recombinant DNA technology.
By growing vast quantities of E.coli, large-scale production of human insulin was achieved. The resulting insulin was abundant, inexpensive, low immunogenicity and free from other animal pancreatic substances. Recombinant insulin was approved for use for the US FDA in 1982.
Explain Erythropoietin as an example of recombinant protein therapy.
Erythropoietin (EPO) is a hormone essential for the production of red blood cells, it is mostly present in human kidneys. Deficiencies in this hormone results in anaemia leading to fatigue, impaired mental function and respiratory distress (particularly in patients suffering from chronic kidney failure)
Recombinant human EPO (rHuEPO) is currently being used to treat patients with anaemia associated with CKF or decreased RBC production.
A decrease in oxygen transport from RBC sensed primarily by the kidney leads to an increase in renal EPO production, stimulating the proliferation of erythroid progenitor cells in bone marrow. This results in an increase in the number of circulating RBS capable of carrying oxygen to correct the tissue hypoxia.
Explain thrombin and coagulation as an example of recombinant proteins
Under normal conditions, coagulation proteins are inactive when they circulate in blood. Vascular injury induces a coagulation cascade. This cascade activated several protease that lead to the activation of the Xa factor. This factor catalyses the conversion of pre-thrombin to thrombin which can lead to the production of a fibrin clot, which can lead to stroke.
Anti-thrombin is used an as anti-coagulant.
How can protein drug therapy by improved?
- Route of administration (IV, IP, Oral)
- Dose (Frequency and amount)
- Absorption
- Distribution
- Metabolism and excretion
- Plasma half-life and protein stability via protein modifications such as mutation, glycosylation, encapsulation etc to prevent enzymatic degradation
- Increase specificity of binding to the receptor to improve targeting and reduce side effects
- Increase binding efficacy to the receptor to amplify the effect.
Explain the hypothesise and rules associated with protein degradation. I.e. PEST hypothesis and N-end rule
PEST hypothesis - Polypeptide and protein sequences that are rich in proline, glutamate, serine or threonine are more rapidly degraded than others.
N-end Rule - The identity of the N-terminal residue of a protein determines its in vivo half life. Eg Phe/Leu/Asp/Lys/Arg 20hrs.
The ubiquintin proteasome pathway targets proteins for degradation through destabilising the N-terminal residues allowing it to control many physiological processes.
How do you develop and improved erythropoietin drug?
The sialo-glycoprotein-sialic acid residues on EPO are responsible for maintaining the biological activity in vivo. Hence the direct correlation between the amount of sialic acid and the in vivo biological activity. The half-life of native EPO can be increased by increasing the glycosylation of the protein. There have also been several dozen analogues produced to create more carbohydrate addition sites, hence increasing the potential sialic acid residues, which in turn increases the biological activity.
What were the steps involved in the creation of Darbopoetin-Alpha?
1) Site-directed mutagenesis: Change the nucleic acid sequence encoding one or more amino acids of a human EPO cDNA clone.
2) The clone encoding each new candidate analogue was transfected into mammalian cells and the expressed protein analysed.
3) Testing: Only a few of the analogues were fully glycosylated, had the proper tertiary structure and retained biological activity.
These EPO analogues have either one or two extra N-linked carbohydrate chains.
What is the difference between Recombinant human EPO (rHuEPO) and Darbepoetin?
rHuEPO can contain up to 14 sialic acids whereas Darbepoetin can contain up to 22 sialic acids.
Darbepoetin has a higher carbohydrate content than rHuEPO, hence there is an increased sialic acid content, making it Darbepoetin has a higher MW and a greater negative charge associated with it. This negative charge leads to greater metabolic stability.
Darbepoetin has a 3-fold longer circulating half-life (>24hrs) than rHuEPO (=4-9hrs)
Darbepoetin has less affinity to the EPO receptor than rHuEPO, but is compensated for this by its increased potency. (3.6-fold more potent than rHuEPO for a dose administered twice weekly or 13 to 14-fold more potent if this dose is reduced to once weekly.)
