Dr Edwardson - Binding Flashcards
What effects can activation of a receptor have on a cell?
- Changes in membrane permeability (and therefore membrane potential)
- Generation of second messengers
- Protein phosphorylation
What is a receptor?
A receptor is a signal transduce and usually respond to neurotransmitters, hormone and autacoids
How do drug receptor complexes form?
- Needs very close complementarity between surface topography of drug and receptor
- Energy barrier may be reduced by ‘induced fit’
Give an example of induced fit between and receptor and a substrate.
Glucagon and its receptor
Why are drug said to be selective but not specific?
Because they often interact with a number of receptors at different concentrations
What is the therapeutic index of a drug?
An indicator of how toxic a drug is compared to its effectiveness
Toxic dose / Therapeutic dose
(TD50/ED50)
TD50 - The dosage that would be toxic for 50% of the population
ED50 - The minimum dosage that would be effective for 50% of population
Why is a semi log plot used to plot a [drug] response curve?
Pseudo linear region
Competitive antagonist gives a parallel shift
(can accommodate large shifts)
What is efficacy?
The ability of a drug to activate a receptor
What is affinity?
The concentration of the drug to the proportion bound to the receptor
What is the difference between an agonist and an antagonist?
Agonist has both affinity and efficacy, whereas an antagonist only has affinity
Name an agonist, antagonist and the receptor type of smooth muscle.
Muscarine
Atropine
Muscarinic
Name an agonist, antagonist and the receptor type of striated muscle.
Nicotine
d-tubocurarine
nicotinic
What effect on the drug response curve will the use of racemate have, if only one of the isomers has affinity?
It will cause a parallel shift as the concentration of the effective isomer is half that of the racemate
What effect on the drug response curve will the use of a racemate have when one isomer is an agonist and the other an antagonist?
There will be a reduced gradient and right shift as you are both increasing the concentration of the effective isomer and that of the antagonist
What is the receptor occupancy equation?
a = [D]K1 / 1+[D]K1
What is the disassociation constant equal to?
50% occupancy!
What are the relative affinities of specific and non specific binding?
Specific has high affinity but low capacity
Non-specific has high capacity but low affinity
Why is Kd not equal to the EC50?
Because there is not a direct proportionality between receptor occupancy and effect
What is a Scatchard plot?
A graph of B (x axis) against B/[D] (y axis)
What information can you calculate from a Scatchard plot?
The Ka (- dy/dx) and the Bmax (the x intercept)
What does it mean if the Scatchard plot has two gradients?
That there are two receptors (with different affinities)
That it is a G protein coupled receptor (as these have two affinity states)
What is the receptor occupancy equation for when there are two ligands present?
a = [D]K1 / [D]K1 + [A]K2 + 1
What is the dose ratio?
[D]2 / [D]1 = 1 + [A]K2
where [D]2 / [D]1 is the dose ratio
What can be calculated from the Schild plot?
The logK2 (from the x intercept)
What is a Schild plot?
A graph of the log(Dose ratio - 1) against -log([antagonist])
What can be calculated from a radioligand binding graph?
Specific and non specific binding and the IC50
What equation can be used with the radioligand binding graph? And how is this equation different if the U and L are the same molecule?
Ku = 1 + [L]KL / IC50
If Ku = KL then….
K = 1 / (IC50 - [L])
What effect does addition of the unlabelled ligand have on the non specific binding
None
Why is there initially a maximal response upon the addition of a reversible inhibitor?
Because there is a receptor reserve present (not all of the receptors need to be stimulated in order to achieve a maximal response)
How does benzilylcholine mustard cause irreversible inhibition of the muscarinic ACh receptor?
By alkylation of the ACh binding site and of an allosteric site that stabilises its inactive state
Why is having a receptor reserve useful?
It allows a large response to a small receptor occupancy therefore increasing sensitivity
What is a partial agonist?
An agonist that cannot cause a maximal response
What is the Hill equation?
n.log[D] - log[Kdiss]
where n is a measure of cooperativity (the number of ligand needed to produce a response)
What can be calculated from a Hill plot?
The cooperativity (from the gradient)
What are the stages of affinity chromatography when purifying a receptor?
Receptor binds to substrate (which is bound to the gel bead)
Receptor is retained
Addition of excess substrate cause the dissassociation from the beads
What are the three structural types of ionotropic receptors
Pentameric (cys-loop family) - eg NAChR
Tetrameric - Glutamate receptor
Trimeric - P2X
What are the four receptor families?
The ligand gated ion channels
The G protein coupled receptors
Enzyme linked receptors
Receptors that bind to DNA
What are the features of the tetrameric ionotropic receptor ?
3 TM domains (M1, 3 and 4)
Re-entrant M2 domain
Extracellular N-terminus
Intracellular C-terminus
What are the structural features of the GPCRs?
- 7 TM domains
- Extracellular N-terminus
- Intracellular C-terminus
How does cholera toxin work?
ADP ribosylation of the alpha subunit
Inhibits the hydrolysis of the GTP by alpha-s = persistent activation
This causes an increase in cAMP
How does pertussis toxin work?
ADP ribosylation of the alpha-i subunit
Prevents stimulation of alpha-i
Blocks inhibition of adenylyl cyclase = ^ cAMP
What are the relative affinities of the ligand when GTP is bound and unbound?
GTP bound = lower affinity for ligand
GTP unbound = higher affinity for ligand
Where does cAMP bind to PKA?
The regulatory domain
What is the role of AKAP in a signalling cascade?
It acts as scaffolding protein, anchoring the signalling molecules a membrane
This acts to organise the reaction chain
What does production of IP3 cause and how?
It cause the release of Ca2+ from the ER via an IP3 receptor (needs 4 IP3 molecules to activate)
What are the two mechanism by which desensitisation can occur?
Via a change in the coupling to G protein (a decreased response)
By internalisation of the receptor and their degradation
What is the role of receptor desensitisation?
It allow cells to adjust their sensitivity to prevailing range of stimulation
What is homologous receptor desensitisation?
It is when activation of the β2-receptor results in the recruitment of β-adrenoceptor kinase (βark)
βark then phosphorylate a site at the end of the C-terminal domain
This phosphorylation then increase the affinity of the β2-receptor for β-arrestin
The binding uncouples the receptor from the α-subunit thus causing desensitisation
What does AlF4- do?
It mimics to gamma-phosphate of GTP and cause persistent activation of G proteins
What is heterologous desensitisation?
This is when activation of the β2-receptor causes an increase in cAMP which in turn activates PKA
If there is a large rise in PKA (due to excessive stimulation) then PKA will then phosphorylate the β2-receptor at its C terminus and its 3rd cytoplasmic loop
This causes uncoupling of the a-subunit and thus prevents further stimulation
What are the difference between homologous and heterologous desensitisation?
Homologous at high conc (requires greater stimulation to phosphorylate more sites)
Heterologous at low conc
Heterologous acts upon other receptors (PKA is less specific)
Which protein domains interact with activated RTKs?
SH2 domains
What class of drugs can be used to inhibit tyrosine kinases (selectively)?
Tyrphostins
What protein are steroid receptors commonly complexed with when inactivated?
hsp90
What are the four domains of a steroid receptor?
Transcription activating domain
DNA binding domain
Hinge domain
Steroid binding domain
Which protein is an essential chaperone for steroid receptors?
hsp70
What is hydrocortisone?
A glucocorticoid agonist and anti-inflammatory used for the treatment of asthma
What is beclomethasone?
An immunosuppressive glucocorticoid agonist
What is mifepristone?
A glucocorticoid antagonist used as an abortifacient
What is fludrocortisone?
A mineralocorticoid agonist used as replacement therapy in Addison’s disease (replaces aldosterone)
What is spironolactone?
A mineralocorticoid antagonist used as a diuretic
What is estradiol?
An oestrogen agonist used in hormone replacement therapy
What is ethinylestradiol?
An oestrogen agonist used as a contraceptive
What is tamoxifen?
A oestrogen antagonist used in the treatment of breast cancer
What is norethisterone?
A progesterone agonist used as a contraceptive
What is mifepristone?
A partial agonist of progesterone used as an abortifacient
What is danazol?
A progesterone antagonist used in the treatment of endometriosis, menorrhagia and gynacomastia (man boobs)
What is stanazol?
an androgen agonist used as an anabolic agent
What is flutamide?
An androgen antagonist used in the treatment of prostate cancer
What is the MODA of local anaesthetics?
They block voltage-gated Na+ channels in all excitable tissues
What is the general structure of a local anaesthetic?
Aromatic group–Ester of amide link–Tertiary amine
Why are local anaesthetics selective to pain fibres?
They target fibres with small diameters and therefore block Type C fires and Type Aδ first
In addition to this they act upon fibres with a high rate of firing (pain fibres have this)
Pain fibres also has a long AP meaning there is an increased chance of binding
What is the difference in the properties between a local anaesthetic with an ester link and one with an amide link?
The ester link is stronger and therefore a local anaesthetic with an ester link will have a longer half life
What are the three states of a Na+ VGC?
Open
Closed
Inactivated
Why is the pH of the solution surrounding the cell important for the action of local anaesthetics?
Because the local anaesthetics charge is important to action (may need to be uncharged to pass through the membrane)
What is use dependence and what cause it?
Use dependence is when an increase in the rate of stimulation of a nerve fibre increases the effectiveness of a local anaesthetic
Why is benzocaine not use dependent?
Because it is uncharged
Which local anaesthetics show use dependence?
Lidocaine and novacaine
Is the hydrophobic or hydrophilic pathway use dependent?
Hydrophilic
Why is rate dependence important in Na+ channel blockers?
Because it determines which tissue the anti-dysrhythmic drugs act upon
Eg slow in/out = low frequency tissues
What are TTX and STX? and where do they act?
Tetrodotoxin and satitoxin
They both bind to the outer mouth of the Na+ VGC pore to Glu residues
What is batrachotoxin?
A Na+ VGC activator, causing it to activate at lower potentials = ^ excitability
What is aconitine?
A Na+ VGC activator, causing it to activate at lower potentials = ^ excitability
How does scorpion α toxin act?
It is a Na+ VGC activator, causing it to activate at lower potentials = ^ excitability
What are the four types of Ca2+ channel? and where are they found?
L-type is found in smooth muscles and glands
N-type, P-type and T-type are found in nerve cells
What are the three types of drugs used as Ca2+ blockers?
dihydropyrimidines, phenylalkylamines and benothiazepines
What is nifedipine?
A dihydropyridine Ca2+ VGC blocker
What is verapamil?
A phenylalkyamine Ca2+ VGC blocker
Class IV antidysrhythmic (reduces Ca2+ entry)
What is diltiazem?
A benzothiazepin Ca2+ GVC blocker
Upon which channel type do most Ca2+ VGC blockers act?
Upon L-type
What is mibefradil
It is a benzimidazolyl tetraline Ca2+ VGC blocker that acts upon L and T-type channels
What are the three modes of a Ca2+ VGC and how are these established?
0, 1 and 2
0 - Ca2+ antagonist
1 - no drugs
2 - Ca2+ agonist
Which Ca2+ VGC blockers show use dependence and what are the implications of this?
Verapamil and diltiazem
This means that they are cardiac selective
What feature of DHPs means that they are effective in the treatment of angina and hypertension?
They bind preferentially to the inactivated form of the channel
smooth muscles undergoes long periods of depolarisation
Therefore they are vascular selective
What are the two main families of K+ channels?
VGCs and Inward rectifiers
What are the features of K+ VGCs?
They have 6 TM segments
Functional channel is a tetramer
S4 is the voltage sensor
S5 and 6 form the selectivity filter
What are the features of Kirs?
Tetramer of 2TM helices
Linked by a pore helix and a selectivity filter
What are the three subfamilies of K+ channels?
Ca2+ activated
G protein-activated
ATP sensitive
What is cromakalim?
A KCO used as an antihypertensive
What is diazoxide?
A KCO used as an antihypertensive
What is minoxidil?
A KCO used to treat baldness and hypertension
What is the MODA of KCOs?
They act upon ATP-sensitive K+ channels in vascular smooth muscles
Inhibit ATP binding = channel stays open
= relaxation
What are the negative side effects of KCOs as antihypertensive agents?
KCOs decrease glucose tolerance
Hyperglycaemia
How do ATP sensitive K+ channels function in pancreatic cells?
ATP produced by metabolism cause the channel to close
This results in depolarisation
= Ca2+ enters
= insulin release
What is tolbutamide?
A sulphonylurea used to treat diabetes by inhibiting the opening of the KATP channel
What is glibenclamide?
A sulphonylurea used to treat diabetes by inhibiting the opening of the KATP channel
How do sulphonylureas cause insulin release?
They bind to a site on the KATP (the sulfonylurea receptor) and decrease the opening of the channel leading to depolarisation
What are the three types of SUR and where are they found?
SUR1 in β cells
SUR2A in the heart
SUR2B in blood vessels
What are the properties of ion channels?
Voltage sensitive
Ion selective
Time depend (takes time to come to equilibrium with new membrane potential)
