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

Question

What is a disadvantage of radiolabeled binding assays?

Answer 1

requires radioactive materials, may not be suitable for all receptors

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

Measures the heat released or absorbed during a binding interaction

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

requires computational expertise, may not always accurately predict experimental results

Answer 14

- neurotransmitters - hormones - growth factors -cytokines/chemokines - drugs - toxins

Answer 15

- receptors - ion channels - enzymes - carrier molecules

Answer 16

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

Answer 17

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

Answer 18

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.

Answer 19

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.

Answer 20

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

Answer 21

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)

Answer 22

- Radioactive hydrogen (3H) - Radioactive iodine (125I)

Answer 23

- 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)

Answer 24

- specialised labs required - labelling is expensive& difficult

Answer 25

- 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

Answer 26

- more readily degraded - biological activity of ligand can be reduced (i.e. not functionally 'invisible') - short half-life (67 days)

Answer 27

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

Answer 28

- 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

Answer 29

take away the non-specific from the total: Specific = Total bound - nonspecific binding

Answer 30

semi-logarithmic scale

Answer 31

50% occupany

Answer 32

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

Answer 33

Less drug required = better binding Lower KD = higher affinity

Answer 34

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.