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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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June 7, 2013

Making Peroxides, Quietly And Unhelpfully

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Posted by Derek

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.

Comments (7) + TrackBacks (0) | Category: Drug Assays


1. noname on June 7, 2013 8:42 AM writes...

might want to check that DTT name again.

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2. barry on June 7, 2013 8:54 AM writes...

got a screening surprise once when the resynthesized sample lacked the hit's activity. Turned out that storage in cold DMSO was enough to oxidize a dihydropyrimidine (with interesting stereochemistry, but no binding) to a pyrimidine (which of course is flat, but that's what this binding site wanted)

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3. Practical Fragments on June 7, 2013 9:13 AM writes...

Ben Davis and I mention these in our recent review on things that can go wrong (Figure 7):

The problem is that some of the molecules don't look so bad, and there are some rather embarrassing papers reporting redox cyclers as "small molecule probes."

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4. rtw on June 7, 2013 10:12 AM writes...

I am not at all surprised. As #2 Barry has found DMSO is a bad actor in solution sample storage. I have particularly been critical of it when used to solubilize acid salts of compounds. After all one of my favorite oxidation methods involves the use of DMSO. Also Sulfoxides can undergo some very interesting chemistry. Take a look at the Pummerer reaction sometime. It can be used to even form carbon-carbon bonds.

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5. NJBiologist on June 7, 2013 12:00 PM writes...

Worrying about this in the context of cysteine proteases makes sense, but I wouldn't rule out other targets... aren't there some ion channels like NMDAR that are regulated by redox/peroxide?

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6. We on June 7, 2013 8:34 PM writes...

Think aromatase inhibitor and cox inhibitor

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7. ty on June 9, 2013 5:42 PM writes...

There are a whole lot more enzymes/proteins that are sensitive to redox cyclers. Much bigger problem is that cellular assays tend to dominate in academic small molecule screenings. Chances are, you will see what you want to see with a little bit of hydrogen peroxide in the cell...

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