GPCRs drug binding kinetics Flashcards
What is a closed vs an open system
A closed system is where the drug and target are at constant concentrations, allowing an equilibrium to be established, between the bound/free drug and the target.
In an open system, the concentration of drug (and target to a lesser extent) is in constant flux.
As such, equilibrium measures of drug-target interactions may not be predicative of what would occur in an open system. Here, residence time likely plays a large role too.
Law of mass action, what is Kon and Koff and what order reactions are they
Law of Mass action states that the rate of drug binding are proportional to concentration.
Kon is the association rate constant (second order), describing the rate of drug and receptor binding with each other. estimates drug-target complex formation. It is proportional to [drug] and [target]. Kon can be diffusion limited, or encounter limited (indicates that some sort of reaction needs to occur to enable drug-target binding). Unit: M-1s-1.
Koff describes the dissociation of a single species (the drug-receptor complex). It is a first order rate constant. Independent of local [free drug]. Unit s-1.
KD = Koff/Kon
Method of experimentally measuring dissociation rate
Mix known [labelled drug] and receptor together and incubate until equilibrium.
Do either an isotopic dilution or infinite dilution.
Isotopic dilution adds a very high conc of unlabelled ligand. This will instantly bind to unoccupied receptors, and you can then measure the rate of dissociation of the labelled drug.
Infinite dilution significantly dilutes the assay, meaning that the very low [labelled drug] would not be capable of new binding. Then you can measure the dissociation of the labelled drug from the receptors.
To quantify the dissociation, separation of the bound and free drug must be done at regular time points.
Dissociation half-life is used as a measure of the drug’s residence time.
Method of experimentally measuring association rate
Mix known [labelled drug] and receptor in an assay.
Stop the assay and take samples from regular time points until equilibrium is reached.
The rate at which the equilibrium is reached indicates the observed rate (Kobs). Kobs depends on Kon, Koff, and the [drug]. Faster Koffs cause the equilibrium to be reached faster.
The Kon can be calculated using Kobs, Koff and [drug]
Kon = (Kobs-Koff)/[Drug]
Alternative way of calculating Kon experimentally
Doing an association experiment with multiple [Drug]
As Kobs will vary with [drug], where as Kon and Koff are constant, this method can identify Kon.
Doing a global fit of the data can give the Kon
How to measure binding kinetics of unlabelled ligands
Using competition assay.
Using a labelled ligand with known kinetics, a competition experiment can identify the kinetics of an unlabelled drug competing with it.
As such, by knowing the Koff and Kon of the tracer ligand, you can analyse the data to determine the Koff and Kon of the unlabelled drug. There are equations that can be used to quantify the kinetics of the competition drug.
what limitations are there to traditional techniques, and mechanism of TR-FRET
Classically radioligands were used. The high number of time points, meant that for each experiment there were numerous reaction tubes, a laborious, time consuming, and expensive method.
TR-FRET commonly utilises terbium as a donor, which has long lived fluorescence. this enables there to be a time delay between excitation and measurement. This reduces auto-fluorescence, and improves the signal to noise of the assay.
SPR: Alternative label free approach for drug binding experiments
Surface plasmon resonance (SPR).
A protein is immobilised on a coated gold chip. It is then continually exposed to a flow of the compound of interest. The receptor imobilized on the chip undergoes a conformational change that alters its refractive index upon ligand binding.
Difficult to use on GPCRs, as they rapidly deteriorate when they are taken out of the membrane environment. This assay requires an isolated protein.
Further limitations and features of equilibrium binding
Drug binding and subsequent receptor signalling pathways will all have their own kinetic profiles. individual pathways will have different kinetics. e.g., Ca2+ signalling is very fast, gene transcription changes very slow.
Equilibrium conditions will not match the open system of the body.
Dose affects equilibrium rate. High dose - quick equilibrium.
Drugs with slow dissociation rate need longer to reach equilibrium, so if the assay time point is wrong, it would underestimate its effect.
These effects must be considered when assessing biased agonism.
Hemi-equilibrium explained
Occurs primarily when antagonists have a slow dissociation rate.
Here when a very high conc of agonist is added to the assay, and an equilibrium point has been reached, if the antagonist has a slow dissociation rate it can mean that some of the receptors are still occupied by antagonist, despite the competition favouring agonist binding. This can make it appear that the antagonist is insurmountable, however the assay has not been incubated for long enough.
Role of drug binding kinetics in LAMA use
long acting muscarinic antagonists e.g., tiotropium
M1 receptors mediate bronchoconstriction. M3 receptors mediate bronchoconstriction and mucous secretion. M2 autoreceptors inhibit ACh release.
COPD treatment would benefit from M1/M3 selective drugs. Difficult to create due to high homology between mAChRs. As such, mAChR antagonists tend to also inhibit M2, leading to bronchoconstriction and systemic effect of tachycardia
Of the used LAMAs, they tend to have similar Ki for all mAChR subtypes, but they have a much more rapid dissociation rate from the M2 than the M3. This leads to lower M2-signalling, and as such, a better side effects profile.
Antipsychotics mechanism and side effects
Primarily through blockade of D2R. Mesolimibc hyperactivity causes schizophrenic positive symptoms.
On target side effects include EPS (nigrostriatal blockade) and hyperprolactinaemia.
Second gen have reduced EPS risk compared to first gen. Third gen are D2R partial agonists - atypical.
Fast off hypothesis description
Proposes that dissociation rate is associated with side effects profile (EPS).
Due to the high dissociation rate, the antagonists can be displaced by endogenous dopamine when it is in transiently high concs (following synaptic release). This maintains some level of dopaminergic signalling, thus avoiding EPS.
Correlation of Kon and Koff with side effects and proposed hypotheses
Prolactin correlated with Koff
EPS correlated with Kon
Drug rebinding is more likely to occur with drugs that have a high Kon - they are more readily able to reassociate with the receptor before diffusing.
The local environment of the receptor largely determines the degree of rebinding. The hypothalamic-pituitary portal system means that free diffusion of DA occurs here. As such, Koff more so determines the level of D2 blockade - hence the correlation of Koff and hyperprolactinaemia. in the striatum however, there is slow diffusion out of the compartment, leading to higher Kon-driven drug rebinding.
