Drug affinity (Receptor theory 1), L2

Explain the term ligand

molecule that binds to a specific receptor site on another molecule. This binding may or may not trigger a biological response.

Explain the term affinity

tendency for a drug to bind to a receptor, drugs of high potency generally have a high affinity for their receptors

Explain what Kd is

measure of the affinity of a ligand for its receptor, represents the concentration of ligand at which half of the receptors are occupied

What is the most basic test for whether a binding reaction has reached equilibrium? 

fraction of complex formed between two molecules does not change over time - as many complexes are formed as dissociated 

How do you find Kd ? (equation)

rate of reverse reaction over rate of the forward one

K_D= k_-1 / k₊₁ = [A_eq]*[R_eq]/ [A_eqR_eq]

What is the unit of Kd ? 

moles 

What is the efficacy of agonists vs antagonists? Define efficacy

significant efficacy ie over 0 // 0. tendency for a drug, once bound, to activate a receptor

Explain the concept of receptor occupancy

proportion of receptors occupied will vary with the drug concentration, varies between 0 and 1 (all receptors occupied)

Name some quantitative methods used in pharmacology to measure drug affinity

• radioligand binding assays 

• fluorescence polarisation assays 

• surface plasmon resonance 

• isothermal titration calorimetry 

• computational modelling

Name some limitations and potential problems encountered when performing drug-binding experiment

Radioligand binding assays: Requires radioactive materials, may not be suitable for all receptors.

FPA: Requires a fluorescently labelled drug, may be less sensitive than radioligand binding assays.

SPR:  Real-time measurement, can be used for both small and large molecules, label-free.  

ITC: Requires specialized equipment, may be less sensitive for low-affinity interactions.

Modeling: Requires computational expertise, may not always accurately predict experimental results.

What is a similarity between agonists and antagonists?

both bind to receptors

What is a difference between agonists and antagonists? 

only agonists activate the receptors to induce signalling and a response

What is the forward rate of a reaction?

k₊₁[A]*[R]

What is the reverse rate of a reaction?

k_-1 [AR] , governed by the disassociation rate

What governs occupancy? What values does the latter vary between?

affinity

Occupancy varies between 0 (no drug present) and 1 (all receptors occupied)

Define occupancy

proportion of receptors occupied will vary with the drug concentration

Equation to determine occupancy

nb of receptors occupied / total nb of receptors ie [AR]/[R]

When does measuring/quantifying biological responses to a drug not necessarily work?

when studying an antagonist bc by def doesn’t produce measurable biological reaction

What is the relation between affinity and occupancy?

High affinity generally leads to higher occupancy

Briefly explain the principle of Radioligand Binding Assays to measure drug affinity

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

Briefly explain the principle of Fluorescence Polarization Assays (FPAs) to measure drug affinity

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

Briefly explain the principle of Surface Plasmon Resonance (SPR) to measure drug affinity

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

Briefly explain the principle of isothermal titration calorimetry (ITC) to measure drug affinity

Measures the heat released or absorbed during a binding interaction.

Briefly explain how computational modelling can be used to measure drug affinity

Uses computational methods to predict drug-receptor interactions based on molecular structures.

Name four practical considerations for measuring receptor affinity

• source of receptors and incubation conditions (tissue/cells with correct receptors, incubation must conserve ligands and receptors)

• radioligand (pure, degradation, labelling)

• separating bound from free (filtration or centrifugation, dialysis, column chromatography - rate is main consideration)

• non-specific binding (reduced by anti-absorbants like albumin, collagen for peptides, o-catechol for catecholamines)

How is non specific binding determined?

by addition of excess non-radioactive drug bc Specific ‘bound’ radioactive drug which is present in lower concentrations will be displaced, non-specific bound radioactive drug will remain

What are advantages and disadvantages of 3H as a radio label?

+: 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)

-: Specialized labs required; Labelling is expensive & difficult

What are advantages and disadvantages of 125I as a radio label?

+: If compound has an aromatic hydroxyl group (eg. tyrosine residues in peptides) can be incorporated at very high specific activities (> 2000 Ci/mmol); Iodination easy in most labs and cheap

-:  More readily degraded; Biological activity of ligand can be reduced; (ie. not functionally "invisible"), Short half life (67 days)

What are types of ligands used in radioligand binding assays?

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

What are types of protein targets in radioligand binding assays?

receptors, ion channels, enzymes, carrier molecules

How do you find the amount of specific binding that occurs for a ligand and its receptor?

Specific binding = Total bound – nonspecific binding

What does the following graph of radioligand binding show about the limits of non specific binding?

there is no limit, non specific binding is infinite 

What does the following graph of radioligand binding show about the limits of specific binding? 

once all receptors are occupied, they are termed “saturated“ and the limit of specific binding is reached

What type of scale is binding data usually plotted on?

a semi-logarithmic scale

Specific Binding shows ‘saturation’ while non-specific does not. Why? 

specific binding relies on specific receptors - once all of these are occupied, they are saturated. non specific binding refers to all other types of binding ie can even be to the tube, etc so there can always be more

Why is it recommended when doing experiments on binding data in multiples of 1 and 3?

binding data is usually plotted on a semi-logarithmic scale so multiples of 1 and 3 are easier to read on that type of plot

Which equation shows the relationship between receptor occupancy, affinity and drug concentration? 

langmuir/scatchard equation

If a drug has high affinity for its receptor, will the concentration of the drug needed be higher or lower? And its Kd?

lower

lower

When a drug has high affinity for its receptor, will the forward or the reverse reaction be faster?

forward ie more complexes made than dissociated

Describe the binding capacity for a drug

B max is capacity - saturation of receptors ie every receptor is bound to a ligand, forming a complex AR = no more can be made

What is Kd a measure of? What is it equivalent to?

Kd is measure of affinity

Kd is equivalent to concentration of drug required to occupy 50% of receptors at equilibrium

On the following graph, which drug has the highest affinity for the receptor? What shows this? 

drug a bc lowest Kd = lowest amount needed to reach 50% occupancy ie highest affinity

What does this graph show about the affinity of the drug for muscarinic receptors in sympathteic ganglia and sublingual gland resepctively? What does this tell us abut the structure of muscarinic receptors?

drug is clearly specific for muscarinic receptors in the symathetic ganglia (less drug needed to reach Kd) ie muscarinic receptors have different structure subtypes - drug can bind to both but binds better to the SG receptor

How was it first discovered that muscarinic receptors have different subtypes?

experimentally by pharmacologists testing drugs for affinity with various receptors like muscarinic ones - different affinity to a same ACh ligand implies different subtypes exist. later confirmed by new molecular techniques 

How does this curve show an absence of selectivity of the ligand for the receptors studied?

both ligands/drugs bind to the receptors at the same rate - one doesn’t have a higher affinity than another 

Define ligand competition

inhibitor competes with the ligand for the same binding site on the receptor

What is the inhibition constant?

Ki is a measure of the potency of a competitive inhibitor in preventing the binding of a ligand to its receptor.

What does a lower Ki value indicate?

more potent inhibitor - better competition against other ligands

In competitive inhibition, where the inhibitor competes with the ligand for the same binding site on the receptor, what is the relationship between Ki and Kd?

Ki ≈ Kd

inhibition constant (Ki) is approximately equal to the dissociation constant (Kd) of the ligand because the inhibitor competes with the ligand on a one-to-one basis.

Explain what the following graph demonstrates about the the result of increasing amounts of naloxone being added to mu-opioid receptors already bound to H-DAMGO

with increasing concentrations of naloxone, the % of ‘bound’ ³H-DAMGO decreases ie. It is displaced from the receptor binding sites - example of ligand competition

Which opiate has the highest affinity for the mu opioid receptor? 

Which has the lowest affinity?

sufentanil (curve most to left)

tramadol (curve most to the right)

If a drug is made with a high affinity, how would this effect the concentration of antagonist needed to displace them?

high concentrations of antagonists would also be needed to displace the agonist

What important aspects of drug function does the affinity and potency of drugs not inform on?

this tells you nothing about the action of the drug in terms of cell signalling and physiology!

What Law does the binding of drugs to receptors obey? Explain

Law of Mass Action 

rate of a reaction is proportional to the product of the concentrations of each reactant (duh)

What 2 factors does agonist potency depend on?

affinity and efficacy

What does the binding of an agonist to its receptor trigger?

biological response ie change in cell function

Explain potency

amount of drug needed to produce a given effect

What distinguishes potency from efficacy? 

amount of drug needed for an effect // max possible effect 

Does a partial or full agonist have higher efficacy?

full agonist, partial agonist has lower efficacy (it can't produce the same max response as a full agonist)

Does a partial or full agonist have higher potency?

depends, a partial agonist can bind to the receptor with higher affinity, allowing it to achieve a larger effect at low concentrations than a less potent full agonist - just won’t be able to achieve max response