Mitochondrial production of reactive oxygen species 2

Now I want to move on to looking for molecules which are able to influence these sites and prevent them from making super oxide or hydrogen peroxide. So, what we've been talking about when we discussed the capacities of the sites was the assays using isolated mitochondria where we could run individual sites and find out how fast they could go. But of course you can use the same logic to run a screen for small molecules that might stop individual sites from making superoxide. Of course, for many sites that's trivial. For example, we can have Rotenone to block the IQ site. We can add Myxothiazol, a very good inhibitor of the IIIQo site. But of course when we do that, we also stop ATP production. We stop electron flow, we stop oxidative phosphorylation, we prevent ATP generation. A therapeutic molecule is very undesirable to prevent oxidative phosphorylation because that's the main function of the mitochondria. So, what we're looking for here is molecules which have a particular property of being able to prevent superoxide or hydrogen peroxide production from different sites without preventing oxidative phosphorylation. So, we run a screen of a small chemical library in-house, three and a half thousand molecules, against the superoxide and hydrogen peroxide production from different sites on isolated muscle mitochondria. We looked to the IQ site, IF site, IIIQo site and IIF site. Those are the four major sites that we think run in vivo. And also, the glycerol phosphate dehydrogenase site because that was convenient and easy to do. And then we did the counter screen against mitochondrial membrane potential. Looking for anything that decreases the mitochondrial potential of the proton motor force is potentially an inhibitor of electron transport and that would be discarded from the hits in the screen. So, the counter screen is oxidative phosphorylation. The on target screen is the ROS production from particular sites. And surprisingly, even in this rather small library we found plenty of hits. The universe is populated by quite a lot of molecules which are able to specifically prevent ROS production by the IQ site without preventing oxidative phosphorylation and without affecting the ROS production by any other site in the electron transport chain. And based on that initial screening, we subsequently screened a much larger library where we found much better compounds, and I'm going to talk about those in the next slide. So, these are the complex three suppressors.