L3 - Receptor Theory 1- Drug Affinity Flashcards
What is affinity?
A chemical property that defines how well 2 molecules can come together
What is a Forward reaction?
When a drug and agonist come together
What is a Backward reaction?
When a drug and agonist come apart
What is occupation? and what is it governed by
The number of cells that are occupied, governed by affinity
What is activation? and what is it governed by
The “turning on” of a receptor governed by efficiacy
How is forward rate of reaction measured?
K+1 [A] * [R]
How is reverse rate of reation measured?
K-1 [A] [R]
What is the equation at equilibrium?
K+1 [Aeq] x [Req] = K-1 [Aeq Req]
What is Kd?
direct measure of affiinity a constant that defines the affinity of a receptor
(where D= dissociation)
How is Kd measured?
K+1/K-1 = [Aeq] x [Req] / [Aeq Req]
When is the Kd at a low number?
At high affinity
How is occupancy measured?
number of receptors occupied divided by the total number of receptors
Varies between 0 (no drug present) and 1 (all receptors occupied)
How do we practically consider tissue and incubation for measuring receptor affinity?
Tissue - selected to condition the recognition sites and receptors, can be isolated
Incubation - temperature must be kept cold so enzymes arent active
How do we practically consider the radioligand for measuring receptor affinity?
molecular structure of the ligand must not be changed as the ligand must be active
purity - ligand must be chemically pure
degradation - solved by avoiding light, storing and low
temps and antioxidants
labelling - labelling with radioactivity must achieve
specific activity, to allow low tracer
concentrations
Some advantages and disadvantages of H3 as a radio label
Advantages - indistingushable from native compounds
- long half life
- good stability
Disadvantages - specialised labs required
- expensive and difficilt to label
Some advantages and disadvantages of I125 as a radio label
Advantages - if compound has an aromatic hydroxly
group (eg tryosine residues) it ca be
incorporated at high specific activities.
- iodonation very easy and cheap.
Disadvantages - easily degraded
- biological activity of the logand can
be reduced.
- Short half life.
How do we practically consider separating bound from free for measuring receptor affinity?
- Usually done by filtration or centrifugation
- Issues is rate of dissociation of ligand-receptor complex, speed of separation must be compatible with affinity of ligand for receptor.
- lower affinity or higher Kd requires more efficient separation.
How do we practically consider non-specific binding for measuring receptor affinity?
- lignad may also bind to other things in the test tube or other proteins in the separtation.
- can be reduced by measureing the proportion of specific to non-specific binding. (rinsing only removes unbound radioligand from incubation medium)
What is the shape of radioligand binding curves?
Rectangular hyperbola
What does the lagmuir equation meaure and what is it?
Describes the relationship between receptor occpancy, affinity and drug concentration.
Bound = Bmax x Xa / (Xa + Kd)
What does transformation of the langmuir equation give?
Scatchard Equation - Commonly used for analysis of binding data
Why do we need to measure receptors?
- To define them
- to understand the rate of drug/ligand action
- to find how many receptors are ina given tissue
- to find how receptor numbers change in disease
