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.