antagonists in functional assays

Explain how you would test experimentally whether a compound is a competitive antagonist. Include a description of how the data would be presented and what result you would expect to see. (10 marks)

Experimental method:

• perform a functional cAMP assay with fixed concentration of a known full agonist

• use increasing concentrations of the compound being tested

• measure the cAMP produced for each concentration compared to that for full agonist

How the data would be presented:

• log concentration of compound on x axis, cAMP functional response on the y axis

• if the compound is a competitive antagonist it will create a parallel shift to the right with no change of Emax

• Schild analysis will show linear regression with slope=1.0

• F test will be greater than 5%

Explain how you would test experimentally whether a compound is a non competitive antagonist. Include a description of how the data would be presented and what result you would expect to see. (10 marks)

Experimental method:

• perform a functional cAMP assay with fixed concentration of a known full agonist

• use increasing concentrations of the compound being tested

• measure the cAMP produced for each concentration compared to that for full agonist

How the data would be presented:

• log concentration of compound on x axis, cAMP functional response on the y axis

• if the compound is a non competitive antagonist it will have a lower Emax as less receptors available but the EC50 will be unchanged.

• Schild analysis will show non straight line or will not be equal to 1

• or F test will be less than 5%

Purpose of F test

• affirms whether the Schild analysis is by chance

• if <5% then uncompetitive antagonism

• of >5% then competitive antagonism

Purpose of Schild Analysis

• a method for analysing antagonist data fdrom functional assays using the EC50 and Ki produced

• gives linear regression

Schild analysis and the type of antagonism seen

• If it is not a straight line plot or there is a slope different to =1 then it is not competitive antagonism

What is used to measure antagonist potency?

• pA2

• higher pA2 more potent the antagonist

Definition for pA2

• negative algorithm of antagonist concentration (-log[B]) which requires double agonist dosage to reach the same effect

What will pA2 be equal to if there is competitive antagonism?

• -log Ki