This recent paper asks the question “Can Invalid Bioactives Undermine Natural-Product Based Drug Discovery?” And as any of you who’ve done any natural product screening can attest, the answer is “You betcha”. There are any number of natural products (here’s one!) that, although active in a given screening assay, turn out to be active in a lot of screening assays. They are, in other words, pan-assay interference compounds (PAINs), and their origin shouldn’t be allowed to confuse that issue. Quercetin, gossypol, berberine, genistein, curcumin. . .the list goes on, and this paper does everyone a favor by providing just such a list, derived from the Napralert database.
You will notice many a quinone and polyphenol on it, and that is no coincidence at all. Natural products have a reputation (often earned) for exquisite selectivity, but they have also, in other instances, earned a reputation as fragmentation grenades. A compound like celastrol can show up as everything from an antioxidant to a protease inhibitor to an insecticide, and you know what? It’s all of those and more, thanks to that quinone methide functional group. Such compounds are almost certainly going to react promiscuously with sulfur nucleophiles inside a cell, show redox activity, and wreak all forms of havoc. There are covalent functional groups that are weakly reactive (epoxides), and there are covalent functional groups that are ready to party, and a quinone methide shows up already lurching to one side, with sloshing red party cups in both its hands. Everyone’s pal resveratrol is there, too, which probably won’t surprise people who’ve worked with it. Its polyphenol nature has raised eyebrows from the moment it was proposed as a sirtuin inhibitor, and its winding, frustrating, unresolved story since then shouldn’t come as too much of a surprise. What you want to do, though, is to avoid running the next project that’s heading in the same direction.
This would be a timely place to mention this open-access book chapter, just out from Jayme Dahlin, Jonathan Baell, and Michael Walters, on assay interference by reactive compounds. It’s a comprehensive guide to the subject, with recommendations for counterscreens at every step, and it’s well worth a look. It’s especially strongly recommended for groups that are just getting into a big screening campaign for the first time – the experience contained there can save you from a lot of wasted time and effort
As this paper shows, you’ll need to pay attention both to functional group problems and to things like aggregation. A good number of the compounds on the watch list are notorious aggregators under common assay conditions, and this behavior can throw false positives all over the place. There are also a lot of fluorescent interference compounds (emitters and quenchers) on the list, so if that’s your readout, caution is indicated. Luciferase-based readouts are all over the place in assay development, and plenty of natural products find ways to interfere with that system, too.
The paper also raises some bigger questions. How are these activities related to these various compounds’ actions in vivo? How do cells handle them, and why are they so common and biologically important? In many cases, their true functions in cells aren’t well understood at all. And for screening, what (and where) is the line between “very active compound that should be ignored” and “very active compound that will save your project”? Choose wisely!