A cluster of chlorination… our latest paper in AJP Heart

Our paper on autophagy is finally out in AJP Heart. It’s essentially the story of a follow up on a high throughput screen we did several years ago, to find molecules that protect cells (and hearts) from ischemia-reperfusion injury.

One of the hits was the molecule cloxyquin, an 8-hydroxyquinoline with striking structural resemblance to another drug, clioquinol. The latter has been proposed to stimulate autophagy, and also causes mitochondrial uncoupling (it’s also known as “chinoform” in the older literature). Notably, Roberta Gottlieb’s group has shown that stimulation of autophagy occurs in ischemic preconditioning, and it’s also known that mito’ uncoupling stimulates autophagy.

So, we hypothesized that the protective effect of cloxyquin might also be due to mito’ uncouping stimulating autophagy.  First we showed that cloxyquin, like its cousin clioquinol, does indeed uncouple mito’s. It also stimulates autophagy in cardiomyocytes (for this we made cells from GFP-LC3 mice). We also showed that cloxyquin is protective in an in-vivo model of IR injury.  Lastly we showed that protection by cloxyquin could be blocked by the autophagy inhibitor cloroquine.

Cloxyquin, clioquinol, chloroquine, what a cluster!

An important thing we learned during this work, is that combining an autophagy activator and inhibitor in-vivo is not a good idea. Apparently this is well known in the field, but not to us…. when you stimulate autophagy and inhibit it at the same time, things go downhill very fast (aka the mouse drops dead).  We learned about this verbally at a conference but I can’t find a reference in the literature for it.  I guess nobody likes to report when experiments don’t go as planned.

Another key thing we learned during the review phase was that drugs can often be had from obscure suppliers for less money.  Specifically, we had argued in early reviews that doing a complex series of experiments using Bafilomycin A1 (another autophagy inhibitor) would be prohibitively expensive, because the drug is $150 for 10 micrograms, and we would have required several thousand dollars’ worth for this experiment.  A reviewer kindly alerted us to the same drug from $198 for 5 milligrams from LC Biosciences. Literally, 500 times more drug for about the same price!  The experiment ended up not working out anyway, so is not lost to the ether (aka figures for reviewers only), but it’s always good when a reviewer offers to help out in this way with practical advice rather than just crapping on your work.

Finally, does this get us any closer to a therapy for MI?  Probably not, because the protection by cloxyquin is prophylactic – we need to guess who’s going to have a heart attack and load them up with drug. Not easy from a clinical perspective. BUT – cloxyquin does appear to be an interesting tool compound.  If you’re looking for something other than rapamycin to stimulate autophagy then give it a try (and no, we haven’t tried to see if it extends lifespan yet)!

Moving forwards, we’ve recently done another screen with a more relevant cell type and adding drugs at the moment of reperfusion (equivalent to delivery during percutaneous coronary intervention in the cardiac catheterization lab’).  This has yielded some very interesting hits which we’ll be reporting on soon.

Kudos here goes to Jimmy Zhang, MD/PhD student in the lab who led this project (and who also BTW just passed his PhD qualifier exam last month). Congratulations Jimmy!