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Are Your Compounds Ugly? Do You Know?

A reader sends along this paper, on some small molecules targeting the C2 domain of coagulation factor VIII. It illustrates some points that have come up around here over the years, that’s for sure. The target is not a particularly easy one: a hit would have to block the interaction of that protein domain with a membrane surface. There is something of a binding pocket down in that region, though, and there were some hits reported from a screen back in 2004. Overall, it looks like a lot of targets that show up, especially these days – you’re trying to affect protein conformation by going after a not-necessarily-made-for-small-molecules cavity. Possible, but not something that’s going to light up a screening deck, either.
And many of the things that do show up are going to be false positives of one sort or another. That’s always the tricky part of doing low-hit-rate screening. The odds are excellent that any given “hit” will turn out not to be real, since the odds are against having any hits at all. This is especially a red flag when you screen something like this and you get a surprisingly decent hit rate. You should suspect fluorescence interference, aggregation, impurities, any of the other myriad ways that things can be troublesome rather than assume that gosh, this target is easier than we thought.
It’s often a chemist who’s in charge of dumping these buckets of cold water (if you have the help of the people who set up the assay, so much the better). Traditionally, it’s one of the biology project champions who gets enthusiastic about the great list of compounds, but if you have someone who’s been burned by false positives a few times, then so much the better, too. It’s not fun to knock down all these “hits” and “leads”, but someone’s got to do it, otherwise everyone’s time will be wasted to an even more painful extent.
And you should be especially worried when your screen turns up compounds like some of the ones in this paper. Yep, it’s our old friends the rhodanines, everybody’s cheap date of the screening deck. These compounds have come up around here many times, because they keep on showing up in the flippin’ literature. In this case, the authors did some virtual screening over the ChemBridge collection and then moved on to assays against the protein itself, eventually finding a number of active compounds in the micromolar range. The compounds look a lot like the ones from 2004, since those were used as the template for screening, and that was a pretty ugly rhodanine-infested set, too.
Indeed most of the compounds they found are pretty unattractive – the aforementioned rhodanines, lots of nitroaromatics, some other heterocycles that also hit more often than one would like. I would feel better about these sorts of papers if the authors acknowledged somewhere that some of their structures are frequent hitters and might be problematic, but you don’t often see that: a hit is a hit, and everything’s equally valid, apparently. I would also feel better if there were something in the experimental section about how all the compounds were assayed by LC/MS and NMR, but you don’t often see that, either, and I don’t see it here. Implicitly trusting the label is not a good policy. Even if the particular compounds are the right ones in this case, not checking them shows a lack of experience (and perhaps too trusting a nature where organic chemistry is concerned).
But let’s cross our fingers and assume that these are indeed the right compounds. What does it mean when your screening provides you with a bunch of structures like this? The first thing you can say is that your target is indeed a low-probability one for small molecules to bind to – if most everything you get is a promiscuous-looking ugly, then the suspicion is that only the most obliging compounds in a typical screening collection will bother looking at your binding site at all. And that means that if you want something better, you’re really going to have to dig for it (and dig through a mound of false positives and still more frequent hitters to find it).
Why would you want to do that? Aren’t these tool compounds, useful to find out more about the biology and behavior of the target? Well, that’s the problem. If your compounds are rhodanines, or from other such badly-behaved classes, then they are almost completely unsuitable as tool compounds. You especially don’t want to trust anything they’re telling you in a cellular (or worse, whole-animal) assay, because there is just no telling what else they’re binding to. Any readout from such an assay has to be viewed with great suspicion, and what kind of a tool is that?
Well then, aren’t these starting points for further optimization? It’s tempting to think so, and you can give it a try. But likely as not, the objectionable features are the ones that you can’t get rid of very easily. If you could ditch those without paying too much of a penalty, you would have presumably found more appealing molecules in your original screen and skipped this stage altogether. You might be better off running a different sort of screen and trying for something outside of these classes, rather than trying to synthesize a silk purse out of said sow’s ear. If you do start from such a structure, prepare for a lot of work.
As mentioned, the problem with a lot of papers that advance such structures is that they don’t seem to be aware of these issues at all. If they are, they certainly don’t being them up (which is arguably even worse). Then someone else comes along, who hasn’t had a chance to learn any of this yet, either, and reads the paper without coming out any smarter. They may, in fact, have been made slightly less competent by reading it, because now they think that there are these good hits for Target Z, for one thing, and that the structures shown in the paper must be OK, because here they are in this paper, with no mention of any potential problems.
The problem is, there are a lot of interesting targets out there that tend to yield just these sorts of hits. My own opinion is that you can then say that yes, this target can (possibly) bind a small molecule, if those hits are in fact real, but just barely. If you don’t even pick up any frequent hitters, you’re in an even tougher bind, but if all you pick up are frequent hitters, it doesn’t mean that things are that much easier.

21 comments on “Are Your Compounds Ugly? Do You Know?”

  1. SP says:

    Along the lines of people never learning from the past- I recently learned that there is a paper from 20 years ago, cited over 200 times, that comes up with the brilliant idea of measuring AUCs by breaking the curvy area into a bunch of almost-rectangles then taking the area of the rectangles and adding them together. The author grandly names this method after herself. Talk about being scooped by only a few centuries. (And I checked, the publication date was not April 1.)

  2. alig says:

    I disagree with your statement that finding these types of compounds means “this target can bind a small molecule”. There are a lot of ways these compounds can interfere with the assay without binding to the target. If a group wants to advance a PAINS compound, they should be required to show an xray structure with the compound bound to the target or very robust biophysical data showing 1:1 binding.

  3. UICAlchemist says:

    Yo compound is so ugly that it makes syn-Propanethial-S-oxide cry!
    Yo compound is so ugly that computational chemists think it is attractive!
    Yo compound is so ugly that it makes a microwave assisted Suzuki coupling look clean!

  4. Derek Lowe says:

    #1 SP – I’m surprised that there wasn’t a follow-up paper, where the author noted a relationship between the area under the curve and the change in the slopes of the tangent lines. Sheesh. Perhaps the “EdD” after the name is a warning sign?
    #2 alig – Good point. I’ll change the wording a bit.

  5. watcher says:

    Related to the old axiom: An hour in the library (now, a 30 min search through on-line literature) can save (pick one)an day, week, month year in the lab.

  6. RTW says:

    It all comes down to knowledge management or experience. Too may experienced med chemists put out of work in favor of inexperienced outsources or younger “fresh idea” folks. No once with any kind of actual long term medchem experience took a look at the manuscript. I also fault the editors. If we can pick apart these papers certainly the reviewers should be able to.

  7. anon the II says:

    Well, the good news is that if the rhodanines fail, they’ve got some coumarins to follow up on. 🙂

  8. Anonymous says:

    The journal Blood isnt exactly the kingpin of medicinal chemistry journals and the Netherlands is probably better known for drug discovery at coffee shops than cardiovascular research centers.
    The poor scientists thought they struck gold with their findings- got too excited and forgot to open a book on the evil of rhodanines let alone run a LC/MS or two.
    In simpler times this article would have escaped such public attention, but not anymore!
    Beware of the ITP Inquisition!

  9. Hap says:

    Tool compounds are supposed to be more selective, not less, than drugs (drugs can succeed being partly selective), so the line about PAIN-type compounds not needing to be drug like because they’re just tool compounds isn’t just tiresome, but either dumb or dishonest.
    Perhaps the post should have been titled “Are Your Compounds Ugly? Do You Know? Do You Care?”

  10. DH says:

    #1 SP – The truly insane thing is that the Tai paper continues to get cited — even by authors other than Tai, at different institutions than hers. E.g., from a paper published this year: “…total area under the curve (AUC) was calculated using the trapezoidal method (Tai 1994).”

  11. MedChem says:

    For what it’s worth, ibrutinib was a tool compound for a long time until they realized they couldn’t make a better one.

  12. Teddy Z says:

    We need more of this: PAINS Shaming. (See my first effort in my URL link)

  13. MedChem says:

    For what it’s worth, ibrutinib was a tool compound for a long time until they realized they couldn’t make a better one.

  14. medchemist2 says:

    Having transitioned from Pharma to Academics a few years ago, I run into a lot of naiveté regarding compound structures – with collaborators and while reviewing manuscripts. I am frequently the only chemist reviewing a paper that has a ‘magic compound’ whose structure they either don’t disclose or, when disclosed, it is one of these suspect ‘hits’. Meanwhile, the biological reviewers are picking apart the western blots and choice of cell lines etc. that I sometimes appreciate after they point it out, but wouldn’t flag on my own. My only point being, multidisciplinary science needs to be reviewed by experts from all the disciplines. I see plenty of dubious biological claims in JACS, for example. We all have our blind spots – the editor’s job is critical to the process. There’s an interesting symposium in Cambridge, MA in October on reproducibility in preclinical target validation where some of this will be discussed:

  15. David Borhani says:

    Derek: Paper is behind a firewall for me. Would be helpful if you could add a picture of the PAIN-ful molecule! Thanks!

  16. Anonymous says:

    I ran my PAINS script on my chembridge and off the top of my head there was maybe about 600 in total.

  17. molecular_architect says:

    #5 My PhD advisor switched it around to emphasize the absurdity: “A week of hard work in the laboratory can easily save an hour of time in the library”

  18. Anonymous says:

    #1, #4 This made my day

  19. Anonymous says:

    There are different philosophies about when running medchem in silico filters, how stringent the filters have to be: depending on the target, the stage of the project, the goals etc. There are many debates about some toxicophores like NO2 that can easily be changed; several PAINS have been recently re-investigated in J Chem Inf Model. 2013 Mar 25;53(3):534-44 and some were not found that problematic for the assays. As such several molecules in this coagulation study, considering the data, should indeed bind the right domain of the right target and not bind everywhere as direct binding on several other proteins was carried out with biacore. The molecules were filtered in silico and the authors seem to be perfectly aware of PAINS etc as they have tools flagging these compounds and many other structural alerts. As such, if they kept them it is because they are doing the job, they probe a molecular mechanism, the ones selected do not behave in a strange way as this would be easily seen in several of the functional assays. Chemical beauty is in the eye of the beholder

  20. chemstudent says:

    Am I wrong or is the top compound from this study not a rhodanine at all???
    Just looked up Epalrestat (a rhodanine), looks like this is an approved drug….

  21. Bob says:

    #19, sorry but I think you’re wrong, it’s not about philosophy. The J Chem Inf Model paper you refer to missed the point about nonspecific inhibitors. It doesn’t matter if a given compound only inhibits 0.01% of assays nonspecifically, but if it does it in your assay it’s still a problem. In my company we try to run every screening hit progressed through some kind of direct binding assay as dose-response. That’s a lot of work and we wouldn’t do it unless there was a real need.
    SPR can flag up nonspecific binding, but not when used as it was in the coagulation paper. Rather than monitor direct binding of compounds to proteins, they used it to monitor binding of proteins to membrane. The compounds were used as competitors. In that format I don’t think SPR says anything about binding specificity.

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