Receptors and Drug Action Flashcards
1
Q
What compound do most drugs target?
A
Proteins
2
Q
What are the 4 common proteins that drugs target, which is the most common?
A
Receptors (most common), ion channels, enzymes, transporters
3
Q
What is an a)agonist b)antagonist?
A
a)Drugs that bind to a receptor producing a response
b)Drugs that prevent the response of an agonist.
4
Q
What are 6 examples of agonists that bind to mu-opioid receptors and where are these receptors found?
A
> Morphine, heroin, methadone, codeine, fentanyl, beta-endorphins
Brain, gut, spinal cord.
5
Q
What is an example of an antagonist for a mu-opioid receptor and what is its use?
A
> Naloxone
To help someone in an overdose.
6
Q
What is a ligand?
A
General term for a binding chemical, can be an agonist/ antagonist.
7
Q
What does activation of a receptor by an agonist do?
A
Causes a Change in behaviour of the cell
8
Q
What are the 4 types of receptors?
A
1)Ligand-gated ion channel (ionotropic receptors)
2)G-protein coupled receptors (metabotropic receptors)
3)Kinase-linked receptors
4)Nuclear receptors.
9
Q
How many transmembrane domains do the 4 families of receptor contain and what other proteins do they contain?
A
1)Ligand-gated ion channel (ionotropic receptors)
>Around 2
>Binding site
2)G-protein coupled receptors (metabotropic receptors)
>Always 7
>Intracellular G-protein coupling domain.
3)Kinase-linked receptors
>1
>Kinase (catalytic domain)
4)Nuclear receptors.
>None, as not anchored into the plasma membrane.
>DNA binding site.
10
Q
How quick are the 4 receptor families?
A
1)Ligand-gated ion channel (ionotropic receptors)
>Very quick (0.5 milli seconds)
2)G-protein coupled receptors (metabotropic receptors)
>A bit quick (10-100 milliseconds)
3)Kinase-linked receptors
>Slow (100s milliseconds- hours) due to phosphorylation.
4)Nuclear receptors.
>Very slow (hours) due to transcription of proteins.
11
Q
What is a shared feature between ligand-gated ion channels, GPCRs and Kinase linked receptors?
A
> Transmembrane-spanning segments composed of 20-25 hydrophobic amino acids.
Possess extracellular ligand binding domain.
12
Q
What is a feature of a ligand needed for nuclear receptors.
A
Ligand must be able to cross the plasma membrane.
13
Q
What is the difference between receptors of the same family found in different tissues?
A
> Different tissues express different combinations of genes
As receptor families have multiple genes which code for them.
14
Q
What are 3 examples of drugs that target voltage-gated ion channels?
A
Lignocaine, Apamin, Dihydropyridines
15
Q
What is the difference between receptors and ion channels?
A
The receptor is the ion channel, the aqueous pore is in the middle of the receptor where the ions flow.
16
Q
What is an example of ligand-gated ion channels and where are they found?
A
> Nicotinic receptors
In reward pathways in the brain, peripheral system (neuromuscular junction).
17
Q
What effects the composition of receptors and what effect does this have?
A
> Subunit composition effects the function.
Receptors in different areas have different compositions, these require different doses of a ligand to have an effect.
18
Q
What receptor family takes up the most of our genome?
A
G-protein coupled receptors
19
Q
What is conserved on extracellular loops on GCPRs and what do they do?
A
2 cysteine residues that form disulphide bonds to stabilize the structure of the receptor.
20
Q
What 3 subunits make up the G-protein domain in GCPRs?
A
alpha, beta, gamma subunits
21
Q
What receptor types can ACh interact with?
A
1) Nicotinic (ionotropic/ ligand gated)
2) Muscarinic (metabotropic/ GPCRs)
22
Q
What happens when a agonist binds to GPCRs?
A
1) Alpha subunit exchanges GDP for GTP
2) Alpha separates from Beta + Gamma
3)Alpha and Beta + Gamma interacts with downstream effector proteins
4)Alpha becomes nucleotidases and breaks GTP into GDP, or agonist becomes dissociated, and Alpha is now attracted to Beta + Gamma and rebinds.
23
Q
What subunit has GTPase activity and why?
A
Alpha subunit to break down GTP to GDP and release a phosphate group.
24
Q
What are the 3 different types of G-proteins?
A
1)Gq
>activates phospholipase C-Beta
2)Gs
>Activates adenylyl cyclase which regulates kinase A which activates Ca2+ channels.
3)Gi
>Inhibits adenylyl cyclase
25
What does knowledge of the G-protein the receptor is coupled to help us do?
Predict the effect through the signalling pathways.
26
What method can we use to asses if a drug is an agonist or an antagonist?
>Assays can be used to asses drug actions on receptors based off signalling pathways
>E.g. FLIPR
27
What is a) Kinase b) Tyrosine kinase?
a) enzyme that adds phosphate to proteins
b) causes phosphorylation of tyrosine residues.
28
What are the 2 types of tyrosine kinase enzymes, what is an example of each and a disease which effects them?
1) Built into receptor so is already there when agonist binds
>Receptor has enzymatic part
>E.g. insulin receptors which is effected by Type II diabetes.
2)Attract an enzyme to receptor when agonist binds
>These are used to regulate transcription of genes allowing for growth
>E.g. growth factor receptor effected by cancers.
29
How do Kinase-linked receptors work on the nucleus?
Indirectly work on nucleus for transcription regulation.
30
How do growth factor receptors work?
>Takes 2 proteins to come together (dimer) and usually needs more than one ligand bound
>Recruits kinase enzyme causing cascade downstream of kinase enzymes to alter trasncription.
31
What are cytokine receptors an example of and what do they mediate?
>Tyrosine Kinase linked receptors
>Regulate inflammatory response.
32
What is a similarity and a difference between Kinase-linked receptors and Nuclear receptors?
Both have DNA binding sites to regulate transcription but Kinase-linked receptors act indirectly while Nuclear receptors act directly on DNA.
33
What property do ligands need to be able to bind to Nuclear receptors?
Need to be lipid soluble molecules that can diffuse through the lipid bilayer.
34
What are 2 effects of mutations in receptors involved in signal transduction?
1)Excessive activity of receptors in absence of stimulus
>E.g. cancers
2)Increase or decrease activity of receptor when ligand is bound.
35
What is an example for a disease caused by mutations in each of the 4 classes of receptor?
1) Ligand-gated ion channels
>Epilepsy
2)GPCRs
>Endocrine disorders
3)RTKs
>Associated with many cancers.
4)NRs
>Inflammations cancer
>Diabetes
>Cardiovascular
>Obesity
>Reproductive disorders.
36
How would a mutation in beta 2 adrenoreceptors effect the treatment of asthma?
Would reduce the efficacy of asthma drugs.
37
What is an autoantibody?
Autoantibodies are antibodies (immune proteins) that mistakenly target and react with a person's own tissues or organs
38
What are 2 examples of diseases due to autoantibodies and their symptoms?
1) Myasthenia gravis
>Lose muscle
2) Thyroid hypersecretion
>Also called Graves' disease, high metabolic rate, increased skin temperature, tremor, tachycardia, bulging eyes, nervousness.
39
What is the a) Occupation b)Activation of a drug governed by?
a) Affinity
b) Efficacy
40
What is the efficacy of a) Agonist b) Antagonist c) Partial Agonist
a) 1
b) 0
c) Around 0.5
41
What observation reflects the affinity of a drug?
The amount of complexes formed (occupation).
42
What is the KD constant and what does it define?
> the concentration of ligand, which half the ligand binding sites on the protein are occupied in the system equilibrium
>defines the affinity of a drug for a receptor.
43
What is the forward rate of reaction determined by?
> K+1[A]*[R]
>Forward rate constant * (conc of ligand +conc of receptors)
44
Are most drug-receptor interactions reversible?
Yes
45
What is the reverse rate of reaction defined by?
>K-1[AR]
>????????????????? What does this mean (lecture 3)
46
What is KD and its units?
>Dissociation constant
>This is equal to: reverse reaction/ forward reaction (K-1/K+1)
>Molarity
47
What is KD a direct measurement of?
Affinity (how well 2 things fit together).
48
What is the KD at a) high affinity b) low affinity and what does this mean?
a) High affinity= small KD number
>Forward reaction is quick, chances of complex coming apart is low/ slower
b) Low affinity= high KD number
>Reverse reaction will be bigger
49
How is equilibrium reached for ligands forming complexes?
When forward rate is matched by reverse rate of reaction.
50
What is occupancy and what is it governed by?
>Occupancy= number of receptors occupied/ total number of receptors
>Number of receptors occupied by a drug is governed by the affinity of the drug for that receptor.
51
What values does occupancy vary between and what does it show at these values?
Varies between 0 (no drug present) and 1 (all receptors occupied).
52
What method can we use to measure the occupancy (showing us the affinity) of an agonist?
Can use radioligand binding assays, will not show antagonists however.
53
What are the 5 steps to measuring occupancy using radioligand binding assays?
1. Prepare cells or membranes e.g. guinea pig ileum – detergent treatment and centrifugation
2. Aliquot out membranes onto filters
3. Add radiolabel at different concentrations and equilibrate
4. When equilibrated remove unbound drug by filtration (bound drug remains attached to filter) all unbound ligands are washed out.
Count radioactivity of filter
54
What is important about the tissues and incubation conditions using radioligand binding assays?
>Tissues must be selected to contain the recognition sites (receptors) of interest
>Incubation must try to perverse integrity of both ligands and receptors
55
What are 2 safety measures used during radioligand binding assays?
1) Keep enzymes cold so receptors aren't digested.
2)Antioxidants (e.g. amine neurotransmitter) are used so oxidation doesn't occur when exposed to air,
56
What are 2 issues with radioligand binding assays?
1)Ligand must be extremely pure chemically
>So radioactive element can't change chemical properties too much.
2)Radioactivity breaks down molecules quickly (degradation)
>Can be solved by keeping in dark or cold conditions.
57
What are the 2 choices of radio-labels and their advantages/ disadvantages for a radioligand binding assay?
1) 3H (hydrogen)
>Advantages: small so keeps chemical pure, long half life, stable when stored.
>Disadvantages: Dangerous due to strong radioactivity, labelling is expensive.
2) 125I (iodine)
>Advantages: Gives off strong signal, cheap and easy to store.
>Disadvantages: more readily degraded, Bigger than hydrogen so effects structure/ affinity of chemical (less pure), short half-life.
58
What is an issue when filtering tissue from their bound ligand?
>Low affinity drugs (reverse rate= quick) so when rinsing the tissue they fall off straight away.
>This makes it hard to measure low affinity interactions.
59
What is non-specific binding and how does it occur?
Non-specific binding occurs= ligand binds to wanted receptors but also other things in the test tube too (e.g. plastic of test tube, other proteins in the preparation).
60
How do we measure the proportion of specific and non-specific binding during an assay?
1) Set up a test tube with Tissue + radiolabelled ligand to get the total binding value
2)Set up a test tube with unlabelled ligands which bind to the non-specific binding sites, then place in the radiolabelled ligands to get the specific binding value.
3) Minus the specific binding value off of the total binding value.
Check this on lecture 3?
61
How do you work out the specific binding value off of a radioligand binding curve?
Minus the non-specific binding off of the total binding to get the specific binding.
62
Why is data usually plotted on a semi-logarithmic scale?
On linear graphs its hard to see vast change in concentrations so we use semi logarithmic graph (x-axis on log scale) to allow us to plot a wider range on numbers.
63
What curves do a) specific binding b) non-specific binding have and why?
a) Sigmoidal curve (S-shaped)
>Plateau occurs as specific binding shows saturation (as only limited number of specific receptors).
b) No plateau
>Non-specific binding is practically infinite so doesn't show saturation (as so many random unspecific binding sites).
64
What does the Langmuir equation describe?
The relationship between receptor occupancy, affinity and drug concentration
65
What is KD (inverse to affinity) equal to, and what does a higher KD mean?
>The concentration of drug needed to get 50% occupancy.
>The higher the affinity of a drug for the receptor, the lower the concentration needed to produce 50% occupancy, the lower the KD
66
What does a higher affinity mean in terms of occupancy and specificity of a drug?
>The higher the affinity of a drug for its receptor, the lower the concentration will be needed for a given level of occupancy
>Also the higher the specificity of that drug for the receptor.
67
Why is it useful to know the affinity of a drug to a receptor, give an example of when this was used?
>As we can identify and define a receptor and where it is found.
>The values of affinity of opiates in the brain matched the affinity in the gut, this showed that the opioid receptors in the gut is the same as in the brain.
68
What is the KD equivalent to?
the concentration of a drug required to occupy 50% of receptors at equilibrium.
69
What does a) low KD b) high KD show?
a) high affinity
b) low affnity
70
What can you learn from binding data of the same type of receptor in 2 different tissues?
>We can see if a ligand is selective or non-selective (if a drug will have side effects on a tissue we don't want to target).
>Also allows us to compare the consequences of a mutation on a receptor
71
Why do we need to measure receptors?
>Allows us to quantify the number of receptors in a tissue
>Helps us diagnose a disease as we can do a binding experiment to see if a loss of receptors is a part of their problem.
72
What measurement shows if a drug is an agonist or an antagonist?
Efficacy (not affinity)
73
How does an agonist induce a response when bound to a receptor?
When an agonist binds, it stabilises the protein structure in a way that favours its signalling e.g. opening ion channel or activating G-protein.
74
What is the EC50 of a drug?
The concentration needed to give 50% of the maximal response.
75
What does "receptor reserve" mean and why is this possible?
>Not 100% of the receptors on a tissue need to be occupied to give a maximal response.
>Due to amplification of signals.
76
Describe the relationship between response and occupancy?
>It is not a 1:1 ratio.
>Many tissues have an EC50 shifted to the left of occupancy the tissue is more sensitive (don't need 50% of receptors to be occupied by drug to get 50% of a response).
77
What is an advantage of the EC50 of most drugs requiring less than 50% occupancy?
We can make lower amounts of a drug to produce a response due to how sensitive they are.
78
How does amplification of signalling occur at GPCRs?
When an agonist is bound, it stays and activates multiple G-proteins and these can activate more than one enzyme, so every step of a signalling cascade can be amplified which changes cell function.
79
What is the advantage ,while making drugs, that signals are amplified?
We don't need to occupy lots of receptors to cause a large response to tissue.
80
What is the hill equation used to work out?
Used to plot concentration-response curves.
81
What is the hill equation formula?
Response= max.[Xa]^n / ([Xa]^n + [EC50]^n)
>where max = maximum response e.g amplitude of contraction
>Xa = concentration of agonist
>n = slope factor (often called the Hill slope)
>EC50 = concentration of agonist evoking 50 % of the response
82
What do square brackets mean in biology?
Always means concentration.
83
What does the slope factor show?
The number of molecules needed to bind to a receptor to activate it.
84
What does it mean if a drug has a high potency, what would this show on a concentration response curve?
>A drug with a high potency requires a lower concentration to produce a 50% maximum response.
>A drug with a high potency is shifted left on a concentration response curve.
85
What 3 factors does the potency of a drug rely on?
Affinity, efficacy and spare receptors.
86
Why is the EC50 a bad measurement of maximal response of a drug?
As agonists with different efficacies produce different maximal responses but can have the same EC50.
87
What happens if you mix partial agonists in with agonists, what would this show on a concentration response curve?
>The proportion of receptors available to the full agonist is reduced by presence of partial agonist
>The concentration response curve would shift to the right as a the efficacy of partial agonists is lower so a weakened response is caused.
>Partial agonists behave like an antagonist in the presence of a full agonist.
88
In what situation are the KD and EC50 equal and why?
>When a partial agonist is present
>As a partial agonist needs to achieve 100% occupancy in order to produce a maximum response it is capable of.
89
What is efficacy the measurement of?
Efficacy is a measure of a single agonist-receptor complex's ability to generate a response
90
What are the 3 factors which determine the effect of a drug in a living suystem?
1) Specificity
2) Affinity
3) Efficacy
91
What is potency and why is it a relative term?
>The amount of drug (usually expressed in milligrams) needed to produce an effect, such as relief of pain or reduction of blood pressure.
>It's relative as different drugs have different strengths so require different concentrations to cause their desired therapeutic effect.
92
How can potency be measured?
>In biological assays
>Require a full dose response curve at different concentrations to measure potency.
93
What does dose mean?
When we give a drug to an animal it's in a dose, a certain weight relative to their weight.
94
Are the majority of clinically useful drugs antagonists or agonists?
Antagonists
95
What is the definition of an antagonist drug?
A drug that prevents the response of an agonist
96
What are the 5 classes of antagonism?
1. Chemical antagonism
2. Pharmacokinetic antagonism
3. Physiological antagonism
4. Non-competitive antagonism
5. Competitive antagonism
97
How does Chemical Antagonism work?
The antagonist binds to a ligand altering the shape so it can't bind with a receptor to give a response.
98
What are 2 examples of chemical antagonism used clinically?
1. The inactivation of heavy metals
>Mercury, lead and cadmium toxicity is reduced by adding a chelating agent (e.g. dimercaprol)
2. Monoclonal antibody based therapies
>E.g. preventing cytokine from binding to its receptor.
99
How does Pharmacokinetic antagonism work?
Controls how long a drug stays in the body by affecting processes in the which alter the concentrations of drugs; absorption, metabolism (enzymes) and excretion (filtration, urine flow, tubular secretion).
100
What is an example of Pharmacokinetic antagonism effecting a. absorption b. metabolism c. excretion ?
a. Opiates reduce absorption of drugs taken through the oral route by decreasing absorption from the GI tract (reduce gut motility).
b. Antibiotics stimulate the metabolism of Warfarin (blood thinners) decreasing its concentration.
c. Diuretics increase urine flow which would increase secretion of agonists.
101
How does Physiological antagonism work?
When 2 drugs with opposing actions interact in the body.
102
What is an example of physiological antagonism?
Noradrenaline raises arterial blood pressure, while histamine causes vasodilation lowering arterial pressure.
103
How does non-competitive antagonism work?
Antagonist competes indirectly to stop receptor signalling by inhibiting the function of the signalling molecule by binding to the ligand or the receptor at a different location (doesn't compete with the agonist for the receptor site).
104
What is a clinical example of non-competitive antagonism?
Dihydropyridines are an anti-hypotension drug that block Ca2+ channels so smooth muscle cells can't contract, reducing pressure.
105
What is an effect of non-competitive antagonism which could be an advantage or a disadvantage?
Non-competitive antagonists would interfere with signalling of a range of receptors as the ligand they inhibit may bind to a range of receptors.
106
How does competitive antagonism work?
A drug competes with an agonist to bind to a receptor to form a complex, this complex is unable to stimulate any downstream signalling.
107
In the presence of a competitive antagonist, what is the effect on the occupancy of the receptor by the agonist?
The occupancy of the receptor by the agonist is REDUCED
108
What are the 2 types of competitive antagonism?
Reversable and Irreversable
109
What would happen if there was a higher conc of agonists than competitive-antagonists?
There would be higher occupancy of agonists than antagonists as there would be a higher chance of an agonist binding.
110
How do we measure the effects of competitive antagonism
We have to look at the response curve of an agonist alone with increasing conc of itself until max response is reached, and then add increasing concentrations of competitive antagonists
111
What should be seen on a response curve of a competitive reversable antagonist?
>The conc curve should shift right in the presence of the competitive agonist but it will always reach the max response when a certain amount of agonists are added.
>The gradient and maximum response stays the same, as if we add enough agonists we will get there
>But the EC50 will increase in the presence of increasing conc of competitive antagonists (more drug required for 50% response)
112
How do you work out the dose ratio of an antagonist off of a response curve?
Conc of agonist at EC50 in presence of antagonist / Conc of agonist at EC50 in absence of antagonist = DR
113
What does the dose ratio mean?
The fold increase of the agonist needed to achieve the same response at a given concentration of antagonist.
114
What it useful about knowing the dose ratio?
Allows us to do a Schild analysis.
115
Why is a Schild analysis useful?
Allows us to measure the affinity of a competitive antagonist for a receptor (affinity constant/ KD)
116
What is the Schild equation?
dose ratio = conc of antagonist +1 / antagonists affinity constant (KD)
117
What is the a. Y axis b. x axis labelled as on a Schild plot and what type of scale is it.
a. Y = Log10 (Dose Ratio - 1)
b. X = Log10 (conc of antagonist)
>Both are on a linear scale as are log values.
118
What will the line look like on a Schild Plot if it is a competitive antagonist?
A straight line.
119
What is the PA2 value and how do you work it out from a Schild Plot?
>The affinity of an antagonist to a receptor
>Where the line crosses the x-axis, multiply this concentration of antagonist by -1 (to give positive number).
120
How does PA2 differ from KD?
>The larger the PA2, the higher the affinity. While the lower the KD (dissociation constant), the higher the affinity.
>PA2 is just for antagonists, KD is for both agonists and antagonists
121
How do you work out the PA2 from the KD and the KD from the PA2
>Log10(-1*KD) = PA2
>The inverse log (-1*PA2) = KD
122
What is the effect if partial agonists in a. presence of agonists b. absence of agonists?
a. While other agonists are present, partial agonists act like competitive antagonists as compete to bind to receptors but have a lower efficacy.
b. In the absence of other agonists, partial agonists produce some signalling as still have an efficacy of 0.5 (half a normal agonist).
123
Why are partial agonists so often used clinically?
As we have endogenous agonists in our body, if we give partial agonists it will act as an antagonist.
124
What is the effect of partial agonists added to normal agonists on a response curve?
>The partial agonists will act as antagonist when there is normal agonists present and will shift the curve right while still reaching the max response.
>But when the conc of the normal agonists are 0 (at the left side of the graph) the partial agonists will cause some response due to their slight efficacy.
125
How does the response curve of a partial agonist differ to when a competitive antagonist is added?
>Different as when an competitive antagonist is added, the response is 0 until the agonist is added, but partial agonist have a slight response before the normal agonist is added
126
How does an irreversible competitive antagonist work?
They compete to bind to the receptor but once bound the bond is permanent (cannot be washed out)
127
What is the only way to stop the effects of a irreversible competitive inhibitor?
By making a new receptor after the drug has been metabolised or excreted (is absent).
128
Why is irreversible antagonism time dependent?
As over time more and more receptors will have permanently bound antagonists that have competed, so the max response decreases over time.
129
What does a response curve when irreversible antagonists are added look like and why?
>Initially is a parallel shift to the right with the max response staying the same, as increasing agonist conc means they can compete to bind to spare receptors before the antagonists.
>Eventually the irreversible antagonists will permanently bind to the spare receptors and the max response will decrease
130
Can a Schild analysis be used to measure the KD for an irreversible competitive antagonist?
No as the reaction does not reach equilibrium (max response is not reached).
131
What are 2 examples of irreversible competitive antagonists?
1. dibenamine
2. Aspirin
