Well, I’m back in the Eastern Time Zone after flying in from Basel (and Amsterdam) yesterday. And the first thing I wanted to mention was this article from Jonathan Baell and Michael Walters inNature, on the PAINS compounds. It’s good to see the journal cover this issue (and I was impressed that they got New Yorker cartoonist Roz ChastRoz Chast to illustrate it).
PAINS are, of course, nasty frequent-hitting compounds that should be approached with great caution in any sort of screen for activity. This topic has come up many times on the blog (for someone writing about chemistry and drug discovery, there’s no way it couldn’t have), most recently just a few weeks ago. There are a lot of these things out in the literature (and the catalogs), and they just keep on coming. Now a wider audience gets to hear about the problem:
Academic researchers, drawn into drug discovery without appropriate guidance, are doing muddled science. When biologists identify a protein that contributes to disease, they hunt for chemical compounds that bind to the protein and affect its activity. A typical assay screens many thousands of chemicals. ‘Hits’ become tools for studying the disease, as well as starting points in the hunt for treatments.
But many hits are artefacts — their activity does not depend on a specific, drug-like interaction between molecule and protein. A true drug inhibits or activates a protein by fitting into a binding site on the protein. Artefacts have subversive reactivity that masquerades as drug-like binding and yields false signals across a variety of assays.
That’s the problem, all right. It’s not like ugly-looking compounds can never become drugs, and it’s not like they can’t be starting points for research. But the odds are against them, and you have to realize that, and you also have to realize why this “hit” you’ve just uncovered may well be spurious (at worst) or need a lot of extra work (at best). Far, far too many papers from less experienced research teams seem to be oblivious to these concerns. Compound hits? Compound good!
Appropriately, this piece calls out the rhodanines as perfect examples of the problem:
Rhodanines exemplify the extent of the problem. A literature search reveals 2,132 rhodanines reported as having biological activity in 410 papers, from some 290 organizations of which only 24 are commercial companies. The academic publications generally paint rhodanines as promising for therapeutic development. In a rare example of good practice, one of these publications (by the drug company Bristol-Myers Squibb) warns researchers that these types of compound undergo light-induced reactions that irreversibly modify proteins. It is hard to imagine how such a mechanism could be optimized to produce a drug or tool. Yet this paper is almost never cited by publications that assume that rhodanines are behaving in a drug-like manner.
Very occasionally, a PAINS compound does interact with a protein in a specific drug-like way. If it does, its structure could be optimized through medicinal chemistry. However, this path is fraught — it can be difficult to distinguish when activity is caused by a drug-like mechanism or something more insidious. Rhodanines also occur in some 280 patents, a sign that they have been selected for further drug development. However, to our knowledge, no rhodanine plucked out of a screening campaign is in the clinic or even moving towards clinical development. We regard the effort to obtain and protect these patents (not to mention the work behind them) as a waste of money.
Yeah, I wouldn’t spend much on trying to stake a claim to these things, either. If you haven’t done much screening, you may not appreciate just how many false positives are out there (and for difficult targets, how few real positives there may be). I see people in the literature screening little libraries of a few thousand compounds from a catalog and reporting hit after hit, even in very tricky systems, while in industry we’re used to running hundreds of thousands of compounds past some of these things and coming up with squat. Well, after checking the “hits” for purity, aggregation behavior, reactivity, and profiles from past screening campaigns, that is.
Here’s the sad truth: If you’re doing a small-molecule screen to affect transcription factors, protein-protein targets, or anything in general that doesn’t have an evolutionary optimized small-molecule binding site, you’d better assume that the vast majority of any hits you get are false positives. There’s almost no way that they can be anything else. The true hit rate for some of these things against any sort of typical compound collection is damn near zero, which means that the ways your compounds can be wrong far outnumber the ways that they can be right.
Every single hit, for any assay, should be regarded with appropriate suspicion. Purity check first, LC/MS and NMR. Is it what it says on the label? You might be surprised how often it isn’t (or isn’t any more, even if it started out OK). If you have solid material and DMSO stock, check both of them, because things diverge on storage. It’s a very good idea to take your interesting hits, run them through a plug of silica gel, and test them again. That’s especially true if they have any color to them (but keep in mind, some assay-killing contaminants are completely colorless). The gold standard is resynthesis: if you can make the compound again and purify it, and it still works, you at least know you can trust it that far. If you can’t, well, how exactly is this compound going to do anyone any good?
Note that we haven’t even gotten to the PAINS yet. There are a lot of clean, accurately labeled compounds that should be chucked into the waste can, too, which is where the Baell PAINS list comes in. You’re going to want to check for aggregation: run your assay with some detergent in it, or do some dynamic light scattering or any of several other techniques. A lot of false-positive compounds are aggregators, and you can’t completely predict which ones they might be (it varies according to assay conditions).
You’re also going to want to run your hits through some other assays. How promiscuous are they? If you have access to data from multiple screening campaigns with the same compound collection, good for you. If you don’t, you should strongly consider sending your hot compound(s) out for a commercial screening panel. Don’t just pick the similar targets to screen – you want those, of course, but you want all kinds of other stuff. If a compound hits against widely disparate protein classes, it’s a PAIN, and is set to cause trouble. Don’t assume that they’re clean – don’t assume that any compound is clean, because it almost certainly isn’t. That goes for marketed drugs, too – the question is, does it have selectivity that you can live with, or not?
Those are the big tests, and believe me, they’ll clear out your initial list of screening hits for you. If your target is a tough one to start with, they may well clear out everything. Better that, though, than working on (and publishing) crap.