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
G-Protein Coupled Receptors
STRUCTURE: 7 transmembrane helixes back and forth through the membranes
- The largest family of integral membrane protein, integral protein = is a protein that passes all the way through a membrane
- They recognise a large range of ligands e.g. proteins, lipids, amino acid, photons (light)
- GPCR’s work by activating a G-protein via transduction (hence 2ndary messenger) and beta-arrestin (they tend to activate protein kinase and cell desensitisation)
- Share a common architecture (GPCRs are also referred to as 7-TM receptors)
Ligand binding
- LARGE LIGANDS: make contact with extracellular regions
- SMALL LIGANDS: Sit in the hydrophilic pocket within the transmembrane helices e.g. ACh or NA
Forces in Ligand Binding
- Ionic interaction (full +ve and -ve charge)- this is long range, non-directional (non-specific, drug can be in any rotation) and strong interaction
- Hydrogen bonds between -OH and (-NH2 ), medium range, fairly weak bonds individually but collectively is stronger, Directional- has to be in a straight line (this gives more specificity)
- Hydrophobic interactions- between uncharged amino acids, short range, non-directional: ALTHOUGH- important in specificity because if a hydrophobic molecule is next to the hydrophilic pocket this could cause repulsion and so change the way drugs bind
Forces in ligand binding 2
- STERIC FACTORS: basically the shape of the drug and the shape of the receptor have to be similar to allow the ligand to dock; short range but very important in specificity e.g. very big drug in a small receptor pocket
- Lock and key model AND induced fit model (both right but induced fit is more applicable)
Binding of adrenaline to Beta-AR
- Look at notes for the picture
- It won’t fit into the binding site of the muscarinic receptors because of the hydrogen bonding and hydrophobic interaction arent there
- NH3+ group forms an ionic bond with TM3 (transmembrane protein 3)- long range pull
- There are 3 OH groups: the 2 on the benzene ring form H binds with TM5; the OH on the aliphatic chain forms an H bond with TM7
- The benzene ring forms 2 hydrophilic bonds with TM6 and 7
How does the agonist cause receptor to change shape
1) agonist binds to receptor causing flowering
2) Agonist alters receptor state- this is accompanied by a conformational change
- This is usually accompanied by a conformational change in the receptor
- TM5,6,7 movement has been noted in the cytoplasmic face of GPCRs ‘flower effect’
- Governed by a multiple molecular switches
Desensitisation
- If a receptor is treated for too long (agonist is bound) with high conc of agonist the response fades away. DESENSITIZATION: stops receptor being over activated
- During desensitisation, receptor uncouples from G-protein and is internalised (moved into the cell)- During this process, the G-protein falls off and beta-arrestin binds instead; B-arrestin then drags into the cell; the receptor can signal in a different way when attached to beta-arrestin
- Desensitisation is caused by specific protein kinases called G-protein coupled receptor kinases (GRK’s).
Desensitisation 2
- This leads to binding of arrestin to block G-protein binding and switching signalling to phosphorylation (MAPK)
- Phosphorylation involved in G-protein-coupled desensitisation
- 3rd or 4th loop are phosphorylated on the receptor (c-terminal) this is where B-arrestin associates
- Phosphorylated by GRK’s
GPCR families
- FAMILY A: (usually) small N-terminals- most common e.g. muscarinic receptors; Histamine receptors
- FAMILY B: medium-sized terminals- these bind peptide hormones e.g. glucagon; corticotropin
- FAMILY C: Large N terminus- bind small ligands e.g. GABA, n Ca2+
G-proteins
-Be careful GPCR’s and G-protein are different
- Trimeric peripheral membrane proteins
- Relay signal from GPCR’s to 2nd messenger producing enzymes and ion channels
- Activated by GTP/GDP exchange
- B+Y always stay together and always attached to the plasma membrane
- a- always stays attached to the membrane but not always associated w/B+y units this is determined by whether a- is bound to GTP or GDP exchanged
G-protein 2
- When GDP is bound the a-subunit is inactive and associated wBeta and gamma units (trimeric G-protein)
- When activated the GDP- falls off and GTP attaches which signals to the G-protein to become active
- The alpha then separate and they can go and activate other proteins
- This process is catalysed by the GPCR’s
G-proteins activation
- GPCR binds to inactive trimeric G-protein (Alpha GDP beta gamma) see BB replay
- The Alpha subunit binds (has a finger-like part which sticks into the GPCR), as the finger moves up it nudges another part of the Alpha unit which removes GDP off the unit, allowing GTP to bind
- This is switched off because a-subunit can act as an enzyme and convert GTP –> GDP
- This causes the a-unit to become inactive and causes high affinity for beta and gamma units and makes them inactive
G-protein diversity
- 20 a-sub units; 5 b-units and 12 y-units
- Individual receptors have a strong preference for a combination of particular subunits
- a-subunit seem to take a primary role in activating effector
- B and Y subunits can also activate or modulate the activity of a wide range of effectors such as adenylate cyclase, phospholipase C, ion channels
- This gives a wide range of action
Family of G-protein
- alpha s: activates adenylate cyclase, Ca2+ channels, activated by cholera toxin (stimulatory)
- Alpha i: inhibit adenylate cyclase, open K+ channels (nerve inhibited) or close Ca2+ or inhibit pertussis toxin-outside nerve system
- Alpha O: inhibit adenylate cyclase, close Ca2+ channels- inside nerve systems
- Alpha q: activate phospholipase
- Alpha 12,13: activate shape change (motile cells by activating kinases) also links to MAP kinase for release of prostaglandins
