It’s safe to say that the concept of “pan-assay interference compounds” evokes some strong feelings in medicinal chemists. And those feelings run in several directions: some people are very glad to have a tool with which to winnow down their screening hit lists (or at least to prioritize them), while others are infuriated by the idea of tossing out what could actually be useful compounds on the basis of structural similarities alone. (Here’s a recent overview (open access version) from the originators of the concept). I myself land somewhere in the middle of this one. I think the idea of such a list is a sound one, as long as it’s seen in shades of trouble rather than a sharp dividing line. If you want to try to develop one of these structures, you’d better have a good reason, and you should be prepared to give it extra scrutiny. It’s a sliding scale – some of the PAINS structures are particularly prone to give false positives with some assay technologies but not so much with others, for example, so things might not be so bad. But if you want to proceed with (say) a rhodanine that has a quinone hanging off it, you have to realize that you’re running a huge risk of wasting your time.
This new paper examines well-known PAIN motifs in the X-ray crystallographic literature, which is an angle that hasn’t gotten as much attention. Digging through the PDB, the authors find 2784 structures of such ligands – but those represent 1107 separate molecules, which tells you that some of them are showing up in several proteins. That’s one flag, binding promiscuity at the X-ray structure level, but it’s not the whole story. The paper also identified a number of structures where it appears that reactive groups have formed covalent adducts with the protein binding sites – but that’s not the whole story, either.
That’s because there are also many “normal”-looking structures, where the compounds are making reasonable interactions with their binding pockets. In many cases, these interactions don’t actually involve the problematic parts of the structures – it’s not the PAINful part of the molecule that’s feeling the protein. This emphasizes that you can’t take the fact that a compound’s (sub)structure is on a PAINs list and just cross it off immediately as an artifact. (Nor, of course, should you proceed blithely ahead with it!) There are marketed drugs with some of these structure in them, remember.
But there are a lot more marketed drugs that don’t have them. I very much believe that if you take a randomly selected similarly-sized list of functional groups and run it through a Protein Data Bank ligand search, you will find far fewer problematic results. And remember that an X-ray structure search certainly doesn’t cover all the mechanisms that can make a compound troublesome. For example, as the paper shows, you can find aminoacridines making perfectly legitimate binding interactions in X-ray structures – but that doesn’t mean that they’re not interference hits in fluorescent assays, because believe me, they are.
This paper, at least to me, reinforces the “yellow caution light” approach to these structures. It’s lazy and bad practice to immediately throw all such structures into the compost pile, but it’s just as bad (or worse) to ignore the potential problems and carry on as if everything is going to be fine.