Practical Fragments has repeatedly warned about the dangers of what Jonathan Baell dubbed pan-assay interference compounds, or PAINS (see for example here, here, and here). These are compounds that hit numerous unrelated targets through mechanisms that can charitably be described as “non-druglike”. Regrettably, many people still do not recognize these nuisance compounds for the artifacts they are, and PAINS continue to show up in high-profile fragment libraries. An unintentional illustration of why this is a problem was recently published in J. Med. Chem.
The researchers were interested in STAT3, a popular oncology target. They used a computational approach to extract fragments from reported inhibitors and then recombined them into new molecules, a few of which were made and tested. Unfortunately, some of the previously reported inhibitors were PAINS, and, like HeLa cells contaminating cell cultures, the resulting pathological fragments contaminated this research. The most active molecule out of this exercise, compound 8, is a para-quinone:
Quinones are troublemakers for two main reasons. First, they can nonspecifically react with thiols (see figure), and STAT3 does indeed have several free cysteine residues. Second, quinones are well-known redox-cyclers: they can be reduced and then re-oxidize in air, generating reactive hydrogen peroxide in the process.
The researchers showed that compound 8 is active in several cell assays and a mouse xenograft tumor model, but of course any generic alkylator could also show these effects (mustard gas, anyone?) and hydrogen peroxide is itself an important second messenger. It is impossible to say whether the activity of compound 8 is due to interaction with STAT3 on the basis of the experiments reported in the paper. The only evidence that compound 8 interacts with STAT3 at all comes from a fluorescence-based assay which appears to show 70% inhibition at >100 micromolar compound 8, a concentration far higher than the cell experiments.
In other words, what this paper shows is that a quinone has modest but ill-characterized biological activity. Of course, just because a compound can be a bad actor doesn’t necessarily mean it is behaving as one, but in the case of PAINS it is best to assume guilty until proven innocent. Indeed, a figure in the Supporting Information shows that the compound also inhibits STAT5 phosphorylation, supporting the notion that it acts through multiple mechanisms.
We can do better than this.
I hesitated before writing this post – I don't want to come across as a mean-spirited vigilante – but one of the strengths of science is its self-correcting nature. Researchers should learn to recognize PAINS when they inevitably show up as screening hits. If and when they don’t, editors and reviewers evaluating manuscripts and grants have an obligation to hold them to account.
It is easy to ignore or shrug off sloppy science, perhaps with a cynical chuckle, but papers like this fill me with a mixture of sadness and outrage. This research consumed the time and efforts of four scientists, not to mention scarce funding from the NIH and Alex’s Lemonade Stand Foundation, a charity founded by a young girl who subsequently died of her cancer at the age of eight.
We owe it to society to stop wasting resources chasing artifacts.