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Molecular Pharmacology > Lecture 7 & 8 > Flashcards

Lecture 7 & 8 Flashcards

1
Q

What is the difference between AMPA and NMDA receptors?

A
  • NMDA opens slower and has a slower current
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2
Q

Describe the ligand binding domain in iGluRs?

A
  • 2 subunits
  • Forms a dimer
  • Linkers join LBD to ATD
  • Linkers join LBD to TMD
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3
Q

What effect does isolating the LBD from the receptor have?

A
  • Retains native-like agonist binding behaviour
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4
Q

How does glutamate interact with LBD?

A

Via strong ionic and H bonds through the carboxyl group

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5
Q

How can we stabilize the glutamate and LBD complex?

A

By interactions between residues in glu and LBD domains:

  • D1 and glu
  • D2 and glu
  • D1 and D2
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6
Q

Describe the difference between glycine and glutamate binding in NMDA receptors

A

Glutamate:

  • Hydroxyl group in T690 sidechain of GluN2A subunit forms an H bond that stabilises the carboxylate group
  • Y730 has a smaller side chain in van der waal contact with carboxylate sidechain

Glycine:

  • Methyl group of V689 in GluN1 is not able to form H bond so less stable.
  • W731 sterically hinders the glutamate sidechain carboxylate group
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7
Q

How can we study the behaviour of protein structures?

A
  • Using crystal or cryo-EM structures
  • Embed structure in solvent environment
  • Use Isaac Newtons law of motion
  • Use algorithms
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8
Q

What is glutamate funneling and what is it for?

A
  • Glutamate guided into binding site by clusters of charged residues in mouth of LBD.
  • It is needed to increase risetime of AMPAR

(In experiment, mutated AMPAR to remove charged residues = slow risetime of AMPAR)

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9
Q

What is the apo state?

A

Structure of receptor without ligand

with ligand = clam shell like closure

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10
Q

How does the channel open after agonist binding?

A

Coupled linkers between D1 and D2 subunits of LBD change conformation = channel open/close

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11
Q

What is iodo willardiine?

A

It is a partial agonist of AMPAR that binds to GluA2. It causes less closure than glutamate.

(Different ligands causes different conformational changes of LBD)

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12
Q

Why does IW give less current than glutamate?

A

Bulkiness of ligand affects the cleft closure and activation of the receptor. IW is less bulky than glutamate.

When replacing iodine with bromine or fluorine, could see the different effects.

Bulkiness:
Glu > HW > FW > BRW > IW

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13
Q

What is the relationship between linker separation and ligand efficacy?

A

Greater separation is positively correlated with ligand efficacy.

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14
Q

How can we compare potencies/EC50s of different agonistic compounds?

A

By giving each one it’s maximum response so we can easily compare the EC50.

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15
Q

Describe the effects of partial agonists on KAR and GluN1 subunit of NMDAR?

A

Effects are similar to those of full agonists

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16
Q

What is a competitive antagonist?

A

It keeps the ligand binding domain cleft open - competes for the orthosteric site with full agonists and does not cause a conformational change. Whilst, a full agonists leads to a higher degree of closure.

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17
Q

What is the difference between a full agonist and a partial agonist?

A

Full agonist increases the freq of channel opening but does not change the conductance whilst partial agonists reduce the freq of channel opening, also doesn’t change the conductance.

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18
Q

What drugs reduce AMPAR desensitization and how?

A

Diazoxide, Benzothiadiazides and Cyclothiazide

Opens K channels, leading to hyperpolarisation which will prevent the activation of Ca channels. This lead to depressing synaptic transmission.

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19
Q

Describe cyclothiazide

A
  • It is a positive allosteric modulator of AMPAR
  • Reduces desensitization
  • CTZ dimer bind at an allosteric site between the D1 and D2 subunits of LBD
  • It increases the dimer affinity by 5000 fold (ultracentrifugation)
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20
Q

How can we see the relationship between LBD dimer affinity and desensitization?

A
  1. Mutation in LBD dimer = D1s separates whilst D2s don’t = channel still closed even when ligand is bound. This is desensitization.

= Found that increased dimer affinity is correlated with reduced desensitization of the intact receptor.

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21
Q

What are flip and flop and what do they do?

A

Exons that are removed from LBD of AMPAR (GluA1-4) that modify:

  • Contacts across dimer interface
  • AMPAR desensitization
  • Sensitivity to cyclothiazide
22
Q

What is aniracetam?

A

It is a PAM that occupies the A subsite between D1 and D2

23
Q

Where so classical thiazide (CTZ) compounds bind?

A

B and C subsites in LBD

24
Q

Where does PEPA bind?

A

To all subsites in LBD

25
Q

What is the relationship between the different binding sites in the LBD and their modes of action?

A

Means that PAMs of AMPAR form varying degrees of contact with:

  • Interdomain hinge to stabilize glutamate bound conformation
  • Dimer interface of both upper D1 lobes to stabilize the dimer
26
Q

What is the difference between aniracetam and ampakines?

A

Ampakines also bind to A subsite but are more rigid, this increases the efficacy of ampakines.

27
Q

Side notes

A

PAMs have not received FDA approval due to toxicity, side effects and doesn’t work at safe doses.

28
Q

What iGluR has the highest rate of desensitization?

A

AMPAR > KAR > NMDAR

same for deactivation rates

29
Q

What are are the different pathways for NMDAR desensitization?

A
  • Gly-dependent desensitization
  • Zinc-dependent desensitization
  • Gly/Ca independent desensitization
  • Ca dependent inactivation
30
Q

Describe the affinity of different AMPAR agonists

A

2-Me-Tet-AMPA > Quisqualate > AMPA > Glu

Opposite looking at rate deactivation slide 42

31
Q

Describe the relationship of potency and deactivation rates of agonists

A

Negative correlation

positive correlation between potency and affinity

32
Q

What iGluR has the most mutations and what does this lead to?

A

NMDA > AMPA > KAINATE > DELTA

Can lead to seizures and variety of mental and developmental disorders.

33
Q

How can we study the affect of mutations on iGluR function?

A

Heterologous expression systems to establish how the mutations affect iGluR function

34
Q

Give an example of personalized therapy

A

Inhibition of NMDARs in patients with GOF GluN2A mutations will reduce seizure frequency.

e.g. Memantine: negative allosteric modulator of NMDAR.

35
Q

Would treatment with memantine be equally effective for all patients with epilepsy?

A

No, because different mutations have different effects. E.g. mutation in V619G would decrease the potency whilst, a mutation in N615I increases the potency.

36
Q

What can memantine treat?

A

Moderate to severe AD

37
Q

What is memantine?

A

A dissociative anaesthetic at supratherapeutic doses

It is an uncompetitive antagonist of NMDAR: open channel blocker

38
Q

How can reduce NMDAR current?

A

Apply memantine during NMDA application (blocks channel not the orthosteric site), not between.

39
Q

What is the effect of MK-801 on NMDAR?

A

Slowly reduces current

40
Q

What is better at blocking NMDAR?

A

Memantine > Mg

Mg and Memantine leave upon large depol. MK-801 leaves the channel upon large depol

41
Q

Why do Mg and Memantine leave during depol?

A
  • Due to pronounced voltage dependent block and rapid unblocking kinetics
42
Q

SIDE NOTES

A

AD:

  • Due to too much glutamate
  • Sustained Ca influc through NMDARs
43
Q

How do pathalogical conditions affect NMDAR blocking and shit aejkdfkla?

A

Mg leaves during mild depol (not memantine)

44
Q

Name other targets of memantine

A

Serotonin receptor (non competitive antagonist)

Dopamine (agonist)

NMDAR (uncompetitive blocker)

Sigma1 (agonist)

alpha7 nAChR (noncompetitive antagonist)

45
Q

Name 3 uncompetitive (blockers) allosteric modulators of NMDARs

A

Mg, memantine, ketamine

46
Q

What are Ifenprofil and Zn (in relation to NMDAR)?

A

Non competative antagonists (allosteric modulators) of NMDAR

47
Q

What is perampanel?

A

It is a noncompetitive antagonist of AMPAR.

FDA approves for partial onset siezures in adults and now children and also for 75% of epilepsy patients

48
Q

What are the parampanel binding sites on AMPAR?

A

Hydrophobic pocket on extracellular side of ion channel on each subunit in tetramer.

Interactions between residues in preM1, M3 and M4. This prevents conformational changes that are needed for channel opening.

49
Q

What are the side effects of perampanel?

A

Motor deficits at high doses

50
Q

What is a better way to prevent seizures other than perampanel?

A

By targeting AMPAR complexes that have auxiliary proteins bound. E.g. TARP gamma 8

Examples of drugs: Lilly and Janssen

Protected rats from seizures without side effects of perampanel or memory deficits

51
Q

What is an uncompetitive blocker of AMPAR?

A

Polyamine toxins

Molecular Pharmacology (5 decks)

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