Advanced drug ligand binding properties

Question 1

Influence of receptor reserve on transducer coupling

Answer 1

When receptor reserve is low, an agonist may only produce a response in the pathway to which the receptor couples strongly. Conversely, when receptor reserve is high, the same agonist may produce a response in all pathways at submaximal receptor occupancy

Question 2

True Partial Agonism ?

Answer 2

• As receptor reserve decreases CR curves will displace dextrally until reserve is 0. After this point the maximal response will decrease successfully, which can give the appearance of partial agonism

Question 3

In vivo vs Ex Vivo

Answer 3

A common approach to studying agonist responses is by evaluating receptor signalling in recombinant cell lines that overexpress the receptor of interest (e.g. in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells).

Although receptors may signal promiscuously under these high receptor density conditions, they may be less promiscuous in vivo where receptor expression is typically lower, and where other receptors may compete for the same pool of G proteins.

Question 4

Temporal Promiscuity

Answer 4

Receptor phosphorylation can lead to a switch in transducer coupling. For example B2 AR stimulation leads to the accumulation of cAMP via a Gas pathway and activation of PKA. PKA subsequently phosphorylate the receptor which results in a change in coupling preference to to Gai which inhibits cAMP.

• also B-arrestin dependent signalling pathway can result in changes to signalling outcomes as a function of time
• Receptors transducer preference may change depending on if agonist receptor binding is at equilibirum

Question 5

Spatial Promiscuity

Answer 5

Receptors are not expressed only in the cell membrane but also distinct localized microdomains. Different compartments will alter signalling due to availability of different signalling partners

Question 6

Agonist induced Bias

Answer 6

• Distinct ligands can stabilize different receptor signalling states; each with a prefference to interact with distinct subsets of receptors
• Modulators can also induce bias for a particular pathway

Question 7

Detecting Agonism Bias

Answer 7

• A selected standard agonist must be used to account for SYSTEM BIAS and OBSERVATIONAL BIAS
• Tell tale signs include reversals in efficacy and/or POTENCY between two pathways
• BIAS plot: if the bias plot deviate from a straight line it indicates the agonist receptor complex couples to one pathway at the preference of another, must be performed for at least two agonists which must show different results. If they show the same results it is indicative of system bias

Question 8

Allosteric Modulators

Answer 8

Bind to a site topographically distinct from the orthosteri site of the receptor

PAMS: potentiate orthosteric agonist activty

NAMS: augment orthosteric agonist activty

NALS: bind to the receptor but do not alter orthosteric ligand activity

three main mechanisms:

1. They alter the affinity of the orthosteric ligand for the receptor
2. They alter the efficacy (signalling or channel opening capacity) of the orthosteric ligand by altering the energy required to activate the receptor
3. They act as agonists or inverse agonists

Question 9

Detection and quantification of allosteric effects on agonist binding

Answer 9

• Labelled orthosteric ligands, binding in the presence or absence of an allosteric modulator can be measured. As allosteric modulators alter affinity by changing the on/off rate kinetic based

Question 10

Detecting and quantifying effects on orthosteric agonist function

Answer 10

In the isntance where the allosteric modulator does not affect binding, a functional assay such as a second messenger assay

Question 11

Probe Dependence

Answer 11

When allosteric modulators only work where the agonist is present

Question 12

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