GPCRs allostery Flashcards
Orthosteric and allosteric binding advantages and disadvantages
Allosteric sites can be anywhere the orthosteric site is not. Can be found above the orthosteric site (common in rhodopsin class), at the bottom of the receptor (common in secretin/adhesion), or in the venus flytrap domain for mGluRs.
Orthosteric drugs tend to have poor subtype specificity. Furthermore, it will signal at all receptors indiscriminately of endogenous signaling. Overdose risk is also increased for orthosteric drugs.
Allosteric sites are less conserved - more specificity. They maintain endogenous spatial and temporal signaling. Their saturability effect also reduces overdose risk + increases the therapeutic window. Furthermore, there may be a possibility of signaling pathway bias with allosteric modulators.
Parameters defining allosteric kinetics
KB is the affinity of the allosteric ligand for the allosteric site. TauB is the intrinsic efficacy of the allosteric ligand.
alpha (a) is a cooperativity factor. indicates how much the binding of an allosteric drug affects the binding of an orthosteric ligand.
Beta (B) is a cooperativity factors that indicates how much the binding of an allosteric drug affects the efficacy of an orthosteric ligand.
TauB is used to describe the agonistic effects that some allosteric modulators can possess.
Once all the allosteric sites become saturated the effect of the allosteric ligand is limited. Cannot completely antagonise a receptor.
Assay for allosteric modulator binding effects with known ligand
Allosteric modulator titration binding assay.
Uses a single known [labelled drug] with known kinetics (affinity).
Administer allosteric modulator in increasing concentration and assess the change in the binding kinetics of the labelled ligand.
An a>1 is positive cooperativity. PAM. If a=10, then it means the allosteric drug increases affinity of orthosteric ligand by 10-fold. a=0.1 decreases affinity by 10-fold. a=1 indicates neutral cooperativity - antagonist for binding at the allosteric site.
new KA of the orthosteric drug = KA/alpha: e.g., KA = 50 and a=0.1 -> 50/0.1=500 = new KA is 500, a 10-fold decrease in affinity
Assay for allosteric modulator binding effects with unknown ligand
An allosteric competition interaction assay.
Unlabelled allosteric and unlabelled orthosteric ligands. Looks at the modulatory effects on signalling and utilising equations. Don’t really need to know much about this.
Effect of allosteric ligand on the binding kinetics of the orthosteric ligand
It can alter the association or dissociation rate of orthosteric ligands.
This can be detected using a dissociation binding kinetics assay, with and without the presence of an allosteric ligand (infinite or isotopic dilution).
E.g., HMA (an NAM of adenosine A2A receptor) causes increased dissociation rate of equilibrium binding of an antagonist. Through this mechanism is likely how it negatively modulates the antagonist effect.
E.g., PAM alcuronium can slow dissociation of [3H]NMS (an antagonist) from m2AChRs. Proposed to do this by ‘capping’ the orthosteric site, affecting access of the antagonist to the orthosteric binding site.
What effects to orthosteric ligand signalling can occur from PAMs
Can increase potency, increase efficacy (Emax), and a PAM can even be an agonist on its own - an allosteric agonist.
These can be quantified by applying an operational model of agonism to concentration-response data of orthosteric ligands in the presence of different [allosteric modulators]
Kinetics of NAM antagonism
They are non-competitive antagonists.
They tend to cause a limited shift of affinity of the orthosteric ligand, having no effect on Emax. When Schild plot is created they form a curvilinear plot. Unlike a competitive antagonist which has a linear rightwards shift in a concentration-dependent manner.
NAMs are also able to cause decreases in efficacy (and thus Emax), however this effect is often confused with irreversible antagonists or competitive antagonists in hemiequilibrium conditions. Therefore, when these results are attained, further tests are needed to ensure that the MOA is through allostery as opposed to the other 2 possibilities mentioned above.
What is probe dependence
It is when the allosteric effects can change depending on the orthosteric ligand.
As the allosteric modulator promotes a specific conformation of the receptor, it can favour the binding of one orthosteric ligand over the other, with different kinetics compared to control.
E.g., LY2033298 an allosteric modulator of M2AChRs is a PAM of ACh and oxotremorine-M, but an NAM of xanomeline.
