Lec 2- receptor Flashcards
Non-competative or allosteric antagonist
- The antagonist doesn’t compete directly for binding to agonist site but acts elsewhere on the receptors e.g. hexamethonium blocks the ion channel of nicotinic receptors
- Results- Decreased Max response but may also have complex effects on the slope of the curve
other types of antagonists
- Chemical antagonist (antibody- bind to receptor inactivating it (antagonist))
- Physiological or functional antagonist (Calcium channel blocker will just stop the process)
- Inverse agonist- they stop the agonist working but also stop the basal activity
Characteristic of partial antagonist: partial agonist can act as an antagonist
- Partial agonist may occupy receptors normally occupied by endogenous full agonist
- They occupy these sites but do not activate them
- Full agonist cannot get access to the receptors occupied by the partial agonist- the latter appears to be acting as the competitive antagonist
Inverse agonist
- There are systems in which there is constitutive activity: activity in the absence of an agonist
- In these systems, it is possible to decrease the constitutive activity with an inverse agonist (does the opposite effect of the agonist)
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Specificity
- This measures how well a drug can discriminate between different receptors
- Low specificity can lead to many side effects
- e.g. a and b adrenoreceptors- a= vasoconstriction b= tachycardia and bronchial dilation: we may want a drug to block the a but not b receptors, so we need a drug that will just target a this is specific
Yohimbine: conc Vs specificity
No dug is absolutely specific, the danger of toxic effects especially at high conc
(1) At -8 to -7 log[Molar] yohimbine is a a2-adrenoceptor blocker
(2) At -7 to -6 This will do (1) also 5-HT receptor blocker
(3) At -6 to -4 This will do (1,2) also a1-adrenoceptor blocker
(4) At -4 to -3 This will do (1,2,3) also act as a local anaethetic
(5) At -3 to -1 This will do (1,2,3,4) also MAO block
Yohimbine different specificity
Alpha2- adrenoceptor blockage: conc -9 to -1 5-HT2 receptor blockade: conc -7 to -1 Alpha1- adrenoreceptor block: conc -6 to -1 Local anastetic: conc -4 to -1 MAO Block: conc -3 to -1 Cholinesterase: conc -3 to -1
How to measure ligand binding
-K+= rate at which forward rate goes: K-1= rate at which backward reaction goes
b= amount of drug bound
[A]= conc of drug
Bmax= total number of receptors in tissue
- Kd= conc of drug which occupies 1/2 of receptors
- Known as the occupancy equation
- Fractional occupancy = b/Bmax- this is important because with benzodiazapine we may not want to send them to sleep therefore we want to find out the conc of drug that will occuy 60% of receptors

measuring ligand binding- what they stand for
b= amount of drug bound
[A] = conc of drug
Bmax= Total number of receptors in the tissue
Kd= conc of drug that occupies 1/2 of the receptor
Factional occupancy = fraction of receptors occupied by drugs
radioligand binding
- 1st used in 1960’s
- e.g. the binding of atropine to muscarinic receptors in smooth muscle, the binding of labelled a-bungarotoxin to the nicotinic Ach receptor
- Thousands of radiolabelled ligands now available e.g. antagonists, agonists, allosteric modulators, subtype-specific drugs
Advantages of radiolabelled
1) A specific measure of receptor-ligand interactions
2) Direct measure of receptor densities and affinities
3) Characteristics of binding of non-labelled drugs (by-competition- if radioactivity drops non-labelled drug has higher affinity and so is a good competitor
4) The characterisation in absence of functional response- if you have a drug, you cant find out what the drug does just by radiolabelling e.g. agonist or antagonist
5) Useful as probe during receptor purification and characterisation
Radioligand binding: equilibrium Measurements
1) incubate constant amounts of receptors- mash up into individual cells or grind cell membrane; make into a suspension; to the purified add increasing amounts of radioligand
2) allows this to come to Eq; a portion fo radioligand will be bound to the receptor and portion will be in solution
3) Separate bound ligand from the free ligand and measure for bound radioactivity (Centrifuge) (filter paper)
Non- specific Binding
-In practice, there is usually some non-specific binding of the radioligand (to other proteins, membranes, and organelles)
-This is estimated by carrying out the parallel assay in which a high a conc of unlabelled ligand present: -Saturates specific receptor sites, -radioligand now only binds to non-specific sites which are of low affinity and non-saturable
Non-specific binding is approx linear with radioligand conc
How to measure non-specific and specific binding
1) Place radioligand with receptor = specific binding radioligand also binds to other parts of the cell/ test tube = non-specific binding
2) Block radioligand binding but plenty of non-specific binding
specific binding= total binding - non-specific binding
Analysis of radioligand binding
-Direct fit: computer analysis of binding curves by non-linear regression methods
The Scatchard plot
[Bound]/[Free] = ([Rtot]/Kd)- 1/Kd x [Bound]
- [Bound]/[Free] is on Y axis
- [Bound] is on X axis
- [Rtot] is intercept
- -1/Kd is the slope
- N.B. non-linear Scatchard plots can be difficult to interpret
Competition studies
- Using radioactive ligands, saturation analysis allows the affinity and binding capacity for a site to be determined
- Subsequent experiments may be carried out using a non-radioactive competitor for the site (gradual increase in non-labelled conc)
- Determination of the concentration of competitive ligand that displaces 50% of the radioactivity bound (IC50) the affinity of the non-radioactive ligand for the site can be determined
Competitive inhibition
- The Cheng-Prusoff relationship
- Ki = IC50/ 1+([RL]/Kd)
- Ki is dissociation constant for a drug
- [RL] is conc for radio-ligand
- If [RL} and Kd are known can estimate Ki
- NB IC50 depends on the [RL] but Ki doesn’t
Autoradiography
- it is possible to use radioactive compounds to label receptors in sections of tissue (see where receptors/ ligand are)
- Take a slice of tissue and incubate with ligand
- Wash off unbound ligand
- Place on a photographic plate and wait
- Develop film and there will be a photograph
- DISADVANTAGE: can’t do on a living patient
Positron emission tomography (PET scan)
- Positron- emitting isotopes can be used to localise receptors in vivo
- 11C-flumazenil is used to show the distribution of benzodiazepine receptors in human brain
Concentration-effect curves
- This is a technique to measure drug binding without a radio-ligand
- Ec50 - the conc of agonist that cause 1/2 max response
- This increases as you add more antagonist
- The shift of the Ec50 depends on how well the antagonist binds to the receptor
- Weak binding = lower conc of agonist to get the response back and vice versa
- The shifts are therefore a direct measure for how well it binds to the receptor and is called the dose shift/ratio
- Dose ratio=EC50 of the agonist in the presence of the antagonist/ EC50 in the absence of the antagonist
- Large dose ratio = good affinity for antagonist
Schild plot
- Helps you calculate pA2
- Intercept = -LogKd= pA2
- Slope = 1
- Log[Dose ratio (r)] on Y vs Log[antagnonist] on X
pA2
- pA2 = -Log[Kd] (similar to pH)
- The concentration of antagonist needed to halve the response seen with an agonist
- Measure how well a drug binds to its receptor: higher the pA2 = better it binds
- e.g. 1x10-9 = Kd = pA2= 9
- This is also the amount of drug you need to add to reduce response by 50%
Families of membrane receptors
- GPCR’s
- Ion channels
- Enzyme-linked receptors