Small but PAINful

We’ve written previously about the phenomenon of compound aggregation, and how this can lead to false positives in high-concentration screening (see also here). Unfortunately, aggregation is not the only thing that can trip you up. Earlier this year Jonathan Baell and Georgina Holloway published a description and systematic catalog of “Pan Assay Interference Compounds,” or PAINS, and Baell has now followed up on this with a Perspective in the October issue of Future Medicinal Chemistry. (For those of you without journal access, he has also summarized some of this material here.)

PAINS are compounds that frequently show up as screening hits, but that act through non-specific mechanisms such as covalent attachment to proteins or generation of hydrogen peroxide. Sometimes it is not the compound itself that is problematic but a breakdown product or leftover reactant; most readers with experience in hit-to-lead discovery will experience a painful sense of déjà vu reading about hits that did not confirm when the compound was resynthesized or repurified. This problem is exacerbated when trifluoracetic acid (TFA, which is toxic to cells and can accelerate compounds’ decomposition) is used in reverse-phase purification of compounds; apparently Pfizer has systematically repurified hundreds of thousands of their screening compounds to remove traces of TFA.

The problem with PAINS is that they may show convincing biochemical and even cell based activity, but mechanistically be useless for further advancement to drugs or even chemical probes. Unfortunately this does not prevent them from being published, where they are picked up by other researchers and identified as “privileged pharmacophores” by computational chemists. This leads to more (wasted) research and more (useless) publications in a vicious circle Baell terms “pollution of the scientific literature.”

Although the focus of the Perspective is high-throughput screening, fragments can be PAINS too, as the sampling below shows.

Compound 1 is obviously a Michael acceptor and compound 2 is unsurprisingly reactive with cysteine, but the problems with compound 3 are less obvious, and compound 4 generates hydrogen peroxide in the presence of reducing agents and oxygen – a mechanism it likely took some time to track down.

One of the fun aspects of reading this paper is that Baell is not shy about calling out high-profile publications that are likely to report false positives, though he readily admits that he too has been misled. The question is what to do: using computational filters to get rid of the worst offenders only eliminates 5-12% of commercial compounds, but more stringent screens can eliminate upwards of 95%!

Baell calls for better stewardship of the scientific literature:

Journals therefore have a responsibility here because if misleading information is published, unintentional though it may be, it is more likely to propagate and be taken as fact by others who may then initiate flawed research projects. Research resources are too precious for this to be acceptable.

I agree whole-heartedly with this, though given what I see published on a weekly basis I despair of this happening any time soon. Ultimately of course the onus is on researchers to carefully follow up and fully understand the mechanism of their hits, particularly those with dubious structures: don’t contribute to the pollution yourself, and don’t let friends (or papers you review) pollute!