19th Oct - Radioligand binding Flashcards
Wen were radioligands developed?
1970s
Outline the procedure for a radioligand binding assay
- Choose and make tissue/cell preperation containing the receptor
- Select suitable labelled ligand
- Incubate receptor preparation with appropriate concentrations of labelled ligand for a definet tim and temperature based on the law of mass action
- Seperate and count bound and free radioligand
- Repeat steps 3 and 4 with addition of unlabelled ligands (to see if it duplicates it) or modulatory agents as dictated by the experiment
- Analye the data to extract quantitative estimates o max and Kds of labelled and unlabelled ligands
What are the three parameters measured on a radio-ligand binding assay?
Total Bound
Specific Binding
Non-specific binding
What is the equation for the concentration of the drug-receptor complex at equilibrium?
[DR]= [D][Rt]/ Kd+[D]
What is the langmuir fit?
The sigmoidal curve of receptor ligand binding is fit using DBmax/(Kd+D) meaning that you can define the Bmax by the y intercept and the Kd by 1/2 bmax.
What are the minimum criteria expected for binding to a genuine receptor?
- Binding should be saturable i.e. a finite number of receptors
- The specificity of agents in competing with the radioligand D* binding to R should parallel biological specificity
- The kinetics of binding should be consistent with time course of biological effect
What are the advantages of a radioligand binding assay?
Determine the number of receptors in cells
Localise receptors with tissue
Refines structure-activity relationships
Identify new receptors from unexpected results
Allows early dmonstration of affinity and efficacy
Allows pharmacoloigcla characterisation of receptors rapidly, simply and cost-effectively
Why is AR*G stablised in the absence of ATP?
Because without GTP the alpha subunit can not bind GTP and thus dissociate from the complex.
How is the affinity of the receptor for the ligand altered when the g protein leaves the GPCR?
The affinity is reduced, allowing the ligand to dissociate
How is g-protein and agonist binding interlinked?
When an agonist binds to the receptor it causes a conformational changed which increases the affinity of thr receptor for the g protein
When the g protein dissociates there is a conformational change which reduces the affinity of the receptor to the ligand, allowing the ligand to dissociate.
How do agonists bind to the receptor in the presence of GTP?
Agonists will bind in a simple manner when GTP is present i.e. to R with low affinity
How do agonists bind to the receptor in the absence of GTP?
Agonists will bind in a complex manner when GTP is absent i.e. to R* with high affinity and to R with low affinity
Which conformation of the receptor do antagonists preferentially bind to R or R*?
Either, antagonists bind with equal affinity to both conformations as no conformational change will take place
What is the equation for agonist efficacy?
Agonist efficacy = R*/R affinity
Outline the experiment which demonstrated agonist complex binding
Agonist (isoprenaline) and antagonist (propanolol) binding to beta adrenoceptor in the presence/absence of GTP
Measured ligand displacement of 125IPIN, with increasing concentrations of our drug
The antagonist (propanolol) had near identical curves with or without GTP
The agonist (isoprenaline) is alters depending upon whether GTP is present. In the presence of GTP the curve is shifted to the right (i.e. lower affinity)
