Pan assay interference compounds – PAINS – have received quite a bit of attention at Practical Fragments. In addition to being a fun topic, the hope is that publicizing them will allow researchers to recognize them before wasting precious resources.
But not all PAINS are created equal. Some, like toxoflavin, simply do not belong in screening libraries due to their tendency to generate reactive oxygen species. I would put alkylidene rhodanines in the same category due to their ability to act as Michael acceptors, their tendency to undergo photochemistry, and their hydrolytic instability. The nice thing about these sorts of molecules is that their clear mechanistic liabilities justify excluding them.
But things are not always so simple, and in a recent paper in J. Med. Chem. Martin Scanlon and co-workers at Monash University, along with J. Willem Nissink at AstraZeneca, describe their experiences with a more ambiguous member of the PAINS tribe: 2-aminothiazoles. (See here for In the Pipeline’s discussion of this paper.)
That 2-aminothiazoles (2-ATs) should be PAINS is not obvious: at least 18 approved drugs contain the substructure. Thus, it was not unreasonable to include 2-ATs in the 1137-fragment library assembled at Monash. But after screening 14 targets by STD-NMR and finding a 2-AT hit in every campaign, the researchers started to become suspicious. They gathered a set of 28 different 2-ATs and screened these against six structurally diverse proteins using surface plasmon resonance (SPR). Many of the 2-ATs bound to 5 of the proteins, and a couple bound to all six. The researchers used 2D-NMR (HSQC-NMR) to further characterize binding and found that the 2-ATs bind to multiple sites on the proteins rather than the desired one-to-one binding mode.
A common source of artifacts is the presence of reactive impurities, so the researchers resynthesized some of the 2-ATs and showed they behave the same, ruling out this mechanism. Solubility was also not a problem. Finally, the ligand-based NMR experiments revealed that the 2-ATs really did appear to be binding to the proteins, ruling out interference from unreacted starting materials or decomposition products.
One structure-activity relationship did emerge: acylation of the amino group dramatically reduced promiscuity of the 2-ATs. However, in the case of 2-ATs with a free amino group, there was little meaningful SAR. Thus, the researchers propose calling these molecules PrATs, or promiscuous 2-aminothiaozles.
Further analysis of high-throughput screening data from the Walter and Eliza Hall Institute and AstraZeneca revealed that 2-ATs were also over-represented among hits. What’s spooky about this result is that most of the screens were done at 10 micromolar – far lower than typical fragment screens.
The researchers freely admit that they have no mechanism for why PrATs bind to so many proteins. I suspect there is something fundamental to be learned about intermolecular interactions here, though how to extract these lessons is beyond me. One gets the impression that the authors themselves have been burned by pursuing PrATs, as they conclude:
On the basis of our findings reported here and our unsuccessful attempts to optimize these fragments against different targets, we have removed 2-ATs from the fragment library.
This paper serves as a thorough, cautionary analysis. As evidenced by multiple approved drugs, PrATs can be advanceable, and we certainly won’t be PAINS-shaming papers that report them as screening hits. If you can advance one to a potent lead, then bless your heart. But be warned that this is likely to be even more difficult than normal.