Cocaine (and Methylphenidate) are DAT Inverse Agonists, not just Inhibitors

[aced126]

Do you have a source that cocaine binds to an allosteric site? I didn't know this until now. I'm assuming all dopamine reuptake inhibitors would bind to this site then? Since the structures of some reuptakors and releasers are very similar (eg methylphenidate & amphetmaine) then what is the interaction when both the dopamine substrate site and the allosteric site are occupied by ligands? And if this is so, how is the proposed neuroprotective effect of mph against amphetamine explained?

 

[serotonin2A]

Do you have a source that cocaine binds to an allosteric site? I didn't know this until now. I'm assuming all dopamine reuptake inhibitors would bind to this site then? Since the structures of some reuptakors and releasers are very similar (eg methylphenidate & amphetmaine) then what is the interaction when both the dopamine substrate site and the allosteric site are occupied by ligands? And if this is so, how is the proposed neuroprotective effect of mph against amphetamine explained?

See for example, the link below. But there are many other citations -- it is pretty clear that the SAR of amphetamine and cocaine are completely different. The cocaine binding site will tolerate a large amount of distance between the amine and the aryl group, but that doesn't seem to be the case for the substrate binding site.

Not all DAT inhibitors work by the same mechanism. Some may bind to the substrate site.

MPH blocks transport by DAT so it would block the trsnsport of amphetamine into dopamine terminals.

http://www.sciencedirect.com/science/article/pii/S109332631200068X

 

[aced126]

Wow, I can't believe I am only just finding out about this. So in fact cocaine and methylphenidate have a different mode of action despite being commonly classed together? Could it not be so that this allosteric site shares some similarities to the substrate site, given the similar overlays of cocaine, methylphenidate and amphetamine? Surely each molecule will have a certain (non-negligible) affinity at each of the sites?

In that study it shows that Y156 is not involved in the binding of dopamine. Is this truly the case? In another study you linked it showed Y156 playing a binding role in DAT for substrates like fenfluramine.

 

[serotonin2A]

Cocaine and MPH act via the same mechanism. I know it is possible to overlay MPH and amphetamine but I think those similarities are purely superficial. Indeed, amphetamine is a DAT substrate whereas MPH is not transported and has a completely different mechanism of action.

I think you have to consider that the pharmacophore for MPH and cocaine is similar, and is different from that of amphetamine. In both cocaine and MPH, there is an aryl ring and an amine, with an ester linkage located 2 methylene units from the amine. By contrast, there is no ester in amphetamine, and adding an ester to the beta-position in amphetamine doesn't seem to make it more potent. So the pharmacophore is different. Of course, you could argue that the ester at C2 can be omitted from cocaine and still leave some stimulant activity intact. That may be the case, but the presence of the ester definitely increases the affinity of cocaine. The fact that the same isn't true for amphetamine strongly argues that there are differences in their pharmacophores.

It isn't true that there is necessarily overlap of affinities of ligands at different sites on DAT -- cocaine doesn't bind to the substrate site (at least not at concentrations that are physiologically relevant).

Binding clefts are composed of the side chains of many residues and each ligand can potentially interact with a different subset.That is one reason why different ligands have different efficacies and different functional selectivity. Ligands with similar structures will often adopt similar binding modes but you can't assume that they interact with exactly the same residues.

 

[Nagelfar]

I can't find a good citation, but I know cocaine has a "high-affinity" and a "low-affinity" binding site on DAT; certain phenyltropanes turn this around: High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims.

"Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine"

Chronic IV treatment with cocaine, cocaethylene, and a water control was accomplished for 7 days using osmotic minipumps attached to jugular catheters. Animals were then challenged with an IV bolus of cocaine. RESULTS: With acute treatment, the IV bolus of cocaethylene at the beginning of the infusion period resulted in an initial behavioral activation equivalent to that caused by cocaine, after which there was a striking difference in that the cocaethylene group displayed a return to predrug levels of activity, while the cocaine group showed high levels of activity throughout the 3-h period. Both cocaethylene and cocaine resulted in an initial increase in the extracellular concentration of dopamine. However, after that initial increase, levels of dopamine dropped in the cocaethylene group while the cocaine group levels remained elevated.

^The only difference is a methyl to an ethyl! Makes one wonder why exactly benzoylecgonine has no efficacy/affinity but benzoylMETHYLecgonine (i.e. cocaine) does. Also seeing how both amphetamine and cocaine are "indirect" agonists for dopamine, and that direct agonists don't seem to be reinforcing, makes me question the dopamine hypothesis altogether, perhaps it has something to do with just the ligand docked itself. Also, there's CART which may have a lot more to do with the anticipation of "incentive salience" reward.

In Vitro Characterization of Cocaine Binding Sites in Human Hair.

^this is interesting, race skewed possible test results; cocaine actually adheres better in 'kinky' Africoid hair; possibly making test results appear that more has been taken than has.

 

[serotonin2A]

I can't find a good citation, but I know cocaine has a "high-affinity" and a "low-affinity" binding site on DAT; certain phenyltropanes turn this around: High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims.

The existance of high and low affinity binding isn't necessarily evidence that cocaine binds to multiple sites. Cocaine preferentially stabilizes the outward facing conformation of DAT, which means that it has lower affinity for the inward-facing conformation. So when they run binding assays with DAT, some of the transporters will be in the outward facing conformation (high affinity for cocaine), and some will be in the inward facing conformation (low affinity cocaine binding).

"Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine"

That's true, but they cite five other studies that came to the opposite conclusion. It's a molecular modeling study, not crystallography, so their model may be wrong. Until the crystal structure is published, we are kind of stuck relying on the consensus of modeling and homology studies and mutagenesis experiments, which is that cocaine binds to an allosteric site.

 

[farmacista]

I can't find a good citation, but I know cocaine has a "high-affinity" and a "low-affinity" binding site on DAT; certain phenyltropanes turn this around: High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims.

"Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine"



^The only difference is a methyl to an ethyl! Makes one wonder why exactly benzoylecgonine has no efficacy/affinity but benzoylMETHYLecgonine (i.e. cocaine) does. Also seeing how both amphetamine and cocaine are "indirect" agonists for dopamine, and that direct agonists don't seem to be reinforcing, makes me question the dopamine hypothesis altogether, perhaps it has something to do with just the ligand docked itself. Also, there's CART which may have a lot more to do with the anticipation of "incentive salience" reward.

In Vitro Characterization of Cocaine Binding Sites in Human Hair.

^this is interesting, race skewed possible test results; cocaine actually adheres better in 'kinky' Africoid hair; possibly making test results appear that more has been taken than has.

i think benzoylecgnonine won't pass the blood brain barrier due to the carboxylic group...the experiment sounds strange cocaethylene is supposed to last longer than cocaine

 

[Nagelfar]

i think benzoylecgnonine won't pass the blood brain barrier due to the carboxylic group...the experiment sounds strange cocaethylene is supposed to last longer than cocaine

I figured that they have binding values at all means they had it in the brain; perhaps it was an in vitro study.