Principles of receptor binding Flashcards
1
Q
Whats Pharmacokinetics?
A
Its abbreviated as Pk, it is a branch of pharmacology dedicated to determining the fate of substances administered to a living organism
2
Q
In pharmacokinetics, what does drug concentration depend on?
A
Absorption –> distribution –> metabolism –> excretion e.g. urination
3
Q
Whats pharmacodynamics ?
A
A study of the biochemical and physiological effects of drugs to their target
4
Q
What do drugs bind to ?
A
They bind to specific targets, when this happens it effects the activity of a target it is “true” receptor
5
Q
When drugs can activate receptors what are they known as?
A
When drugs activate a receptor they are known as agonists
6
Q
When drugs inhibit the receptor what are they known as?
A
When drugs inhibit the receptor they are known as antagonists
7
Q
When was the concept of receptors introduced?
A
In the late 19th century bu Paul Ehrlich, he introduced the concept of receptors to explain the selective toxicity of some early chemotherapeutic agents
8
Q
What did J.N. Langley do experiments with?
What did he propose?
A
He did experiments with neuromuscular junctions
His proposals:
- there must be a “receptive substance that transmits the stimulus from the nerve to the muscle”
- First example of receptor pharmacology
9
Q
Give an example of an agonist and antagonist
A
Agonist: Nicotine
Antagonist: Curare (a skeletal muscle relaxant)
10
Q
What is a receptor
A
A macromolecular component of a cell with which a drug interacts to produce its characteristic biological effect
11
Q
Give 7 properties of receptors
A
- Present in low concentrations and show saturable binding
- Many drugs have high affinities for their receptors
- Receptors show selectivity
- Drug-receptor interactions are usually fully reversable
- Drugs are usually small molecules
- Receptors have a binding site with a complementary structure to the drug
- Agonists include conformational changes in their receptors
12
Q
Whats meant by saturable binding?
A
The binding of a ligand to a single binding site is definable by the concentration of the binding site (Bmax) and the concentration of unbound ligand at which the binding site is 50% occupied (Kd)
13
Q
When receptors are present in low concentrations, how can the binding be measured?
A
using radioligand binding
14
Q
If drugs have a high affinity for their receptor, is a higher or lower concentration of the drug required?
A
If they bind effectively due to high affinity then only a small amount of the drug is needed to bind to receptor
15
Q
Whats the drug and receptor formula and include K values and their directions
A
16
Q
What is used to measure affinity?
Whats the units?
A
Affinity is measured by the equilibrium constant KD
KD is measured in units of concentration (M)
17
Q
Whats KD formula?
A
KD= K-1 / K+1
18
Q
Here are some values of KD
KD = 1x10-3 M
KD= 1x10-6 M
KD= 1x10-9 M
Place them in order of highest affinity for receptor to lowest
A
High affinity for receptor
1x10-9 M
1x10-6 M
1x10-3 M
Low affinity for receptor
The higher the Kd value, the weaker the binding and the lower tbe affinity. The opposite occurs when a drug has a low Kd
19
Q
Place the following in order of selectivity for ß-adrenoreceptors:
- adrenaline
- noradrenaline
- Isoprenaline
A
ß-adrenoreceptors: isoprenaline > adrenaline > noradrenaline
20
Q
Place the following in order of specificity for α-adrenoreceptors:
- isoprenaline
- adrenaline
- noradrenaline
A
α-adrenoreceptors: noradrenaline > adrenaline>> isoprenaline
21
Q
What do these two structures show?
A
22
Q
When its said that drug-receptor interactions are usually fully reversible, what does this mean?
A
Neither the drug nor the receptor are permanently changed
Drug + receptor <–> DR
23
Q
Drugs are usually small molecules, whats their mw and also whats the rough mw for receptors in comparison?
A
Drugs usually have an mw of 200
receptors tend to have an mw of roughly 250,000
24
Q
Why is a close fit required between a drug and receptor?
A
Because the drugs are held by only a weak binding force
25
What forces are found between drugs and receptors?
Van der waals
Ionic bonds
Hydrogen bonds
(when agonists bind to receptor, these bonds are present)
26
Are receptors rigid?
No
27
Do antagonists cause conformational changes?
No
28
To allow comparison between drugs, their effects need to be quantifies. What 3 ways is this done?
1. Assume the law of mass action
2. Assume that only a negligable amount of the total drug is bound
3. at equilibrium
29
Whats the definition of law of mass action?
The rate of a reaction is directly proportional to the product of the reactants
30
Whats the equation for the law of mass action?
Whats the rate of the forward and the reverse reaction?
Drug (D) + Receptor (R) \<----\> DR
Rate of forward reaction: K1[D][R]
Rate of reverse reaction: K-1[DR]
31
When assuming that only a negligable amount of the total drug is bound, how can we write this assumption?
free drug= total drug
The density of receptors in tissue is low, so amount of drug bound to receptor at one time is really low
32
When quantifying drug-receptor interaction at equilibrium, what equation can we use to support this?
K1[D][R]= K-1[DR]
33
What do each of the following stand for when talking about quantifying drug-receptor interactions?
* RT
* D
* DR
* r
* KD
Using this knowledge, what's the equation for r and what is this equation valid for?
**RT** is the total number of receptors
**D** is the total drug concentration
**DR** is the concentration of the bound drug
**r** is the fractional occupancy of the receptors **(i.e. DR/RT)**
**KD** is the equilibrium dissociation constant of the drug for its receptor (a measure of its affinity) = **[D][R]/[DR]**
Then : **r= [D]/ [D] + KD**
This equation is **valid for a simple bimolecular interaction** between a drug and its receptor
34
What shape would the graph be if r is plotted against [D]?
What shape would the graph be if r was plotted against log[concentration]?
A rectangular hyperbola
35
How is the equation of r altered if the interaction between drug-receptor is more comlex
i.e. Receptors which bind two drug molecules at once (nicotinic)
Receptors which convert between high and low affinity forms
'n' is the hill coefficient
36
What are the two theories that relate to biological effect?
* Occupation theory
* Rate theory
37
Whats the **occupation theory?**
Occupation Theory: the idea that a response emanates from a receptor only when it is occupied by an appropriate ligand (drug)
Response [E] is proportional to the number of receptor occupied
38
Whats the **rate theory?**
Rate Theory: the idea that a response emanates from a receptor in proportion to the kinetic rate of onset and offset of drug binding to the receptor.
Response [E] is proportional to the rate of receptor occupation
39
In the occupation theory it states that:
the response [E] is proportional to number of receptors occupied
infinity symbol on side (horizonal 8 with right cut) means: This means that the terms on each side are proportional to each other. That is that one side is a constant multiple of the other
Whats the equation to support this?
40
Given the occupation theory, this means that graphs of fractional response against drug concentration will have the same shape as....?
Also what shape is the fractional occupancy against log [D] concentration?
Fractional occupancy against drug concentration
The fractional occupancy against log [D] concentration os a sigmoid curve
41
If all the assumptions are correct for the occupational theory then whats the equation for half maximal response of the following graph?
42
Whats pD2?
A useful pharmacological parameter that quantifies the affinity of an agonist for its receptor
(pD2 is a measure of agonist affinity)
43
Drugs with a high pD2 value acts as what concentration?
Drugs with high pD2 values act at low concentrations
44
What value is pD2 always?
Always a positive value
45
According to the occupation theory, pD2 equals what?
However, what can be the issue with the occupation theory and this equation?
pD2 = -log(KD)
BUT the assumptions of occupation theory are not always correct and for many receptor systems pD2overestimates the KD. i.e. the drug appears to bind more tightly than it really does
46
What are the two types of Antagonists?
* Competitive antagonist
* Non-competitive antagonist
47
What are **Competitive antagonists?**
* The agonist and antagonist bind to the same site
* The block can be overcome by increasing the concentration of the agonist
* Antagonists tend to be slightly bulkier molecules: can be overcome by increasing the concentration of agonist
* Binding is driven by the law of mass action
48
What are non-competitive antagonists?
* The antagonist binds to a different site on the receptor, or acts irreversibly
* The block CANNOT be overcome by increasing the concentration of the agonist
e.g. Hexamethonium (a reversible interaction)
49
Give an example of competitive antagonism?
describe the changes that would be seen on the graph in the presence of an competitive antagonist
atropine against acetylcholine in the guinea pig ileum
* Competitive antagonism see this typical dose-response curve shift to the right
50
Give an example of a non-competitive antagonist
Describe the changes on the graph that would be seen
benzilycholine mustard against acetylcholine
51
With **Antagonists,** the ability to block response will depend on what?
* The relative affinity of the agonist (KD) and antagonist (KA) for the receptor.
* * The relative concentrations of the agonist [D] and antagonist [A]
52
Define **Dose ratio?**
the ratio of the agonist concentrations that elicit the same response either in the absence [D0] or the presence [DA] of the antagonist
The dose ratio is DA/D0
53
What would you assume if the response in the presence and absence of the antagonist is the same?
What is this concept used to derive?
The occupancy by the agonist is the same (irrespective of whether there are spare receptors etc or not)
This concept is used to derive the affinity of antagonists from dose-response curves
54
In the dose-response derivations, what does the derivation simplify to and what is this equation known as?
The **GADDUM-SCHILD EQUATION**
55
What is the Gaddum-Schild equation based upon?
What doesnt the equation assume and what does it assume?
This analysis is based on the **law of mass action**
It assumes **simple competitive antagonism**
No assumptions are made about the **relationship between response and the number of receptors occupied.**
56
What is the Gaddum-Schild equation independent of?
The **Agonist** used- so long as it competes with the antagonist for the **same** receptor
57
What happens in the Gaddum-Schild equation when the dose ratio= 2 ?
58
How do you determine the pA2 value for an antagonist?
59
Whats the pA2 value?
a pA2 value determines the important relationship between two drugs "competing" for effect on the same receptor.
The pA2 value indicates the concentration of antagonist when double the agonist is required to have the same effect on the receptor as when no antagonist is present.
60
What do you need to do to distinguish between competitive and non-competitive antagonism using pAx?
pAx is the negative logarithm of the concentration of antagonist that gives a dose ratio of x
**Log(x-1) = pA2 – pAx**
**i.e. pA2 - pA10 = 0.95**
A substantial deviation from this value indicates that the interaction between the antagonist and the agonist at the receptor is not competitive
61
Examples of pA2 values in guinea pig ileum...
62
What assumptions are made about the occupancy theory?
What assumptions have problems with them?
1. There are specific receptors for specific agonists
2. **All agonists for a given receptor can produce the same maximum response.**
3. The drug-receptor interaction is rapidly reversible
4. All receptors are equally accessible to the drug
5. The receptors do not interact with each other
6. **The maximum response occurs when all the receptors are occupied**
63
Whats the problem with the assumption: **All agonists for a given receptor can produce the same maximum response?**
* Some agonist drugs act on receptors and only produce a weak response - PARTIAL AGONISTS (partial agonists act as competitive antagonists of the full agonist)
* It is clear that not all agonists are capable of producing a full response
* We need to introduce the concept that drugs may differ in their ability to induce a conformational change in the receptor, once they have bound
* But this assumes that all full agonists occupy the same number of receptors (assumption 6)
* In fact, a maximum response can be obtained when not all the receptors are occupied
* This is explained by invoking **spare receptors**
64
What is a **spare receptor?**
an agonist with high efficacy may only need to bind to a small fraction of receptors to produce a maximum response
65
How can the occupation theory be modified to account for partial agonists?
1. the effect will depend on the **Affinity** of the drug for the receptor
2. The effect will depend on the ability of the drug to induce a **conformational change** in the receptor
An agonist with high efficacy will preferentially bind to 2
and stabilise the active conformation of the receptor
An agonist with low efficacy may bind to both 1 and 2
An antagonist will only bind to 1
66
What are **inverse agonists?**
a drug that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of the agonist
67
Key points to remember from lectures...
68
At equilibrium, when the forwards and backwards reactions are proportional, write this in terms of KD?
69
You can't measure the amount of free receptor in the tissue which isn't bound to a drug. How is this calculated instead?
The 'free receptor' [R] is the total number of receptors [R]T minus those occupied by the drug receptor complex [DR]
[R] = [R]T - [DR]
in terms of KD ...
70
What does the following terms mean?
## Footnote
**RT**
**D**
**DR**
**r**
**KD**
**RT: **The total number of receptors
**D:** The total drug concentration
**DR:** The concentration of the bound drug
**r:** The fractional occupancy of the receptors (i.e. DR/RT)
**KD: **The equilibrium dissociation constant of the drug for its receptor (a measure of affinity)= [D][R]/ [DR]
71
Whats fractional occupancy?
The number of receptors occupied out of the total number of receptors (the % of receptors that are bound to the drug)
72
What is the name of this equation?
Langmuir Absorption isotherm
73
Whats a micromolar, nanomolar and picomolar in terms of M?
Micromolar: **1x10-6 M**
Nanomolar: **1x10-9 M**
Picomolar: **1x10-12 M**
74
What are the following in micromolar?
1x10-4 M
1x10-5 M
1x10-6 M
1x10-7 M
1x10-4 M: **100 micromolar**
1x10-5 M: **10 micromolar**
1x10-6 M: **1 micromolar**
1x10-7 M: **0.1 micromolar**
75
What is Log10 of 1x10-6 M?
-6
76
To convert between micromolar and molar what do you do?
You divide by 106 (or multiply by 10-6) and then vice versa for molar to micromolar
77
Some DR interactions are more complex, give an 2 examples of this?
78
What is the most likely value for 'n' for the nicotinic receptor?
2 as it has the bind two drug molecules to the receptor
79
What is the most likely value for 'n' for a beta adrenoceptor binding adrenaline ?
0.5
80
Whats an important assumption to make for deriving the equation r=D/D + Kd
That the reaction is in equilibrium
81
When its a bimolecular reaction, does the drug binding always lower the affinity?
Not always
82
What does n\<1 and n\>1 show?
n\<1 is negative cooperativity
n\>1 is positive cooperativity
83
In pD2, what does the p stand for?
**p stands for negative log**
e.g. pH is the negative log of a H ions
In this case, pD2 is the negative log of the agonist concentration that gives half maximal response (it is the negative log of the EC50)
84
Drugs with high values of pD2 act in what type of concentrations?
Drugs with **high values of pD2 act at low concentrations** (note that pD2 is always positive)
85
According to the occupation theory what does pD2 equal?
pD2= -log(KD)
86
Why is the assumptions of occupation theory not always correct for many receptor systems?
The assumptions of occupation theory are not always correct and for many receptor systems pD2overestimates the KD. i.e. the drug appears to bind more tightly than it really does
87
Does pD2 have any units?
No units
88
What is Atropine?
A muscarinic receptor agonist
89
Does pA2 have an units?
What should the value always be?
pA2 has no units and it is always a positive value
90
The higher the pA2 value means what?
The higher the pA2 value the greater the affinity of the antagonist to its receptor
91
What values are used to represent affinity of agonists and antagonists?
pD2: agonists
pA2: antagonists
92
On the straight line graph for detemrining the pA2 value for an antagonist, what does the y-inercept represent?
The value of the -pA2
93
What receptor does acetylcholine and histamine bind to?
Ach: Muscarinic receptor
Histamine: H4 receptor
94
What does Ach and Histamine both cause in Guinea pig ileum?
They both cause contraction
95
How are the 'spare receptors' affected when they are alkylated and 60% of the receptors are 'knocked out'
R is proportional to [D]
96
