Week 2 Flashcards
What is the dose (concentration)-response relationship?
The dose-response relationship depicts the response to an agonist in a cellular and subcellular system as a function of the agonist concentration.
The dose-response relationship is derived from the Langmuir absorption isotherm:
- A = chemical
- p = fraction of target protein bound by A
- k1 = the rate of association with the receptor
- k2 = the rate of dissociation from the receptor
- keq = equilibrium dissociation constant (keq = k2/k1)
p = k1A/k1A + k2 or p = A/A+keq
In pharmacology,
[A] + R <-> [AR]
p = proportion of receptors occupied
[A] = the molar concentration of a drug
[Rt] = the total number of receptors
[AR] = the amount of complex formed between the drug and the receptor
KD = equilibrium dissociation constant of the drug (k-1/k+1)
For a dose-response curve:
- the threshold is where the response begins along the concentration axis
- the slope is the rise in response to changes in concentration
- the maximal asymptote is the maximal response
How are affinity, potency, efficacy, ED50, pD2 and pA2 related to dose-response curves?
Affinity - how tightly a drug binds to the receptor (agonist and antagonist)
Potency - the concentration/dose of a drug at which it produces an effect, EC50 (agonists)
Efficacy - ability of a drug to produce a cellular response, Emax (agonists)
ED50 - the concentration of a drug that produces 50% of a maximum effect (M, mol/L)
pD2 - negative logarithm of EC50 (pD2 = -logEC50)
pA2 - affinity of an antagonist and it is the negative logarithm of the molar concentration of an antagonist that would produce a 2-fold shift in the concentration-response curve for an agonist. To calculate pA2 at least 3 concentrations are needed with the antagonist and one without. If the agonist and antagonist are competitive, the Schild plot will have a slope of 1 and the x-interceptor (pA2) will equal the logarithm of the KB of the antagonist. The pA2 will also equal KD if competition for binding to a single class of receptor site.
What is affinity?
Affinity is the strength of the interaction between a ligand and a receptor. It is governed by:
- the type and number of non-covalent (or covalent if with an antagonist) interactions between the ligand and the receptor
- shape complementarity
Higher affinity means less drug is needed and often enhanced specificity.
What is residence time?
Residence time is how long the molecules stay attached to the receptor.
Residence time = 1/koff
Saturation Binding Assay (affinity)
Measures the amount of ligand bound to the receptor and uses increasing amounts of radioligand with incubation until equilibrium is reached.
Radioligand + receptors solution -> filtration -> gamma counter -> radiation measured
KD is the ligand concentration at which half of the total number of receptors are bound to ligand (M). KD = koff/on = [R] x [L] = [RL].
Bmax is the total density of receptors in a sample of tissue or per cell (sites/cell or fmol/mg protein)
Competition Binding Assay (affinity)
Indirectly measures the affinity of an unlabelled ligand by determining the amount of test ligand required to compete for receptor binding with the labelled ligand. Uses single concentration of radioligand and requires incubation until equilibrium is reached.
IC50 is the concentration of drug required for 50% inhibition of labelled-ligand binding. This parameter is dependent on concentration.
Using Cheng-Prusoff equation IC50 can be converted to Ki which is independent of the amount of labelled ligand added.
Ki = IC50/1 + [L]/Kd
Ki is the equilibrium dissociation constant for binding of the unlabelled drug to the receptor.
Association Binding Assay (kinetics)
Association and dissociation of the labelled ligand is occurring and therefore the kon and koff values are being measured.
kon
R + L <-> RL (complex)
koff
K(observed) = Kon*[L] + koff
Kon is the association rate constant and is concentration dependent (M-1 S-1)
Koff is the dissociation rate constant and is concentration independent (S-1)
Competition Association Binding Assay (kinetics)
Used to calculate the on and off rates of the unlabelled compound. Changing concentrations of the unlabelled ligand are used. Overshoot (bump in curve) is due to the unlabelled competitor dissociating more slowly than the radioligand.
Dissociation Binding Assay (kinetics)
Measuring the dissociation rate of the radioligand. Equilibrium must be reached before the measurement is initiated to prevent labelled ligand from rebinding.
Association = kon + koff
Dissociation = koff
Ways to prevent rebinding are:
- incubation buffer exchange
- >100x dilution of incubation buffer
- vast excess of unlabelled high affinity ligand
Residence time = 1/koff
Half-life = 0.693 (or ln2)/koff
In the curves:
- if there is a deep curve then there is fast dissociation kinetics
- if there is a shallow curve then there is slow dissociation kinetics
How can residence time influence the in vivo pharmacology of a ligand and why is this useful in drug development?
- If plasma half-life < residence time, a drug’s effect will persist beyond its lifetime in the circulation
- Increased residence time can result in kinetic selectivity between receptor subtypes
- Sustained binding (and therefore signalling) could lead to increased adverse effects and toxicity
