Lecture 6

Question 1

What are the different signals that can be measured in screening assay?

Answer 1

Second messenger accumulation (e.g. cAMP, IP3)
MAP Kinase activation (ERK phosphorylation)
Intracellular calcium ion concentration
High content screening-phenotypic screening
Enhanced protein-protein interaction
Reporter gene responses
Label free technologies-mass transcolation
ELISA assay
AlphaScreen assays

Question 2

Describe the signalling pathway of EGFR.

Answer 2

○ Activation of EGFR cause the phosphorylation of intracellular domain’s tyrosine residues
○ Adapter proteins (Shc, PLCγ and Gab1) bind to the phosphorylated tyrosine residue to activate different pathway:
§ Shc and Grb2 pathway lead to activation of ras and ras leading to phosphorylation of ERK and stimulation of gene expression.
§ PLCγ pathway involve:
□ Activation of hydrolysis of inositol phospholipids leading to release of inositol tris phosphate and increases in intracellular calcium.
□ formation of diacylglycerol which can activate protein kinase C and also stimulate NF kappa B signalling.

Question 3

Describe Alpha Screen Assay.

Answer 3

• Used to monitor signalling pathways
  
  • Excitation of alpha donor bead with laser to produce a singlet oxygen
  • Singlet oxygen then interact and excite the acceptor bead that is in close proximity with alpha donor bead
    This only happen if this two bead are in close proximity to each other

Question 4

What are the two propertoes of agonist to consider?

Answer 4

Affinity (ability to bind) and efficacy (ability to activate)

Question 5

What assay is used to measure efficiency of agonist?

Answer 5

• Radioactive cyclic AMP assay to monitor the effectiveness of a beta two agonist in stimulating cyclic AMP formation.
• Radioactive adenine labelled cells make up ATP pool
  These cells are stimulated to produce cyclic AMP from ATP

Question 6

what is the term for agonsit that can’t elicit full maximal response?

Answer 6

Partial agonist

Question 7

What factor determine efficacy of ligand and why?

Answer 7

• Efficacy of an agonist depend on both the agonist itself and the tissue properties.
  This is due to different agonists having different efficacy and the resulting response depend on the signal amplification within the cell.

Question 8

What is the difference between Kd and EC50?

Answer 8

EC50:Concentration of ligand needed to get 50% of the maximum response and determine potency

Kd: Concentration of ligand needed to occupy 50% of the receptors and determine affinity

Question 9

What is the EC50 value if 50% of receptor needed to be occupied to produce 50% response?

Answer 9

EC50= Kd

Question 10

Is the signalling amplification greater when it is measured further down the signalling pathway/cascade?

Answer 10

Yes

Question 11

How can you quantify agonsit efficacy?

Answer 11

• Comparing maximal response of a partial agonsut with a full agonist (experiment dependent)
  -Comparing EC50 and Kd ratio when the maximum responses are the same (experiment dependent)

Question 12

How to measured dissociation constant of competitive antagonist?

Answer 12

• Design an experiment to compare does of agonist in the absence or presence of antagonist and the response to agonist is the same size.
  
  • In the presence of the antagonist, a higher concentration of agonist (A2) to produce 50% of the maximal response.
  • In the absence of the antagonist, a lower concentration of agonist (A1) to produce 50% of the maximal response.
    Despite no knowing the number of occupied receptor, it can be assumed that the number of occupied receptors by agonist to produce a particular response is the same in both condtition (presence and absence of antagonist). This form the basis of the Gaddam equation.
    By designing this experiment and comparing the parallel shift of the dose response curve to produce a ratio of the two agonist concentration that produce the same sized response, this can determine the dissociation constant of the antagonist.

Question 13

How does the dose reponse curve look when measuring non-competitive inhibition?

Answer 13

• No parallel shift with increasing concentration of cediratinib (non-competitive antagonist)
  
  • Reduction in maximal response with increasing concentration of cediratinib.
    But EC50 value remains the same.

Question 14

Describe apparent non-competitive antagonism

Answer 14

See last slide of lecture 6