L5: Radioligand binding Flashcards
what are RLB assays?
RLB assays involve the use of a drug (the ligand) containing a radioactive isotope within its structure to investigate the interaction between the drug and its binding site e.g. on a receptor
Importantly, when considering drug-receptor interactions , RLB assays cannot readily distinguish between agonists and antagonist; the simply tell you how well the drug binds, not whether the drug is able to activate the receptor. In other words, they tell you something about affinity but not efficacy.
Telling us something about affinity. Whether binds to receptor. But not functional assays. Like whether agonist or antagonist.
Determined by binding affinity.
Quantified in terms of Kd, the molar concentration of drug required to occupy 50% of the receptors at equilibrium
what can rlb assays determine?
RLB:
BMAX- NUMBER OF RECEPTORS PRESENT IN A TISSUE SAMPLE
KD- THE DISSOCIATION CONSTANT = K-1/K+1 (UNITS MOLAR, M)
Ka- THE AFFINITY CONSTANT= 1/KD=K+1/K-1 (UNITS M^-1)
K+1- THE FORWARD RATE CONSTANT (UNIT M SEC^-1)
K-1: THE BACKWARD RATE CONSTANT (uNIT: SEC^-1)
RLB and radioactivity
Radioisotopes (e.g. 3H, 125I, 14C) spontaneously disintegrate (unstable) over time, emitting energy in the form of radiation (β or γ particle)
Radiation is quantified by the number of disintegrations per unit time e.g. disintegrations per second (dps)
1 disintegration per second = 1 Becquerel (Bq) the SI unit of radioactivity
1 Tera Bq (TBq) = 1012 dps
1 Bq = 60 disintegrations per minute (dpm)
1 TBq = 60 x 1012 = 6 x 1013 dpm
Typically looking at tera bq. 1 d per sec is very small amount of radioactivity.
You may also come across radioactivity measured in Curies (Ci)
1 Ci = 3.7 x 1010 dps = 3.7 x 1010 Bq = 0.037 TBq
specific activity
Specific activity- the amount of radioactivity emitted by a specified amount of a compound
Units typically TBq/mmol (TBqmmol-1)or Ci/mmol (Cimmol-1)
Usually the radioisotopes incorporated into drugs will be;
3H – Tritium
β-emitter; specific activity < 100 Ci/mmol (<3.7 TBq/mmol)
T1/2 = 12.5 years; low energy; does not alter the properties of the drug
But problem is you dont get much radioactivity from it so difficult to measure it.
Quite a low specific activity. hAlf life 12.5 years time it takes to lose half of its radioactivity.
14C – Carbon 14
β-emitter; specific activity <50 Ci/mmol (calculate how many TBq/mmol)
T1/2 = 5,700 years; low energy; does not alter the properties of the drug, but difficult to synthesise
125I – Iodine 125
γ-emitter; specific activity 2000 Ci/mmol (calculate how many TBq/mmol)
T1/2= 60 days; high energy so easy to detect BUT need to protect workers; can alter properties of the drug it is incorporated into
properties of the ideal radioligand?
Properties of the ideal radioligand
Should have high affinity for the receptor being studied (typically nanomolar affinity)
Should show high selectivity for the receptor (or receptor subtype) being studied (ideally at least 100 fold)
It should take at least 100x higher concentration for it to start binding to other receptors — meaning it prefers your target receptor by a long shot.
Should show low binding to non-specific sites (<10 % of total binding)
Should be chemically stable (as these assays can take quite some time)
RLB and kinetic experiments?
Drug-receptor interaction is governed by law of mass action
The velocity of a reaction depends on the concentration of reactants, and for a reversible reaction, the system will reach an equilibrium where the concentrations of the reactants remains constant.
Binding determine by collisions. Higher drug conc= higher probability of collision happening? Eventually reach equillibrium. Speed which with it reaches equilibrium depends on conc. Hgher drug conc= faster it reaches equillibrium state.
The speed with which the system reaches equilibrium is dependent on the concentration of the drug ([D]) and temperature.
Temperature: diffusion (collisions). Higher temp= quickly moving so collisions more likely.
Most RLB experiments are carried out at constant temperature and at time when the system has reached equilibrium i.e. under “equilibrium conditions”.
RLB can however be used to measure how quickly the system reaches equilibrium; the radioligand is added and the amount bound is measured at different times – so called kinetic experiments .
Because this forward reaction is conc dependent, how lng it reaches equilibrium, important that you leave low conc of drugs long enough to have an effect.
Plot time vs binding. Take sample of tissue and see how much radioactivity is bound. This is a kinetic experiment, looking how quickly reaction proceeds.
how quickly does a system reach equillibrium?
These simulated data show the “association kinetic binding curves” for a radioligand with a Kd of 30 nM (occupying 50% receptors at equilibrium?)
Note that the higher the concentration of drug, the faster the system reaches equilibrium
This is because k+1 is concentration dependent (units M-1sec-1)
k-1 is concentration independent (units sec-1)
GRAPH
Reach plateau quickly. Lower conc taking longer because that forward rate constant dependent on conc of drug. Higher conc= quicker equillibrium.
Receptor molecule undergoing thermal vibrations sitting in membra.e not stable, shaking, drug will eventually be shaken off. Can make drug stay bound longer by cooling temperature so receptor becomes less shaky but eventually comes off. Reverse rate constant is per second. How many shaken off per sec.
It shows how slowly it takes drugs to reach equillibrium conditions
think about
What are the properties of an ideal radioligand?
How many disintegrations per minute does a Becquerel represent?
How many Becquerels (Bq) in a Curie (Ci)?
What are typical units of “specific activity”?
What two things determine how quickly a drug-receptor system governed by the law of mass action reaches equilibrium?
In a drug receptor interaction what are the units for k+1 and k-1 (association and dissociation rate constants respectively), Kd and Ka?
saturation experiments?
The plateau, saturated binding. No further change.
Imagine a hypothetical cell with 1 000 receptors in its membrane
As you increase the ligand (D) concentration, more and more of the receptors will be occupied (DR) at equilibrium
At high concentrations of ligand ALL the receptors will be occupied, i.e. the binding “saturates” – this is the Bmax
The Kd can be determined as the Molar concentration of drug required to occupy 50% of the receptors at equilibrium
GRAPHl DR (y) VS KD (X)
As we increase drug conc (x axis) get to a point where ever receptor in the tissue has a drug molecule attached to it. Saturated all the receptors. (plateu). 1000= conc required to saturate the receptors so tells you n. Of receptors. kd= max conc required to occupy 50%. Now have max total of receptors can find kd from 50%.
So bmax is 1000.
Carrying out a saturation experiment looking at receptors in rat cerebllum (as an example)
- remove the brain, dissect out the cerebellum, place in bugger solution and homogenise to produce a ‘cell-free’ suspension
- determine the weight of tissue per unit volume of suspension
- add equal volumes of suspension to a series of test tubes
- add increasing concentrations of radioligand to the test tubes (one concentration to each test tube) and allow to equillbibrate at a constant temperature
- after a set time, pass the suspension through a ceramic filter and discard the filtrate
- wash the filter, to remove any loosely bound radioligand and discard the washings
- remove the filter, place in scientillation fluid and count the amount of radioactivity (dpm) using a beta or gamma counter
Each of those testubes have a set volume of suspension so same amount of brain tissue. Hope same no. receptors.
The set time has to be long enough to reach equilibrium. As it takes some time. Cold temp.?
Amount of radioactivity stuck on the filter will tell you how much drug has been bound to our set weight of tissue.
Wash to remove unbound radioactivity. Want filter, membranes (cus receptors are on them) radioactivity stuck to it.
So amount of radioactivity relates to amount of drug molecules present and no. receptors. As you increase drug conc get more binding.
give u a measure of total binding
non specific binding
The experiment on the previous slide will give us a measure of a total binding
If we plot the amount of radioactivity bound against the concentration of the radioligand added to the test tubes, we get a graph like that on the right.
Something is wrong. We are not seeing any signs of saturation.
This is because alongside the specific binding (to receptors) we have non-specific binding (NSB).
NSB represents very low affinity binding to high-capacity sites, and increases linearly with ligand concentration
Keeps heading up as this is total binding. Binding includes non-specific to other proteins. This non specific binding is low affinity but high capacity. Higher conc= more non specific binding.
A slight change to the experiment allows us to measure only non-specific binding
We next repeat the experiment, but this time add a high concentration (approx. 100x its Kd) of non-radiolabelled (“cold”) ligand.
Can actually use any non-radiolabelled ligand that binds to the same receptors
The “cold” drug will occupy all the receptor sites leaving only noEach of those testubes have a set volume of suspension so same amount of brain tissue. Hope same no. receptors.
The set time has to be long enough to reach equilibrium. As it takes some time. Cold temp.?
Amount of radioactivity stuck on the filter will tell you how much drug has been bound to our set weight of tissue.
Wash to remove unbound radioactivity. Want filter, membranes (cus receptors are on them) radioactivity stuck to it.
So amount of radioactivity relates to amount of drug molecules present and no. receptors. As you increase drug conc get more binding.
n-specific sites for the radioligand to bind to.
That will produce a graph like that on the right
Cold drug prevents radioactive drug getting to te specific sites. Radioactive drug binds to non specific sites. So only thing left on sample is non specific sites. Getting rid of the specific binding.
GRAPH
