Receptor affinity, Ki, amongst other things...

[MattPsy]

Okay, so i've been interested in pharmcol for a while now. But it's come to my attention that I still don't understand what Ki and a few other values actually /mean/ (but understood the /implications/ of them, so could still design molecules to do things), as well as how they are found for new compounds and the like - quite a deficit, that i'd like to correct to further my understanding.
I can't ask a tutor or whatnot as i'm just a 18yo student and so are still only a first-year (freshman, I believe you call it in the US? I am in NZ), and pharmcol. is unavailable first year.
But it struck me that a few of you here would probably be able to help remedy this lack of knowledge instead, so I don't have to wait.

Can you guys give definitions of Ki and associated values, and how one goes about obtaining said values =) ? I'd assume it's to do with noting perhaps it displacing a radioligand at the binding site at a certain concentration of a certain proportion of receptor, or something similar to that, but I don't see how you'd actually test for that?
Info greatly appreciated =) !
Maybe it will helps some others here gain a better understanding of all this too.

(Oh yeah, and a sort-of relevant question - i've been looking at a paper on some nice new exotic monoamine reuptake inhibitors lately, and I noticed that a few of them had Ki-uptake values for the DAT that were lower than their Ki-affinity values - do I take this to mean they achieve inhibition of DAT more often than they actually manage to bind? I can't get me head around it. And if my deduction is correct, doesn't this mean they'd be /fantastically/ recreational =) ? )

 

[hussness]

You should read Ch. 6 of Lehninger Biochemistry.

 

[BilZ0r]

Hey Matt, what Uni are you going to be Studying Pharmacol at Otago or Auckland?

Eitherway,

I'm not expert on binding, but after you're done reading the drivel I have to say, I suggest you go to http://www.curvefit.com/introduction9e.htm it's a website about binding written by the people who make GraphPad Prism, a graphing/analysis package that a lot of Pharmacologists use. It has quite on bit on actually how to use the program, but also about the pharmacology.

So K is the affinity constant, it describes the interaction between two chemicals, in this case, a ligand and receptor.

Officially K = (Conc. Ligand) x (Conc. Receptor) / (Conc. of Ligand Receptor Complex)

It is also equal to (The rate at which the ligand dissociates from the receptor) / the rate at which the ligand associates with the receptor).

Messing with that first forumlae at bit, and you'll find that:

The Fraction of Receptors bound = (Conc. of Ligand)/(Conc of Ligand) + K

Which from there gives us the final, important definition of K

When the concentration of ligand is equal to it's K value, 50% of the receptors will be occupied.


So it's essentially an EC50 for receptor binding.

The letter that comes after the K i.e. Kd Ki, Kb, Ka, tells you HOW they figured out K, but a dissociated experiment, a competitive binding experiment, using an antagonist, using an agonist, respectively.

The easiest way of ACTUALLY finding out is with competitive binding. You have a fixed concentration of radiolabled ligand, with a known Kd. You apply your unknown drug at various concentrations, to a homogenate containing your receptor (crushed up brain, or something), then count the amount of radioactivity left. Using the 'cheng-prusoff" equation, you can then figure out your drugs affinities constant.

 

[ziddy]

Ah, so "Ki" is really just an equilibrium constant? That makes it a bit easier to understand...

 

[MattPsy]

Thanks Bilz0r. I'm at Auckland.
Will have a look through that site and into getting said book.

 

[BilZ0r]

When you say:

"Oh yeah, and a sort-of relevant question - i've been looking at a paper on some nice new exotic monoamine reuptake inhibitors lately, and I noticed that a few of them had Ki-uptake values for the DAT that were lower than their Ki-affinity values - do I take this to mean they achieve inhibition of DAT more often than they actually manage to bind?"

WTF? You wanna cite the paper or quote something a bit more specific, because that makes so sense. It would be quite common to see an Ki for a transporter that was lower than the IC50 for uptake inhibition, but you can't have a Ki for a process...

 

[MattPsy]

My bad, I was meant to put EC50 reuptake!
It was from: 1-(4-Methylphenyl)-2-pyrrolidin-1-yl-pentan-1-one (Pyrovalerone) Analogues: A Promising Class of Monoamine Uptake Inhibitors .

 

[kidamnesiac]

Yea that confused me for a while too and I thought I was missing something

Ki is basically a ratio of how much of the inhibitor is needed relative to how much effect it has and can be really, really, really complex (at least for enzymes).

 

[hussness]

Thanks Bilz0r. I'm at Auckland.
Will have a look through that site and into getting said book.

Said book is available online as an ebook. If you can't find it pm me.