Lec3: Tyrosine Kinase and How Drugs Work (potency, efficacy, etc)

Question 1

What do tyrosine kinase receptors function as? Name an actual relevant example.

Answer 1

1. Kinase Enzyme (transfers P from ATP to tyrosine residues on intracellular target proteins)
2. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)

Question 2

What are the three steps in the activation of VEGFR2?

Note: In step 2, something associates with something else, clarify each of these

Answer 2

1. Ligand binds = receptor dimerisation
2. Autophosphorylation of tyrosine residues on cytoplasmic domain –> which associate SH2 domain proteins
3. Phosphorylation cascade set off

Question 3

What are the steps in the example of a tyrosine kinase phosphorylation cascade involving Phospholipase C (VEGFR2)?

Answer 3

1. Phospholipase C activated via phosphorylation
2. Converts PIP2 –> DAG + IP3
3. DAG –> Phosphokinase C
4. Phosphokinase C (via Raf and MEK) activate ERK
5. ERK binds to gene (increases gene transcription, this increases cell proliferation/angiogenesis)

Question 4

When are two situations in which you would want to enhance and inhibit angiogenesis respectively?

Answer 4

Wound healing and Cancer

Question 5

What are the 4 kinds of forces that drugs use to bind to other molecules? Roughly rate the strength of each.

Answer 5

1. Van der Waals - weakest
2. Ionic interactions - medium
3. Hydrogen bonding - strong
4. Covalent bonding - essentially irreversible

Question 6

What is the equation for fractional occupancy?

Answer 6

(Drug/ligand conc.)/(Affinity constant + Drug/ligand conc.)

Question 7

What is Kd and how does the size of this value relate to affinity? Also, draw a graph that illustrates this.

Answer 7

1. Affinity constant = The concentration of drug required to occupy 50% of receptor population.
2. Smaller Kd = Greater binding affinity.

Question 8

Why are biological response curves a poor measure of affinity? Give the two reasons for this.

Answer 8

Relationship between receptor occupancy and response is not strictly proportional.

1. Downstream factors from receptor influence final response
2. Considerable amplification = low occupancy may still give max response

Question 9

What is EC50 and how does the size of this value relate to affinity? Also, draw a graph that illustrates this.

Answer 9

The concentration of drug to achieve 50% of max response. It is a measure of potency.
Smaller EC50 = Greater potency.

Question 10

Define efficacy. What unit from a graph is used to quantify this?

Answer 10

The ability of a drug to change the action of a receptor via inducing a conformational change. Seen by how the drug changes the Emax value.

Question 11

Define agonist, antagonist, and inverse agonist.

Answer 11

Agonist = positive efficacy
Antagonist = no efficacy
Inverse agonist = negative efficacy (decreases basal receptor activity)

Question 12

What is basal receptor activity?

Answer 12

Activity of a receptor in the absence of ligand.

Question 13

When would you use an inverse agonist instead of an antagonist?

Answer 13

In cases of hyperactive receptors (increased basal receptor activity), as antagonists won’t reduce BRA, but simply prevent the receptor from being MORE active.

Question 14

Compare full agonism to partial agonism.

Answer 14

Full = drug binding induces max tissue response.
Partial = drug binding does not give max tissue response, even at 100% FO.

Question 15

Why may drugs appear full agonists in one tissue but then partial agonists in another? Illustrate this on a diagram.

Answer 15

Because of the different tissue properties, as different tissues will have different tissue maximums.
$(Maximum response = efficacy + tissue properties)$

Question 16

Describe reverse competitive antagonism.

Answer 16

Drugs binds transiently to orthosteric binding site, competing directly with agonist/natural ligand.

Question 17

How is reverse competitive antagonism:
1. Shown on a biological response curve
2. Is the above a limited effect?
3. Overcome?
Draw an accompanying diagram.

Answer 17

1. Parallel rightwards shift in curve with no decrease in Emax
2. Theoretically limitless
3. Adding more agonist

Question 18

Define irreversible competitive antagonism.

Answer 18

Drug binds covalently to orthosteric binding site (cannot overcome even by adding more agonist).

Question 19

In some cases, LOW DOSES of irreversible antagonist may not show a decrease in Emax of biological response curves for the agonist. Why is this?

Answer 19

Because some agonists do not require full occupancy to achieve max response. You need higher doses of antagonist before the Emax begins to decrease.

Question 20

In terms of R-state and R*-state of the receptor, which state in the equillibrium do these ligands cause the receptor to favour and to what extent?

1. No ligand (basal receptor activity)
2. Full agonist
3. Partial agonist
4. Antagonist
5. Inverse agonist

Answer 20

1. Favours R
2. Strong shift to R*
3. Partial shift to R*
4. No shift in equillibrium
5. Shift to R

Question 21

How are the allosteric and orthosteric binding site related?

Answer 21

Allosteric site is conformationally linked to orthosteric site, and binding to the allosteric site (allosteric interaction) will modulate binding and signalling of orthosteric site.

Question 22

What are the two methods of allosteric enhancement (ie. increasing affinity of binding to orthosteric site), and which is more common?

Answer 22

1. Increase ligand association rate

2. Decrease ligands disassociation rate (more common)

Question 23

Does efficacy have to be altered independently of affinity in allosteric interactions?

Answer 23

No. They can be altered together as well, depending on the type of non-competitive antagonism.

Question 24

Draw a biological response curve and agonist-specific binding curve for each situation and name the relevant receptor and modulator:

1. Increased max response + potency, no change to binding
2. Decreased max response + potency, no change in binding
3. Decreased max response + potency, increase binding affinity

Answer 24

1. Glutamate receptor - CPCCOEt
2. GABA receptor - CGP
3. Cannaboid receptor - Org

Question 25

Why are allosteric binding sites easier to selectively target than orthosteric binding sites?

Answer 25

Ortho all very similar as they must recognise the specific ligand in order to operate –> huge evlutionary pressure to maintain this, otherwise there could be a loss of healthy function. Allo are very different (less evolutionary pressure to maintain these), and so drugs can be created to target receptor SUBTYPES by targeting allosteric binding sites.

Question 26

What are the three reasons allosteric modulators have a therapeutic potential?

Answer 26

1. Only show activity when endogenous ligand is present (greater spatial and temporal resolution of drug effect).
2. Ceiling effect may have therpeutic advantage
3. Allosteric sites enhance selectivity

Question 27

What does it mean by “enhanced spatial and temporal resolution of drug effect” with respect to allosteric modulators? Illustrate this.

Answer 27

Tunes up or tunes down NATURAL response of the body at that particular point in time, because they bind to the allosteric binding site (non-orthosteric), so the endogenous ligand can still act (whether inhibitory or excitatory, depends on the bodies intentions at that time).

Question 28

Why can you inhibit and enhance receptors but only inhibit enzymes?

Answer 28

Enzymes orthosteric binding site is an active site. Substrates that bind here are altered/broken down. It can be blocked with inhibitory drugs, but any “enhancing drugs” are futile, as they’ll simply speed up the process of itself being broken down, rather than targeting other substrates. To enhance enzyme activity, you need to find allosteric ligands, but these are very hard to make.

Question 29

What are two methods by which neurotransmitter is quickly inactivated (removed from synaptic cleft) to prevent overstimulation of post synaptic cell?

Answer 29

1. Reuptake into presynaptic terminal by transporters

2. Degradation by enzymes (eg. Acetylcholine esterase)