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June 7, 2013
Making Peroxides, Quietly And Unhelpfully
Here's a problem with screening collections that I have to admit I wasn't aware of: generation of hydrogen peroxide. This paper (free access) gives an excellent overview of what's going on. Turns out that some compounds can undergo redox-cycling in the presence of the common buffer additive DTT (dithiothreitol - note - fixed brain spasm on earlier name), spitting out, in the end, a constant trickle of peroxide.
Now, for many assays, this might not mean much one way or another. But enzymes with a crucial cysteine residue are another matter. Those can get oxidized, which is irritating in these cases, because DTT is added to such assays just to keep that sort of thing from happening. That link above describes a useful horseradish peroxidase/phenol red assay to detect hydrogen peroxide generation, and its use to profile the NIH's Small Molecule Repository compound collections.
Fortunately, only a limited number of compounds have the ability to hose up your assays in this manner. Of the roughly 196,000 compounds screened, only 37 were true peroxide-generators. Quinones are serial offenders, as any chemist might expect, but if you let you screening collection fill up with quinones you have only yourself to blame. There are less obvious candidates, though: several arylsulfonamides also showed this behavior, and while those aren't everyone's favorite compounds, I'd like to see the large screening set that doesn't have some in there somewhere. It's worth noting, though, that many of the sulfonamides that were identified are also quinon-ish.
So I think the take-home advice here is to be aware if your target is sensitive to this sort of thing. Cysteine proteases are obvious candidates, but Trp can be oxidized, too, and a lot of proteins have crucial disulfides that might get unraveled. Once you've flagged your protein as a concern, be sure to run the hits you get back through this peroxide assay to make sure that you're not being led on. Trying to eliminate compounds by structural class up front is another approach, but the compounds that are first on the list are compounds that you should have trashcanned already.
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