LECTURE 3: RECEPTOR-LIGAND INTERACTIONS – AGONISM & ANTAGONISM Flashcards
Why do we quantify drug:target interaction?
1) 3 reasons
1) Because the therapeutic + toxic effects of a drug depend on the dose
a. Need to know why the dose makes the poison
2) Different molecular mechanisms of action imply different mathematical framework
3) The potency of a drug is intimately linked to the strength of the interaction with their target
a. Achieving potency = big increase in strength of interaction
Molecular Mechanisms of Drug Action:
1) What are the 3 parameters of molecular mechanisms in drug action to consider
1) Parameters to consider:
o Kinetics:
♣ reversible vs irreversible
♣ covalent binding
o Binding site:
♣ Orthosteric vs allosteric
* Allosteric modulators affect the affinity a drug has for a substrate
o Functional effect:
♣ Activation (agonist) vs inhibition (antagonist)
♣ Competitive vs non-competitive inhibition
Receptor: Ligand Binding:
1) what can be used to describe receptor:ligand interactions
2) @ equilibrium, what is equal
3) what is the equilibrium dissociation constant and what is it used for
4) What is the equation for Kd
5) what is the Kd at low affinity vs high affinity
6) what is Kd in a graphical representation of drug response (semi-log plot)
1) A chemical equilibrium equation
2) @ equilibrium: rate of complex association = rate of dissociation
3) Kd –> used to evaluate and rank order strengths of bimolecular interactions
4)
5) Low affinity = high Kd
o Hard to occupy receptor at x concentration of ligand
High affinity = low Kd
o Easier to occupy receptor at x concentration of ligand
6) Midpoint of semi-log plot = Kd
Occupancy:
1) what is occupancy defined as
2) what does it determine
3) what is receptor concentration difficult to find
4) what unit is the value of occupancy
5) what is the value related to
6) what are the equations for occupancy
7) If [R] «_space;[L] or Kd then what
1) proportion of receptor/enzyme occupied by a ligand at any given time
2) Determines the potency + efficacy of a drug
3) Receptor concentration is difficult to find out
♣ Cannot effectively calculate concentration because the same receptor can be found distributed in the body
4) Unitless number
5) Is related to:
♣ Kd
♣ receptor/ligand concentrations
6) Y max = 1, Y min = 0
7) If [R] «_space;[L] or Kd then [L total] ~ [L free]
♣ Amount of ligand bound to receptor is negligible
Cooperativity:
1) When does cooperativity occur
2) what kind of receptors (+ example) display this behaviour and how
3) What value is cooperativity modelled by
4) what indicates positive and negative cooperativity and what does it mean
5) in what equation is the coefficient used
6) what value is no cooperativity
1) Occurs when binding of a ligand at one site influences the binding at another site
2) Oligomeric receptors (GPCRs) frequently display this behavior
- Through allosteric conformational changes
* (communicate to other binding sites via allostery)
3) Modelled using Hill Coefficient (n)
4)
o Positive cooperativity
♣ (n > 1)
♣ Binding of additional ligand is favored
♣ Kd2 «_space;Kd1
o Negative cooperativity
♣ (n < 1)
♣ Binding of additional ligand is disfavored
♣ Kd2»_space; Kd1
5)
6) n=1
Cooperativity: EXAMPLE
1) what has a higher Hill coefficient: hemoglobin or myoglobin and what does this mean
2) why is this the case
3) even if this is the case, how do they compare in affinity for O2
1) hemoglobin has a higher Hill Coefficient than myoglobin
2) This is because myoglobin (as a monomer) does not experience cooperative binding of oxygen like hemoglobin does (tetramer)
3) Myoglobin has a higher affinity for O2
* Binds O2 at very low concentrations of O2
How Do We Measure Interactions Between Molecules:
1) what is the method
2 + 3) what is the main principle of this method and describe it
4) what fluorophores will re-emit polarized light vs non-polarized light
5) what is the degree of polarization related to
1) Fluorescence Polarization/Anisotropy
2) Fluorescent probes are used to determine binding and kinetic constants
3) Addition of fluorophore to ligand
* Taking advantage of polarized light
o Irradiate fluorophore with polarized light excited
♣ Excited state in fluorophore has a specific lifetime (duration)
o When this polarized photon is absorbed, it should be remitted polarized too HOWEVER, molecules rotate in solution
- The molecule can actually re-emit the light in different directions (depolarized)
4) Fluorophores irradiated with polarized light will reemit polarized light
* Only if the molecules didn’t move during the excitation lifetime
o If the molecule rotates then polarization is lost
♣ Small molecules rotate faster than big molecules
* Bigger molecules (moving less) will remit more polarized light compared to the amount of polarized light the smaller molecule will emit
5) Degree of polarization retained is related to degree of molecular binding
* See if ligand is bound vs unbound via amount of depolarized light
o Heavier (i.e. ligand + receptor) = more polarized light then just ligand or just receptor
Choice of Fluorescent Label Based on Lifetime:
1) what does the choice depend on
2) whats an example of how weight changes polarization and how the choice of type of fluorescent molecule is crucial
3) explain how lifetime of fluoresence impacts polarization results
1) The choice of a fluorescent label depends on the expected variation in molecular weight of the already interacting molecules
2) EX: Fluorescein: lifetime ~ 4ns
♣ 2kDa fluorescently-labeled ligand will retain no polarization
♣ However, upon binding to a 50kDa receptor, the polarization will change from nearly ~0 –> 0.4
- Therefore, must choose fluorescent molecule that has a lifetime that matches molecular weight of molecule observed
3) * how much time does the molecule take to randomize its orientation relative to incident plane vs the lifetime of the fluorophore
o If the lifetime is the same time taken by the molecule to randomize itself, then the light will emit depolarized
♣ However, if the lifetime of the fluorophore was shorter than the ligand time to randomize then, light emits polarized (even if the ligand was small)
Binding Analysis with FP:
1) What are the requirements of a fluorescent ligand
2) what is the requirement of the fluorescent interactor (analyte)
3) what else can Kd be measured using
4) what is a binding isotherm and when is it used in this scenario
1)
o Should be small such that binding results in large change in rotational correlation time
o Can be very dilute (0.1-0.001uM)
♣ Because fluorescence measurements are usually very sensitive depending on fluorophore
♣ You don’t want to saturate the signal
2) Concentration range of the analyte should cover 100-fold below and above Kd for proper estimation of Kd
3) Kd can be estimated by using a binding isotherm
4) Binding isotherm: a curve of the number of ligands adsorbed as a function of the concentration of the ligand at a fixed temperature
♣ Used if fluorescent ligand concentration is well below Kd
FP:
1) what does the FP equation look like (write it)
2) what is the key variable in this equation and what does it mean
3) what does max FP mean
1) looks like cooperativity equation
2) ∆mP = different between basal fluorescence polarization of ligand and maximum polarization
3) Max FP found indicates max polarization
♣ Meaning a lot is bound slower movement
♣ Max polarization is usually when ligand concentration is max (more bound)
Competitive Inhibition:
1) what are 2 main types
1) - Reversible Competitive Inhibitors
- Irreversible Competitive Inhibitors (covalent inhibitors)
Reversible Competitive Inhibitors:
1) what rate constant does this use and its equation
2) what is the equation for occupancy in this condition
1) Kapp = function of ligand equilibrium constant + inhibitor equilibrium constant + inhibitor concentration
2)
Competitive Inhibition:
1) what does this inhibition do
2) what is the equation for occupancy in this condition
1) Reduce proportion of active, unmodified receptors without changing KL
2)
Application of FP Screening Competitive Inhibitors:
1) what is the drug inhibition constant and how can it be determined
2) what does the graph look like for competitive inhibitior when there is one curve per compound
3) what does the graph look like when many compounds are screened at once
1) A drug inhibition constant Ki can be determined using a competitive fluorescence assay
2) One curve per compound: max polarization is when inhibitor concentration is lowest
o No binding of ligand to receptor .˙. all depolarized
♣ Ligand is not bound to anything and is too small to re-emit polarized light
3) Many compounds screened at once
o Shows max inhibition as a range and the ones @ 100% are the best hits
2-State Model of Receptor Activation:
1) give ex of receptor with at least 2 conformations and how are is the equilibrium between them described (what is the equation for this)
2) what conformation does an agonist typically bind to
3) what is the consequence of this on Kd app
1) Receptors (such as GPCRs) typically have at least 2 conformations
o Active + inactive
o Described by Kint
Full agonist: active state
2) what conformation does an agonist typically bind to
3) what is the consequence of this on Kd app
4) what is the Kd app equation
2) An agonist typically binds the active conformation of the receptor ONLY
3) High proportion of active receptor and agonist pushes reaction to make an active conformation of receptor
♣ Shifts the equilibrium (Kapp) towards active form
♣ Kapp = midpoint between
minimal response (basal) and maximal response (like Kd but in context)
4)
Inverse Agonist: inactive state
1) what does it bind to and how does it shift the equilibrium
2) equation of Kd app here
1) Inverse agonist binds to the inactive conformation
o Shifts the equilibrium towards to inactive form
2)
APPLICATION: dopamine & 5-HT receptors:
LOOK AT THE GRAPHS
1) what does Aripiprazole act as in each of the 3 conditions
1)
o Partial agonist for 5HT1A
♣ Lower activation @ saturating concentration
o Inverse agonist for 5HT2B
♣ Inactivation
o Effect on dopamine is unclear (no basal activity)
♣ No binding at of dopamine with aripiprazole, line at ~ 0%
