Lecture 3 (Receptor Theory I Drug Affinity) Flashcards
What is occupation of receptor by drug molecule governed by?
Affinity (independant of activation)
-Governs number of receptors activated
What is activation of receptor by drug molecule governed by?
Efficacy
What is affinity regulated by?
Constants (etc.. K+1,K-1)
What is the efficacy during an antagonist reaction
Zero
What is Kd (dissociation constant)
KD is a constant that defines the affinity of a drug for a receptor
What are units of Kd in?
Molarity
How do you find Kd? (without graph)
Divide reverse reaction by forward reaction
KD= k-1 / k+1
How does high affinity affect Kd number?
A high affinity reaction Kd tends to be very small number (inversely proportional)
What is occupancy?
Proportion of receptors occupied will vary with the drug concentration
(varies between 0 and 1)
How do you find occupancy?
Occupancy = number of receptors occupied/
total number of receptors
How can we use occupancy (at equilibrium) to measure affinity?
Use Radio ligand Binding Assays
What us the purpose of tissue in measuring affinity using radioligand binding assays?
Selected to contain the recognition sites (receptors) of interest
What conditions are need to preserve integrity of ligand(s) and binding site in method for measuring affinity? (2)
-Keep temp low as enzymes don’t work well at low temp, stops degradation
-Need to add antioxidant as us may have oxidable molecules
What are the conditions for the radioligand in needed in measuring affinity using radioligand binding assays? (3)
-Radioactive drug must be very pure
-Degradation must be avoided
-Must have very high specific activity to allow very low (“tracer”) concentrations.
How can you avoid degradation of radioligand? (4)
- free-radical scavenger (eg ethanol) in drug solution.
- store at low (not freezing) temperature.
- Avoiding light (dark bottles to store).
- incorporation of antioxidant (eg. ascorbic acid).
How is separating tissue+bound ligand from free ligand remaining in incubation media is usually done? what other techniques are used?
Filtration or centrifugation
Dialysis, column chromatography, precipitation/adsorption
What must the speed of separation of tissue+bound ligand from free ligand be compatible with?
What conditions are required for fast separation?
-Affinity of ligand for receptor
-Low affinity
What is Non-specific binding in test for drug affinity?
Ligand in addition to binding to receptor may bind to other things in test tube such as the plastic it is made up of, or other proteins found in preparation
What is the solution to Non-specific binding in test for drug affinity? (2) How do you take it away?
-Done by setting up 2 test tubes in parallel with the same receptors and tissues in it
-In one test tube add known conc of radiative drug and the same to the other one but you add a huge excess of non-radioactive drug into one of them
-Non-radioactive drug out competes radioactive drug for specific binding as it is higher affinity
-Only radioactivity bound in nonspecific
(Take away non-specific binding from total)
What is the equation for specific binding?
Specific binding = total bound – non specific binding
What kind of scale is used for Radioligand binding curves?
semi-logarithmic
What does it mean when graphs plateaus on radioligand binding curve?
When it plateaus when you know all receptors are bound to ligands
What does higher affinity for drug mean? (in terms of occupancy)
The higher the affinity of a drug for its receptor, the lower the concentration will be needed for a given level of occupancy
What does the concentration of drug at which 50% receptors are OCCUPIED by DRUG provide?
Measure of affinity (Kd)
