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A Pharmacology L2 > Lecture 2 - Drug-receptor interactions 1 (affinity) > Flashcards

Lecture 2 - Drug-receptor interactions 1 (affinity) Flashcards

1
Q

What is a receptors occupation governed by?

A

affinity

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2
Q

What is a receptors activation governed by?

A

Efficacy

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3
Q

What is affinity?

A

A way of describing the tightness of the fit between drug and receptor

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4
Q

What can you can a receptor when it has a ligand associated?

A

occupied

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5
Q

What determines the rate of reaction?

A

‘law of mass action’ - as well as the concentration of the drug (ligand) and receptor

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6
Q

Do the rules for affinity change depending on whether the drug is an agonist or an antagonist?

A

no

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7
Q

What is the efficacy of an antagonist?

A

0

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8
Q

What is a ligand?

A

A ligand is a molecule that binds receptor site on another molecule. This binding MAY or MAY NOT trigger a biological response

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9
Q

Affinity definition

A

The tendency for a drug to bind to a receptor is governed by its affinity.

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10
Q

What type of affinity do drugs of a high potency usually have?

A

A high affinity for their receptors

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11
Q

What is the definition of efficacy?

A

The tendency for a drug, once bound, to activate a receptor

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12
Q

What is KD?

A

a constant that defines the affinity of a drug for a receptor

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13
Q

What is KD expressed in?

A

Molarity

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14
Q

What is the forward rate of reaction?

A

K+1(A)*(R)

A = ligand
R = receptor

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15
Q

What is the reverse rate of reaction?

A

K-1(AR)

AR = ligand-receptor complexes

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16
Q

What is the equation at equilibrium?

A

K+1[A]*[R] = K-1[AR] = KD

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17
Q

What is occupancy?

A

proportion of receptors occupied will have vary with the drug concentration

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18
Q

What do you measure occupancy?

A

number of receptors occupied/total number of receptors (measured between 0 - 1)

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19
Q

Describe the relationship between occupancy and affinity

A

Direct relationship (inverse)

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20
Q

Why is a bioassay not a good way to determine affinity?

A
  • Relationship between occupancy and response isn’t always linear
  • Antagonist doesn’t generate resposne
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21
Q

What could be used to measure affinity?

A

Biochemical techniques

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22
Q

What are 4 experimental approaches used to measure drug affinity?

A
  • Radioligand binding assay
  • Fluorescence
    polarisation assays
  • Surface Plasmon Resonance
  • Isothermal Titration Calorimetry
  • Computational modelling
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23
Q

What is a radioligand binding assay?

A

A radiolabeled drug is incubated with receptor-containing tissue or cells. The amount of radiolabel bound to the receptors is measured

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24
Q

What is an advantage of radiolabeled binding assays?

A

Sensitive - can be used with a wide range of receptors

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25
Q

What is a disadvantage of radiolabeled binding assays?

A

requires radioactive materials, may not be suitable for all receptors

26
Q

What is fluorescence polarisation assays?

A

A fluorescently labeled drug is used. When it binds to a receptor, its rotational freedom decreases, leading to increased polarisation of emitted fluorescence

27
Q

What is an advantage of fluorescence polarisation assays?

A

non-radioactive, high throughput, can be used for both small and large molecules

28
Q

What is a disadvantage of fluorescence polarisation assays?

A

requires a fluorescently-labeled drug, may be less sensitive than radioligand binding assays

29
Q

What is surface plasmon resonance?

A

Measures changes in the refractive index of a surface when molecules bind to it

30
Q

What is an advantage of surface plasmon resonance?

A

real-time measurement, can be used for both small and large molecules

31
Q

What is a disadvantage of surface plasmon resonance?

A

requires specialised equipment, may be less sensitive for low-affinity interactions

32
Q

What is isothermal titration calorimetry?

A

Measures the heat released or absorbed during a binding interaction

33
Q

What are the advantages of isothermal titration calorimetry?

A

provides thermodynamic parameters (enthalpy. entropy), can be used for both small and large molecules

34
Q

What are the disadvantages of isothermal titration calorimetry?

A

requires specialised equipment, may be less sensitive for low-affinity interactions

35
Q

What is computational modelling?

A

uses computational methods to PREDICT drug-receptor interactions based on molecular structures

36
Q

What are advantages to computational modelling?

A

can provide insights into binding mechanisms, can be used to screen large libraries of compounds

37
Q

What are disadvantages to computational modelling?

A

requires computational expertise, may not always accurately predict experimental results

38
Q

What are examples of ligands used as radioligand binding assays?

A
  • neurotransmitters
  • hormones
  • growth factors
    -cytokines/chemokines
  • drugs
  • toxins
39
Q

What are examples of proteins used as radioligand binding assays?

A
  • receptors
  • ion channels
  • enzymes
  • carrier molecules
40
Q

What does an increase in ligand lead to?

A

Increase in occupancy, but also increase in non-specific binding

41
Q

Describe the process of a ligand-binding practical

A
  1. Tissue preparation + radioligand
  2. Mix and incubate
  3. Filter
  4. Rinse
  5. Count
  6. Analyse data
42
Q

Describe the steps involved in source of receptors

A

Selected to contain the recognition sites (receptors of interest)
- can be isolated membranes, slices, synaptosomes, cultured cells or solubilized/purified receptors - e.g. from the total brain or specific brain region, or immortalized cell lines expressing the receptor.

43
Q

Describe the steps involved in incubation

A

Must (try to) preserve integrity of ligands and receptors . “additives” used to protect tissue/ligand (protease inhibitors for peptides antioxidants if ligand is oxidizable.

Temperature: important parameter - usually low room to 0 degrees Celcius. Important as temperature affects enzyme activity.

44
Q

Describe properties of the radioligand needed when measuring receptor affinity

A

Ligand must be biologically active, as it is suppose to correlate with a pharmacological action

PURITY - ligand must be extremely pure chemically

Degradation - major problem

Labelling - labelling of the drug with radioactivity must achieve very high specific activity to allow very low (“tracer”) concentrations

45
Q

Describe ways to prevent degradation of radioligand

A
  1. Free-radical scavenger (e.g. ethanol) in drug solution
  2. Store at low (not freezing) temperature
  3. Avoiding light (dark bottles to store)
  4. Incorporation of antioxidants (ascorbic acid)
46
Q

Describe the 2 choices of radio-labels

A
  • Radioactive hydrogen (3H)
  • Radioactive iodine (125I)
47
Q

What are advantages of using radioactive hydrogen?

A
  • labelled product indistinguishable from native compound
  • high specific activities (>80 Ci/mmol) can be obtained
  • good stability when properly stored
  • Long half-life (12.5 years)
48
Q

What are the disadvantages of using radioactive hydrogen (3H)?

A
  • specialised labs required
  • labelling is expensive& difficult
49
Q

What are advantages of using radioactive iodine?

A
  • if compound has an aromatic hydroxyl group (e.g. tyrosine residues in peptides) can be incorporated at very high specific activities (>2000 Ci/mmol)
  • iodination easy in most labs and cheap
50
Q

What disadvantages of using radioactive iodine (125I)?

A
  • more readily degraded
  • biological activity of ligand can be reduced (i.e. not functionally ‘invisible’)
  • short half-life (67 days)
51
Q

Describe the process of separating bound from free

A

Separation of bound and free ligand in incubation is done via filtration or centrifugation

  • for soluble binding, other techniques are used - dialysis, column chromatography, precipitation/adsorption
  • Major consideration is RATE of dissociation of ligand-receptor complex - speed of separation must be compatible with affinity of ligand for receptor
  • lower affinity (higher KD) requires faster/more efficient separation
52
Q

Describe non-specific binding when measuring receptor affinity

A
  • many ligands bind non-specifically to other proteins in the preparation, plastic, filter paper, glass
  • non-specific binding to filters & glass can be reduced by ANTI-ABSORBANTS -e.g. albumin or collagen for peptides, o-catechol for catecholamines
  • however, this doesn’t reduce non-specific binding to other proteins or lipids in the preparation.
  • Measuring proportion of specific and non-specific binding is a KEY element to assay
  • Rinsing only removed unbound - non-specific binding is determined by addition of EXCESS non-radioactive drug, as specific bound which is present will be displaced, however non-specific bound will remain
53
Q

How do u find the specific radioligand binding curve?

A

take away the non-specific from the total:

Specific = Total bound - nonspecific binding

54
Q

What measurement is concentration shown in?

A

nmol/L

55
Q

What measurement is specificity bound shown in?

A

mol/mg

56
Q

What type of scale is used to plot binding data?

A

semi-logarithmic scale

57
Q

What is KD?

A

50% occupany

58
Q

What is the equation for specific bound?

A

Specific bound =
amount of ligand specifically bound x total number of receptors //// concentration of ligand + KD (dissociation constant)

59
Q

Describe why there is an inverse relationship between KD and affinity

A

Less drug required = better binding
Lower KD = higher affinity

60
Q

What occurs in competition binding experiements?

A

Normal radioligand binding with a known drug.

In competition assays, increasing conc. of an unlabelled ligand is added together with a fixed concentration of a labelled ligand

e.g. Naloxone added to mu-opioid receptors bound with 3H-DAMGO.

increasing concentrations of Naloxone, the % of bound 3H-DAMGO decreases (displaced from receptor binding sites.

The concentration of Naloxone required to displace 50% bound 3H-DAMGO is the inhibition constant K, units Molar

New drug tested to see if it displaces the radioactive ligand.

Concentration of competitor to displace 50% of the known ligand with be the KD of the new drug.

A Pharmacology L2 (15 decks)

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