Intrinsic efficacy assay idea/question

[Cotcha Yankinov]

So if there is effectively a ceiling (or diminishing returns) on partial agonist response compared to a full agonist, then you might just be able to push a low affinity partial agonist to high concentrations and see the maximal response is less than the endogenous agonist, rather than show it has partial activation on its own but still reduces the endogenous agonist's response when given together (meaning it's competitive for the receptor)?

But what about the inverse - using a partial agonist to increase response when given with an antagonist (antagonist being dosed relatively low) but looking for a less increased response than when an endogenous full agonist is given with the antagonist, rather than reducing response to a full agonist with a partial agonist? Would there even be any advantage to that technique, for example, a silent antagonist that wouldn't be directly interfering with surrogate signaling measures or something?

I think the problem you might run into is that a higher affinity ligand is going to be better at binding through the antagonist blockade, and therefore might have the appearance of higher efficacy. But assuming you have pure binding affinity data, maybe you could take that into account?

And I assume that in the same way that you would look for a reduction of a full agonist's response when looking for a partial agonist, you could find super agonism by looking for a full agonist to reduce the response of said super agonist.

But if we investigate the dose-response curve, theoretically to show something is a super agonist, you could just show that it has higher maximal response than the full agonist, and a similar result couldn't be achieved with an extremely high affinity full agonist?

In that same vein, you couldn't achieve the results that a very low affinity full agonist is a partial agonist?

My question being that if it does only take examination of the dose response curve to determine something is a partial agonist, what is the purpose of giving a partial agonist with the endogenous full agonist - to see that reduction of the endogenous full agonist response shows that the partial agonist is competitive for the same site, and/or to achieve more detailed data to calculate it's efficacy?

I think I've heard that there is a name for the phenomenon of a ligand blocking one site increasing binding of a ligand to other targets but heard that it wasn't a significant interaction (given there are many many ligands per receptor). Let's pretend it could be significant in some assay conditions - what if when you see decreased [35s]GTP-g-s incorporation with the "partial agonist" and the endogenous full agonist administered together (relative to full agonist alone), all you're seeing is the binding shifting to other sites that cause decreased GTP-g-s incorporation (see note below), or that dopaminergic D2 binding decreases because the "partial agonist" is changing the molecular conditions in some other manner, and there is no actual competition for the D2 site that would reveal itself in vivo?

*note - This would have to involve a 3rd site. In the case of the LY mGluR 2/3 agonists: 1st site being mGluR 2/3 for the LY compounds, and then the 2nd site being D2 for dopamine, and a 3rd site for one ligand that decreases GTP-g-s incorporation but binding to that 3rd site increases when the two compounds are combined, otherwise the binding but decreased response would be have to be competitive at the site of interest and that would denote partial agonism.

Sorry if my ponderings aren't very clear as of late (if they were ever clear at any time)

Any discussion is welcome.

 

[serotonin2A]

So if there is effectively a ceiling (or diminishing returns) on partial agonist response compared to a full agonist, then you might just be able to push a low affinity partial agonist to high concentrations and see the maximal response is less than the endogenous agonist, rather than show it has partial activation on its own but still reduces the endogenous agonist's response when given together (meaning it's competitive for the receptor)?

But what about the inverse - using a partial agonist to increase response when given with an antagonist (antagonist being dosed relatively low) but looking for a less increased response than when an endogenous full agonist is given with the antagonist, rather than reducing response to a full agonist with a partial agonist? Would there even be any advantage to that technique, for example, a silent antagonist that wouldn't be directly interfering with surrogate signaling measures or something?

With regard to the first question, you don't have to test the partial agonist vs. dopamine. You could run the following experiments:

Partial agonist
Partial agonist + antagonist
Full agonist
Full agonist + antagonist

With the second technique you proposed, you would have to run four experiments:

Neutral Antagonist
Neutral Antagonist + partial agonist
Neutral antagonist + full agonist
Full agonist

But that still wouldn't give you a clear understanding of the efficacy of the partial agonist -- you have to measure that directly.

Another problem is that it is difficult to identify antagonists that are completely neutral; most have some inverse agonist activity

My question being that if it does only take examination of the dose response curve to determine something is a partial agonist, what is the purpose of giving a partial agonist with the endogenous full agonist - to see that reduction of the endogenous full agonist response shows that the partial agonist is competitive for the same site, and/or to achieve more detailed data to calculate it's efficacy?

The point of testing the partial agonist together with a full agonist is to confirm that they have a competitive interaction.

I think I've heard that there is a name for the phenomenon of a ligand blocking one site increasing binding of a ligand to other targets but heard that it wasn't a significant interaction (given there are many many ligands per receptor). Let's pretend it could be significant in some assay conditions - what if when you see decreased [35s]GTP-g-s incorporation with the "partial agonist" and the endogenous full agonist administered together (relative to full agonist alone), all you're seeing is the binding shifting to other sites that cause decreased GTP-g-s incorporation (see note below), or that dopaminergic D2 binding decreases because the "partial agonist" is changing the molecular conditions in some other manner, and there is no actual competition for the D2 site that would reveal itself in vivo?

This is the opposite of "ligand depletion". This condition shouldn't happen in a properly designed assay.

Note that GTPgammaS asssys are often conducted in cell lines, where there is an absence of endogenous neurotransmitter receptors.

*note - This would have to involve a 3rd site. In the case of the LY mGluR 2/3 agonists: 1st site being mGluR 2/3 for the LY compounds, and then the 2nd site being D2 for dopamine, and a 3rd site for one ligand that decreases GTP-g-s incorporation but binding to that 3rd site increases when the two compounds are combined, otherwise the binding but decreased response would be have to be competitive at the site of interest and that would denote partial agonism.

Sorry if my ponderings aren't very clear as of late (if they were ever clear at any time)

Any discussion is welcome.

If conducted in native tissue then you have to confirm specificity by showing that you can block a response with a competitive antagonist.

In the GTPgammaS assay, you are measuring G protein activation by looking at how much GTPgammaS binds. So in your example, the third site cannot do what you proposed (reduce GTPgammaS binding) unless it (1) allosterically modulates D2 receptor responses, or (2) the G protein level is so low that its activstion completely depleted the G protein pool (virtually impossible)

EDIT: I expaned some of my responses to clarify them.

 

[Cotcha Yankinov]

Thank you.

So theoretically if you used homogenized rat brain striata as seen in the CBD Seeman study (http://www.nature.com/tp/journal/v6/n10/full/tp2016195a.html) you would have to show specificity for the D2 receptor by either blocking every receptor (and enzyme, FAAH which could be modulating endocannabinoid metabolism?) that CBD could be interacting with besides D2 (such as cannabinoid receptors and cannabinoid/dopamine receptor heterodimers) and then block effects on immune system related things that may affect results, and/or block D2 to show specificity?

I guess I'm theorizing a lot of immune system related artifacts when homogenizing brain tissue, given that sometimes 90% of the brain is glia and they could be going crazy reacting to tissue damage. Surely a compound like CBD could interact with the microglia/astrocytes (given CBD's supposed anti inflammatory properties), and they could then influence D2 receptor activation in some way? Or am I misunderstanding brain tissue homogenization?

 

[serotonin2A]

Don't forget that the tissue is homogenized, which means that the cells are all broken apart into little pieces. Then they isolate the membrane fraction.

The control for the experiment is to determine whether the response occurs in the presence of a high concentration of a selective D2 antagonist, although that wouldn't address receptor oligomers.

 

[Cotcha Yankinov]

Don't forget that the tissue is homogenized, which means that the cells are all broken apart into little pieces. Then they isolate the membrane fraction

Okay, I was wondering if that was the case.

Membrane fraction meaning they could be examining a piece of the neuron that is "extrasynaptic" or the soma, and not the terminal, or do they check histology to determine extrasynaptic vs. terminal membrane fractions?

It seems like one more step towards in vivo would be to do slices and patch clamping techniques with D2 antagonists vs. the potential D2 partial agonist, but is this just a matter of finances or are membrane fraction studies really preferred in some cases?


At this point I don't know what to believe regarding CBD's possible D2 partial agonism. I understand it needs to be verified by other labs - do you think in this case CBD stands a chance of being verified as a D2 partial agonist by other labs? Or are there any particular issues you see with this study aside from the troublesome history of Seeman?