L5: Radioligand binding part 2 Flashcards
calculating specific binding
Armed with the results from both experiments we can now calculate specific binding
Specific binding = Total binding – NSB
Subtracting this line and this line gets specific binding.
GRAPH
some calculations so better look at notes
and can answer these qs:
Make sure u can answer this:
How do you distinguish between total binding, specific binding and non-specific binding in saturation experiments?
What are the X and Y axes in a Scatchard plot?
How do you obtain Kd and Bmax from a Scatchard plot?
radioligand displacement assays?
Radioligand displacement assays
Not all drugs can be radiolabelled.
When this is the case, we can gain information on the unlabelled drug’s affinity by measuring its ability to inhibit the binding of a radioligand that binds to the same receptor.
The unlabelled drug displaces the radiolabelled drug from the receptors, resulting in less radioactivity bound to the tissue samples.
We can calculate the IC50 for the unlabelled drug; the concentration that reduces the binding of the radioligand by 50%
GRAPH
Ic50 for unlabelled drug
Conc of unlabelled drug displacing 50% of labelled drugs binding
Which can then be used to calc affinity of unlabelled drug for receptor
B0: max binding
and look at: consider the following
hill plot?
The ic50 is more accurately obtained from a Hill plot
A Hill plot has;
The log of the unlabelled, inhibitor drug concentration [I] on the X axis
The log of [B]/([B0] – [B]) on the Y-axis
The IC50 is where [B]/([B0] – [B]) = 1
i.e. where log of [B]/([B0] – [B]) = 0,
i.e. where Y on the Hill plot = 0
GRAPH
The Hill plot on the right had a gradient of
-0.989 (remember a straight line has the equation
y = mx +c, where m is the gradient)
A gradient of -1 (or close by) tells you that the interaction between the drug and the receptor is a simple bimolecular interaction (one drug to one receptor)
A gradient of less than 1 (<0.85) could mean that the receptor can bind more than one drug molecule and that binding of the first, reduces the binding of the second (negative cooperativity)
In displacement binding experiments however, it is more likely to mean that there are two or more distinct receptor types present at which either the radioligand or the displacer has different affinities.
Cheng- Prusoff equation?
The Cheng-Prusoff equation describes the relationship between the IC50 of the unlabelled drug, obtained during displacement experiments, and its binding affinity of the receptor.
The affinity of the drug calculated in this way is given the symbol Ki to denote that it was determined by its ability to inhibit the binding of another drug (the radioligand).
Ki = IC50/(1 + (L/Kd))
Where: L = concentration of the radioligand used in the experiment
Kd = dissociation constant of the radioligand
Ki = dissociation constant for the unlabelled displacer drug
MORE GRAPHS AND NUMBERS!
Make sure u can answer
What is the difference between a saturation experiment and a displacement experiment when considering radioligand binding?
What are the X and Y axes in a Hill plot
How do you obtain the IC50 for the unlabelled ligand from a Hill plot?
What is the name of the equation used to calculate the Ki for an unlabelled ligand from its IC50 obtained during a displacement assay?
the problem with radioligands?
The problem with radioligands
Whilst the use of radioligands has proved invaluable, such experiments, they have their limitations
The do not provide “live” results, especially during kinetic experiments; samples have to be taken and processed for each individual time point
The bound fraction of radioligand has to be separated from the unbound fraction by filtration, which can lead to errors.
Radioactivity is hazardous to staff and costly to dispose of safely
All of these contribute to make radioligand binding of limited use as a “high throughput” screen
scintillation proximity assays (SPA)
SPAs still use radioligands but in this case the receptors are coated onto tiny (approx 5µm) plastic or crystalline beads
The beads contain a scintillant; this is a chemical that emits light when it is close to (in proximity with) a radioactive molecule i.e. when the radioligand binds to the receptors on the surface of the bead
This light can be detected and gives a measure of radioligand binding to the receptors on the bead in real time without having to filter
Radioligand NOT bound to receptors is not close enough to the scintillant to stimulate light emission
SPA assays used widely in high-throughput screening, especially for kinetic measurements
Radioactivity makes the beads light up e.g: a radioligand labelled with 33-phosphorusus binding to receptor on an SPA bead to trigger light emission
But if radioactive ligand not bound to receptor nothing happens. Proximity. Cant go far enough to activate scintiline in the bead. If bind then close to scintiline and bead lights up.
Good for kinetic experiment
A SPA bead
The “core” of the bead might be a plastic (eg polyvinyl toluene, PVT) containing the scintillant diphenylanthracine (DPA) or a crystal such as yttirium silicate (Ysi) with cerium ions trapped in the crystal acting as scintillant
The beads emit blue light (400 – 450 nm) when activated
A typical coating is wheat germ agglutinin (WGA) which captures receptors
The best radioligands are those that contain 3H or 125I as the radioactivity from unbound ligand doesn’t travel far before it is absorbed by the surrounding medium, so only radioligand bound to the beads emits light
GRAPH
conventional RLB?
Conventional RLB (that big process before with the brain filter stuff)
Looking at the association and dissociation kinetics of [3H]DPCPX binding at human adenosine receptors type 1 receptors (hA1R)
Association kinetics measured by addition of 2.4 nM [3H]DPCPX
Dissociation kinetics measured by addition of 10 µM unlabelled cyclopentyl adenosine (CPA), a selective hA1R agonist, at t = 60 minutes
Note how many more data points you can get from the SPA assay, giving a more accurate measure of kinetics
1st is the SPA beads
Far more data points
Resonance energy transfer (RET)
Resonance Energy Transfer (RET) replacing radioligands
What underlies fluorescence?
“creation of an excited electron singlet state”
Absorption of a photon of light energy (excitation)
Lifetime of excited state (brief ; typically 1 – 10 nanoseconds
Loss of a photon of light energy (emission)
Green: absorb enery in form of light? Excites molecule to higher energy level. Bottom line is its baseline. Starts to drop. Lose energy typically in form of heat. Then drop back to baseline non excitated state. When it drops back it will emit light.
So fluorezcent molecular aborb and emit light.
Because lst some in heat energy emitted is less than energy in frm of light.
Lower energy light hsd longer wavelength. So energy absorbed has shorter wavelength than energy emitted. Light has diff wavelengths so diff colour.
SPA still utilises radioactivity, so still has inherent safety issues, with the associated costs
Increasingly, fluorescence techniques, notably, Resonance Energy Transfer, are being used, as the utilise only light
Fluorescent molecules are excited by the absorption of light at a set wavelength and return to their non-excited state by the emission of light of a longer wavelength (less energy)
BRET AND FRET
BRET and FRET
Bioluminescence Resonance Energy Transfer – BRET
Fluorescence Resonance Energy Transfer – FRET
Both techniques are used to study the interactions between fluorescently-tagged receptors and fluorescent ligands
The light emitted from the fluorescent receptor is used to excite the fluorescent ligand (hence Energy Transfer)
However transfer can only occur over a short distance, when the two fluorescent molecules are close by each other (typically< 10 nm), in other words, when the ligand is bound to the receptor.
Ave to be nearby eachither
Bioluminescence Resonance Energy Transfer – BRET
In BRET, the receptor is tagged with a luciferase enzyme (NLuc) which emits light of wavelength 630 nm when it oxidises its substrate (furimazine).
This 630 nm light can excite the fluorescent ligand which will in turn emit light of wavelength 650 nm
But excitation only occurs over a distance of < 10 nm, in other words only when the ligand is bound to the receptor
So measuring light emission at 650 nm provides a measure of ligand binding to the receptor
Red light in turn excites a molecule.
Emit light of longer wvelength of 650nm only if drug bound to receptor. When luficerase and fluorescent mol next to eachother. If far no excitation.
More 650nm light emitted= more drug to receptkr so more being excitated by fluorescence from luciferase.
Using bret for a displacement assay
i f unlabelled ligand binds to recpeotr so labelled comes off then red fluorescent disappears. No resonance enrgy transfer no light. Displacing a fluorescent drug by a non fluorescent drug knocking fluorescent drug into surrounding solution far away from luciferase enzyme so no bret signal.
using FRET to study GPCR dimerisation and G protein activation?
GFP = Green Fluorescent Protein; RFP = Red Fluorescent protein
Dimerisation can be homodimers (two same receptors) or heterodimers (diff receptors)
2 gpcr. One with rfp one with gfp/ if excite gfp with lzser beam with 488nm light, excite, emit light/ longer wavelength. 525nm. If 2 receptors separate nothing happens (if apart in membrane further than 10nm). If sitting by eachother, dimerise, 525nm liht excites rfp. Energy transfer. Rfp emits at 610nm. Emits at longer wavelength.
Tells you about dimeriation. More light emitted= dmore dimerisation
Using FRET to study G-protein activation
This is a functional assay as it can distinguish between agonists and antagonists acting at the receptor
Excite cyan protein. Emit blue light. Nothing would happen if g protein and receptor far apart. But if u have an agonist that activates this gpcr. The g protein and receptor come together. When we excite blue protein some of that light excites yellow protein which causes yellow protein to fluoresce. Tells you something about how able that ligand was able to activate that gpcr.
CFP cyan fluorescent protein
YFP yellow fluorescent protein
using FRET to show activation of protein kinase A
cAMP decreases FRET between PKA subunits labelled with fluorescein and rhodamine
On activation by cAMP, PKA splits into a regulatory subunit plus a catalytic subunit
So receptor activation that stimulates adenyl cyclase to produce cAMP will produce a decrease in the FRET signal as the PKA dissociates.
So this again is a functional assay, that can be used to distinguish between agonists and antagonist acting at a receptor.
Make sure you can answer
What does SPA stand for?
What are the main components of an SPA bead, and how big are such beads?
Why does SPA technology only detect radioligand bound to receptors?
What do the abbreviations BRET and FRET stand for?
Over what distance does “resonance transfer” typically take place in such experiments?
Which has the longer wavelength; the light absorbed by a fluorescent molecule or the light emitted by it?
Which has the higher energy; the light absorbed by a fluorescent molecule or the light emitted by it?
LO:
By the end of this lecture students should be able to:
Describe the main types of radioligand binding assay and the information that can be obtained from them, and how this is used during to the drug discovery process
Describe the principles of saturation and displacement assays and the key considerations that must be taken into account when designing such assays
Explain the terms specific binding, non-specific binding, saturation curve, displacement curve, Scatchard plot, Hill plot
Give examples of technologies that are being used to replace assays that rely on radioactivity
Give examples of how advanced imaging techniques are being used to gain information on drug action
