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Pharmacology > Receptor Pharmacology > Flashcards

Receptor Pharmacology Flashcards

1
Q

In 1980, De Lean et al came up with which model to explain ligand-receptor interactions?

A

The Ternary Complex Model (TCM)

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2
Q

What does the TCM (ternary complex model) say about the affinity of agonists for the different receptor states?

A

Agonist (but not antagonist) has higher affinity for RG than R

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3
Q

Are receptors always quiescent in the absence of agonist?

A

No. Some receptors appear to spontaneously transition between inactive (R) and active (R*) conformations - giving rise to constitutive activity

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4
Q

Give an example of a receptor that appears to require agonist to become active

A

Rhodopsin

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5
Q

After it was found that some receptors possess constitutive activity, the TCM was adapted by Samana et al (1993) and Weiss et al (1996). What were these new models?

A

The extended TCM

The cubic TCM

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6
Q

What did the extended and cubic TCMs allow for?

A

Receptor can spontaneously undergo R/R* conformational changes and for R* to initiate responses agonist-independently

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7
Q

Ligands exhibit differential affinities for the R and R* states. For example, full agonists have a selective affinity for the R* conformation (R*&raquo_space; R). Give some examples of which conformation different ligand have a selective affinity for.

A

Full agonists: R*&raquo_space; R
Partial agonists: R* > R
Antagonists: R* = R
Inverse agonists: R*

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8
Q 
Wild-type GPCRs appear to vary considerably in their intrinsic constitutive activity. Give an example of a GPCR with:
1) VERY low
2) moderate
3) high
constitutive activity
A

1) very low - rhodopsin
2) moderate - H3 histamine
3) high - virally-encoded GPCRs

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9
Q

The degree of constitutive activity for some GPCRs is cell/tissue-dependent. Why is this?

A

Because a variety of proteins can alter this activity through protein-protein interactions

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10
Q

The degree of constitutive activity for some GPCRs is cell/tissue-dependent due to the proteins present. Give an example of this.

A

Homer Proteins alter the constitutive activity of Metabotropic Glutamate Receptors

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11
Q

What is meant by a ‘CAM’ form of a GPCR? When might these be found?

A

Constitutively-Active Mutant

…found in some very rare genetic diseases

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12
Q

Constitutive activity can be induced experimentally by targeted mutation of GPCRs. Which are 3 key regions which can give rise to CAMs?

A

1) D/ERY sequence mutations (TM3/i2 interface)
2) membrane proximal regions of the i3 loop
3) the TM6/e4 interface

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13
Q

Rare, naturally-occurring mutations of GPCRs (eg CAMs) can lead to disease states characterised by increases in agonist-independent (constitutive) activity. Give some examples of such diseases associated with GPCR CAMs.

A

1) >50 point mutations have been found in the TSH (thyroid-stimulating hormone; thyotropin) receptor, many of which lead to HYPERTHYROIDISM
2) male precocious puberty: luteinising hormone (LH) receptor
3) retinitis pigmentosa and night blindness: rhodopsin
4) short-limb dwarfism: parathyroid (PTH) receptor
5) hypocalaemia/hypercalciuria: Ca2+-sensing receptor

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14
Q

Agents previously classified as antagonists actually represent what?

A

A heterogeneous pharmacological group exhibiting varying degrees of NEGATIVE EFFICACY (ie INVERSE AGONISM)

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15
Q

Whilst a neutral antagonist exhibits no efficacy, what do inverse agonists exhibit?

A

Varying degrees of NEGATIVE EFFICACY

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16
Q

With regard to receptors, what is a ‘quiescent’ system?

A

One with very low or no constitutive (ie agonist-independent) activity

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17
Q

In a quiescent system, neutral antagonists and inverse agonists are functionally indistinguishable. How does this differ from a constitutively active system?

A

Neutral antagonists and inverse agonists have very different effects: only an inverse agonist will suppress constitutive activity

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18
Q

Antagonists and inverse agonists cause pharmacologically distinct actions in constitutively active systems. How may this be helpful in the treatment of genetic diseases caused by CAM GPCRs?

A

Inverse agonists will suppress constitutive activity and thus may be useful in treatment

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19
Q

What type of experiments can be used to discern two distinct agonist binding affinities?

A

Radioligand binding studies

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20
Q

The negative efficacy of inverse agonists, in addition to suppressing constitutive activity, may cause further (long-term) changes to occur with respect to receptor regulation. Give an example of such a change.

A

An inverse agonist may cause a change in the cell-surface receptor expression level
Eg for the H2 Histamine receptor, long-term exposure to:
-histamine reduces receptor expression levels, whilst long-term exposure to
-cimetidine increases expression levels

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21
Q

The choice between a neutral antagonist and an inverse agonist is likely to be therapeutically important. Why?

A

Inverse agonists suppress constitutive activity whilst neutral antagonists do not.

The negative efficacy of inverse agonists may cause a change in the cell-surface receptor expression level.

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22
Q

Which two inverse agonists up-regulate H2 Histamine receptors?

A

Cimetidine and Ranitidine

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23
Q

Give an example of inverse agonists that, when used as a chronic treatment, regulate H2 Histamine receptor expression levels. Give an example of a neutral antagonist. What effect does this have on cell-surface H2 histamine receptor expression levels?

A

Up-regulate: cimetidine and ranitidine
Down-regulate: histamine

Neutral antagonist: burimamadine …has no effect on expression levels

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24
Q

Give an example of an experimental approach that may be used to investigate agonist-stimulated conformational changes in GPCRs.

A

GPCRs may be modified with different types of GFP (CFP and/or YFP) to report conformational change

…through the use of FRET

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25
Q

What does FRET stand for?

A

Fluorescence resonance energy transfer

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26
Q

Briefly outline FRET

A

Fluorescence resonance energy transfer occurs between a ‘donor’ and an ‘acceptor’ label; these labels can be attached to a single protein or to two different proteins.

FRET requires spectral overlap.

FRET generally occurs between cyan (CFP) and yellow (YFP) fluorescent proteins.

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27
Q

How can FRET be used to study GPCRs?

A

GPCRs can be modified with different types of GFP labels (CFP and YFP) through the attachment of a label to:

1) a single GPCR (in the third intracellular loop and the C-terminus)

or

2) the C-terminus of one GPCR and one of the subunits of a G-protein

…excitation of a CFP with light at 436nm causes CFP emission at 480nm plus FRET to YFP, which then emits at 535nm. The extent of FRET varies with sixth power of the distance and is thus an exquisitely sensitive indicator of conformational changes or protein-protein interactions

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28
Q

What frequency of light is required to excite CFP? What frequency light does CFP then emit, and what is the effect this might have on a close YFP molecule?

A

436nm
480nm
…this is absorbed by YFP, which emits light at 535nm

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29
Q

What changes the extent of FRET between CFP and YFP?

A

The extent of FRET varies with sixth power of the distance

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30
Q

What can FRET be used to study?

A

Conformational changes

Protein-protein interactions

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31
Q

How many the effect of addition of agonist to a GPCR be studied?

A

FRET: addition of an agonist presumably changes the distances between CFP and YFP; it can induce a rapid reduction in FRET in a single receptor labeled with CFP and YFP, and can promote the interaction between a YFP-labeled receptor and a G protein labeled at its gamma-subunit with CFP

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32
Q

What variation of FRET can be used to study GPCRs?

A

Bioluminescence resonance energy transfer (BRET)

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33
Q

How does BRET work?

A

It uses a light-emitting enzyme, luciferase, as a a donor, and a GFP variant as an acceptor.

34
Q

What technique was used to generate the first three-dimensional structure of a 7TM protein? Which GPCR was this?

A

Electron microscopy

…Bacteriorhodopsin (a high expressed proton pump)

35
Q

Which crystal structure was the first definitive study to provide information on the R* rather than the R state of the GPCR?

A

Scheerer et al (2008), on the 11-cis-retinal-free opsin-Galpha(t) protein fragment complex

36
Q

What is the importance of the work carried out by Scheerer et al in 2008 on the 11-cis-retinal-free opsin-Gat protein fragment complex?

A

It is the first definitive study to provide information on the R* rather than the R state of the GPCR

37
Q

When was the first GPCR crystallised?

A

1999?

38
Q

Due to recent progresses in X-ray crystallography, the number of experimentally solved GPCRs rose from zero in 1999 to approximately what number in 2012?

A

Around 80

39
Q

Which class of GPCRs have been characterised, and which remain to be experimentally solved?

A 
The 'class A'/'family I'/'rhodopsin family' have over 80 characterised strucutures; 17 of which are in complex with ligand
...the remaining four classes remain to be characterised
40
Q

Give some examples of GPCRs (all are Class A) that have been characterised?

A 
Bacteriorhodopsin
Squid rhodopsin
11-cis-retinal-free opsin
11-cis-retinal-free opsin BOUND TO Gat protein fragment
B2-adrenoceptor
B1-adrenoceptor
A2A-adenosine receptor
H1-histamine receptor
41
Q

Describe, using an example, how solved crystal structures can be used to ‘see’ conformational changes in a GPCR

A

Comparison, e.g. of Gat fragment-bound opsin (R*) and dark-state rhodopsin (R) crystal structures reveals how activation caused conformational rearrangement of the TM domains

42
Q

GPCR conformational complexity can give rise to what agonist-mediated phenomenon?

A

Agonist-directed trafficking of signal via distinct signalling pathways

43
Q

Give an example of how different ‘active’ GPCR conformations may give rise to distinct signalling pathways

A

Different conformations may favour receptor coupling to different intracellular compartments. For example, different active conformational states of the receptor (R* and R**) may preferentially couple to distinct G protein subtypes (e.g. Gs and Gq/11) leading to distinct signalling readouts

44
Q

In which two main ways might agonist-directed trafficking of GPCR-signalling occur?

A

1) Different active conformations may favour receptor coupling to different intracellular components (e.g. particular G protein subtypes)
2) Different active conformations may be more or less susceptible to PHOSPHORYLATION and DESENSITISATION (i.e conformational selection by the agonist may determine whether the receptor response is maintained or progressively attenuated)

45
Q

GPCRs represent one of the largest classes of drug targets, with up to 40% of currently marketed drugs acting at these proteins. Give some examples of disorders for which GPCRs are therapeutic targets.

A

Cancer
Cardiac dysfunction
CNS disorders
Obesity

46
Q

GPCRs can signal effectors independent of G-proteins to induce cellular behaviours. Give some examples of these cellular behaviours, and the effectors that mediate them

A

Cellular proliferation/apoptosis, which is mediated by the induction of B-arrestin-dependent signalling

47
Q

What is ADTRS?

A

Agonist-directed trafficking of receptor signalling

48
Q

Give an example of ADTRS (agonist-directed trafficking of receptor signalling)

A

Structurally unrelated orthosteric (competitive) agonists of serotonin 5-HT2A/2C receptors have demonstrated differential phospholipase C (PLC) and phospholipase A (PLA) activation

49
Q

What is the binding site on a receptor for its natural ligand(s) called?

A

The ORTHOSTERIC binding site

50
Q

Receptors may possess additional ligand/drug binding sites that can be pharmacologically distinguished from the orthosteric site. What are these called?

A

ALLOSTERIC binding sites

51
Q

Why are allosteric interactions pharmacologically important?

A

They offer the possibility of POSITIVELY OR NEGATIVELY MODULATING RECEPTOR ACTIVATION, either dependently or independently of ligand binding at the orthosteric binding site

52
Q

What is the orthosteric binding site?

A

The binding site on a receptor for its natural ligand(s)

53
Q

What is the allosteric binding site?

A

The binding site(s) on a receptor that is pharmacologially distinct from the orthosteric binding site

54
Q

What is a prototypic example of allosteric modulation of a receptor?

A

BENZODIAZEPINES acting at ligand-gated GABAa RECEPTOR/ION CHANNELS

55
Q

What are GABA-A receptors?

A

Ligand-gated Cl- -conducting ion channels

56
Q

What are GABA-A receptors responsible for?

A

The majority of inhibitory transmission in the CNS

57
Q

What is the composition of GABA-A receptors?

A

They are functional pentamers made up of alpha, beta or gamma monomers. The usual stoichiometry is (alpha)2(beta)2(gamma)

58
Q

What is the natural ligand for GABA-A receptors? Where does this ligand bind to activate the receptor?

A

GABA (gamma-aminobutyric acid)

It binds to orthosteric sites at the ALPHA-BETA interfaces

59
Q

GABA-A receptor activation by GABA can be modulated by BENZODIAZEPINES (BZDs). How do BZDs do this?

A

They bind at the interface between an alpha (1, 2, 3, or 5) and a gamma (2) subunit

60
Q

Give an example of a benzodiazepine agonist

A

Diazepam

61
Q

What is the effect of a BDZ agonist, e.g. DIAZEPAM, on the activity of the GABA-A receptor?

A

It enhances the activity of the receptor: i.e. it enhances GABA-evoked Cl- conductance at sub-maximal GABA concentration

62
Q

How can the enhancing actions of BDZ agonists (such as diazepam) at GABA-A receptors be blocked?

A

By BDZ ANTAGONISTS (eg FLUMAZENIL)

63
Q

Give an example of a BDZ antagonist

A

Flumazenil

64
Q

Whilst BDZ antagonists act to block the enhancing effects of BDZ agonists, which compounds DECREASE GABA-evoked Cl- conductance?

A

BDZ inverse agonists (eg SAPMAZENIL)

65
Q

Give an example of a BDZ inverse agonist

A

Sapmazenil

66
Q

What blocks the action of BDZ inverse agonists?

A

BDZ antagonists (eg Flumazenil)

67
Q

What is the effect of BDZ inverse agonists, e.g. sapmazenil, binding to allosteric sites on the GABA-A receptor?

A

They DECREASE GABA-evoked Cl- conductance

68
Q

What type of allosteric modulator are:

1) BDZ agonists
2) BDZ antagonists
3) BDZ inverse agonists

A

1) POSITIVE allosteric modulator (PAM)
2) NEUTRAL allosteric modulator
3) NEGATIVE allosteric modulator (NAM)

69
Q

The Class C GPCRs include which 5 subfamilies?

A

1) Calcium-sensing receptor-related
2) GABA-B receptors (GB1, GB2)
3) Metabotropic glutamate (mGlu) receptors (mGlu1-8)
4) A subset of the taste receptor family
5) RAIG (retinoic acid-inducible orphan GPCRs) (RAIG1-4)

70
Q

Where is the ORTHOSTERIC ligand binding site located in Class C GPCRs?

A

Within the extracellular N-terminal domain: in the Venus FlyTrap (VFT) module

71
Q

What does ligand binding cause in Class C GPCRs?

A

A conformational change that is transmitted via the TM domains to the intracellular signal transduction/G protein-coupling domains (i2/i3 loops/C-terminal)

72
Q

How many different families can the GPCRs be classified into based on the sequence phylogeny of a conserved heptahelical transmembrane domain?

A

Five

73
Q

Which two distinct structural features distinguish Class C GPCRs from other GPCRs?

A

1) their unusually large extracellular domain that is responsible for orthosteric ligand recognition
…while the 7TM (which normally contains the orthosteric binding site) has gained many allosteric binding sites

2) they form dimers

74
Q

Where in Class C GPCRs do the majority of allosteric modulation sites reside?

A

In the 7TM domain

75
Q

In which three general ways can allosteric ligands affect receptor function?

A

1) allosteric modulation of orthosteric ligand BINDING AFFINITY
2) allosteric modulation of orthosteric ligand EFFICACY
3) DIRECT ALLOSTERIC AGONISM

76
Q

Do allosteric modulators offer any advantage in the drug discovery process?

A

1) Orthosteric binding sites of receptors are often highly conserved and it is therefore difficult to generate subtype-selective orthosteric agonists/antagonists
2) Allosteric sites are like to be much less highly conserved and therefore more subtype-selective positive or negative allosteric modulators can be discovered
3) Allosteric modulators have the potential to increase (PAMs) or decrease (NAMs) the actions of the endogenous agonist WITHOUT altering the endogenous pattern of stimulation
4) Therefore, complex intermittent/phasic patterns of receptor stimulation can be preserved

77
Q

Why is it difficult to generate subtype-selective orthosteric agonists/antagonists?

A

Because orthosteric binding sites of receptors are often highly conserved

78
Q

Why is it easier to generate subtype-selective allosteric modulators than orthosteric agonists/antagonists?

A

Because allosteric sites are much less highly conserved than orthosteric sites

79
Q

Why is it an advantage that allosteric modulators have the potential to alter the actions of the natural ligand WITHOUT altering the endogenous pattern of stimulation?

A

This means that complex intermittent/phasic patterns of phasic stimulation can be preserved

80
Q

What is phospholipase C?

A

Membrane-bound enzyme that cleaves inositol phospholipids to produce IP3 and DAG in the inositol phospholipid signalling pathway.

81
Q

What are the two main forms of phospholipase C (PLC), and how are they each activated?

A

PLC-beta: activated by GPCRs via specific G proteins

PLC-gamma: activated by RTKs

Pharmacology (13 decks)

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