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Chemical Probe Compounds: Time to Get Real

Update: the chemical probes portal mentioned here has since been updated and re-launched.

There’s a new paper on chemical probes out in Nature Chemical Biology, and right off, I have to disclose a conflict of interest. I’m a co-author, and I’m glad to be one. (Here’s a comment at Nature News, and here’s one at Science). The point of the article is that (1) many of the probe compounds that are used in the literature are inappropriate at best, and junk at worst, and (2) it’s time to clean up this situation.
How bad is it? Try these examples out:

. . .For instance, LY294002 was originally described in 1994 as a selective inhibitor of PI3 kinase and remains advertised as such by nearly all vendors. Yet by 2005, it was already clear that the compound inhibited many other proteins at the concentrations used to inhibit PI3 kinase. In the meantime, a large number of more selective and more well-characterized PI3 kinase inhibitors have become available.
The availability of these new inhibitors certainly obviated the need for LY294002 as a chemical probe, and it should be discarded as a selective research tool. Yet a search
of Google Scholar in 2014–2015 alone for ‘LY294002 and PI3 kinase’ returned ~1,100 documents.

And why not? You can still find people using staurosporine as a PKC inhibitor, even though it’s a kinase blunderbuss. Similarly, dorsomorphin is not a good choice to inhibit AMPK signaling, and chaetocin is a terrible excuse for a selective histone methyltransferase probe. I’ve written about others on this blog, as bad or worse.

But these things are all over the literature. People can’t keep up, or don’t, with the literature showing that these compounds (and many others) are problematic, and the suppliers keep selling them. Far too many researchers look something up in the catalog, see it listed as a “selective XYZ inhibitor”, and believe every word. Both the suppliers and the investigators are at fault, and the result is that the scientific literature ends up with garbage piles and oil slicks floating all over it.

Good probe compounds are not easy to find. Seeing one in somebody else’s paper places you at the mercy of their literature-searching skills if you don’t do some checking of your own. Ordering one up from a catalog proves nothing more than that company’s ability to sell it to you. To try to remedy this situation, this new paper also includes the launch of a web site, a wiki-based compendium of validated probes. The hope is that this will become a resource that everyone can turn to, a one-stop-shop that will save a lot of time, money, effort, and frustration.

It has only a few compounds in it as of this morning, but I plan to send in some suggestions of my own this week. (One of those is for a separate list of probes that are Not Recommended, so that people can find those as well). The plan is to put up editing functions soon so that people can do this themselves. I encourage people to send in feedback – this is an opportunity to try to fix a number of longstanding problems in the literature, and without something like this, these problems will only get worse.

Ideally, I’d like to see references to the site in the supplier catalogs, and attention paid to its listings by reviewers and authors alike. The excuses for using worthless chemical probes have never been good ones, and with any luck, there eventually won’t be any such excuses left at all.

25 comments on “Chemical Probe Compounds: Time to Get Real”

  1. petros says:

    Chembl is a decent source for quick checks on the selectivity of many such “probes”

  2. bhip says:

    Don’t forget the crappy, nonspecific antibodies that are being hustled all over the place. They are frequently used to generate the first piece of misleading data (see Fig 1) prior to misusing the nonspecific compound to block its function, expression, etc. (see Fig 2) & subsequently dosing animals & observing completely unrelated responses (see Fig 3).

  3. Mike G. says:

    IANAC (I am not a chemist), but I think that your Wiki will probably need some very dedicated staff or volunteers, or some vendor(s) will very quickly edit it into uselessness, putting all of their (most profitable?) products on the good list, and all of their competitors’ (most profitable?) on the bad list…
    That’s just a thought – I’m not volunteering. I took one look at organic chem in college and went into Computer Science. I understand chemistry a bit, until C gets involved in anything more complicated than CO2 🙂

  4. John Wayne says:

    “Kinase Blunderbluss” made me chuckle; I have a mental picture of Yosemite Sam in the lab looking for a publishable unit

  5. Vaudaux says:

    Not sure I have ever before seen an author list in strict alphabetical order: 53 authors, from Arrowsmith to Zuercher.
    PS: I liked the text too.

  6. A Nonny Mouse says:

    What’s even worse is when these are being used at 30 micro molar when their IC50 is 0.1; the authors then claim that the activity is due to the stated activity.
    I challenged a couple of authors who have made their career on this stuff but didn’t get any response……. (that’s because they cost use a large amount of money on biological testing of compounds with the same activity which showed no response).

  7. Frank David says:

    I’d love to hear your thoughts on the probes / tool compounds that are available via pharma “open innovation” programs – see here for details: http://www.pharmagellan.com/blog/pharma-academia-collaborations. It’s not quite as simple to get them as picking up the phone and placing an order, but on the other hand, they’re likely to be better-characterized than many commercial alternatives.

  8. Lyle Langley says:

    I find the author list quite interesting (or ironic?) as some of those on the list were some of the biggest offenders during the MLPCN program. When the requirement for ancillary pharmacology was being implemented (why would you want to know the selectivity on a broader panel of receptors?) there were some on this author list that threw quite a fit. Guess it’s always easier to tell others what a selective probe is, rather then actually doing it yourself.

  9. Sam Adams the Dog says:

    Kinda makes you wonder: what would you do if, as a referee, you were asked to review a paper that relied on one or more of these, umm, blunderbi?
    Would you reject it out of hand? Ask the user to modify the discussion section to make his assertions weaker? Pity the poor grad student? Or what?

  10. MoBio says:

    @9:
    I see only 2 that were part of MLPCN (Austin and Schreiber) and my recollection is that they had no beef regarding ancillary pharmacology.

  11. Pliny the Elder says:

    @10 Any biological study that relies solely on the activity of a pharmacological probe (good probe or blunderbus) to understand mechanism is of limited value. Simply chucking a molecule on a biological system, then concluding phenotype z is due to inhibition of target y by compound x (based on some in vitro profile of x’s activity) is not much of a mechanistic insight. With even a “good probe” the mechanistic inferences underlying a phenotype should be considered a hypothesis to be tested rather than standalone mechanistic evidence.

  12. Slurpy says:

    Is it not standard practice to try multiple probes on a target before publishing?

  13. Kenny Powers says:

    The manuscript states that T0901317 (in conjunction with GW683965) is an example of an excellent chemical probe that was used to elucidate important information about LXR biology. However, several publications from a range of laboratories have reported activity of T0901317 on the nuclear receptors PXR, FXR, and the RORs. These findings were not mentioned in the manuscript. More troubling is that many papers are still being published using this compound to describe new biology associated with LXR.

  14. Anonymous says:

    I haven’t read the article yet, so I’m just commenting on the blog, but I don’t think chemical probes need to be 100% selective to be useful. However, I also don’t think they should be the only control in an experiment to inhibit/activate whatever target they’re intended to probe. Chemical biologists aren’t intending to make drugs that are 100% selective when they design probes… they’re not medicinal chemists. They’re just trying to expand the ‘toolbox’ to achieve additional methods to inhibit/activate proteins (and I think these sorts of compounds are particularly useful in vitro/in cell based studies.. I do think they are not as useful in vivo, since they’re not drugs). The problem is that biologists that end up adopting these probes don’t understand that just because it inhibits PI3K or whatever, doesn’t mean that it doesn’t also inhibit other pathways, and they rely on them too heavily as their controls.

  15. Bobby Shaftoe says:

    Interesting piece. I hope this effort engages the community and helps to address the problem as this is one of my pet peeves.
    @14: The manuscript recommends that even “high quality” probes should be complemented by a second compound which engages the same target but is structurally unrelated to the first probe. If possible a negative control closely related in structure to one of the positives should also be employed.
    In the case of LXR, T1317 is not a great LXR probe in itself and should not be used as a standalone. However, T1317 and GW3965 together are a useful probe SET. In fact, the knowledge from this combo of probes has helped in the creation of clinical programs targeting LXR.

  16. TroyBoy says:

    I’m all for people understanding how their probes are functioning and what they’re really doing with their cells. There are too many papers where the scientists don’t understand the compounds they are treating with. Some of them don’t care, as long as it knocks down the activity they’re looking at.
    But I do have a couple beefs with the paper:
    It’s all well and good to have high standards for quality probes, but are the standards too high? It’s difficult enough as it is to find a drug with all the resources that industry puts into it. Finding a probe that meet certain standardized criteria–especially with the typical R01 levels of funding–is arguably even more difficult. So yes, we can have the ideal criteria for a probe and then not be able to publish anything because we’re not there yet.
    The Venn diagram in Figure 1 shows little overlap between probes and drugs. In table 1, the paper then gives examples of high quality probes. The paper that reports PF-04457845–which was discovered by an industry group–says that it is being evaluated in human clinical trials. In other words, PF-04457845 wasn’t developed as a probe. It was developed as a drug, and just so happens to be useful as a probe.
    Another example given in the paper of a high quality probe is GNF-5, which has an in vitro IC50 of 220 nM. It wouldn’t have passed the stated Structural Genomics Consortium criteria (

  17. Hap says:

    Except drugs don’t have to be specific – they just have to work. Lipitor, for example, doesn’t seem to do just what it was supposed to (inhibit HMG CoA reductase) – it’s doing something else as well that people don’t understand. So having a drug is useful, but doesn’t tell you what pieces of the pathway do, which is what a probe is supposed to do. If you can’t separate the effects of individual enzymes, then conclusions on the biology are sort of hard to make, especially if deleting the enzymes is lethal.
    Isn’t probe development what people are getting mucho bucks from NIH to do? If people who are being paid (grant money and publications) can’t be bothered to do it, what’s the point to the grant money?
    On the other hand, if developing probes is so hard, then why is everyone and their dead grandparents claiming to have developed one? This seems like the problem – the people publishing probes either don’t know what their probes do (or should do) or don’t care (as long as they get publications), and people assume from publications that the authors actually mean what they wrote. If this contention is wrong, or doesn’t matter because it’s so hard, then there’s a lot more money than people thought going to waste.

  18. cinnamon says:

    @16: I think the point @14 was making is the manuscript does not mention that T1317 has other activities beyond LXR and that the manuscript being discussed here did not bother to mention that. Bobby adds addition comments that are important but they were not pointed out in the paper. This point is important to whatever database is being created.

  19. Kevin says:

    When you run kinase panels for a living, you quickly learn how much garbage is in the literature.

  20. TroyBoy says:

    @18. Thank you for your comments, Hap.
    My point is that the Venn diagram between drugs and quality probes is such that drugs (almost) fully encompasses probes. The same qualities that make a “high impact” probe are the qualities that you want in a drug, ie., potency and selectivity. I think everyone wants to really develop a drug. They say they are developing probe if they are unsuccessful at dialing out some offending non-drug-like constituent.
    I think that “probe development” is what the NIH calls it to avoid people (on the left and the right) attacking it for competing with industry. And there isn’t a whole lot of money being spent on it. If you go to the NIH Reporter website, and type “probe development” as a search criterion, you get 27 active programs. If you type in “drug development,” you get 2000+ active programs.
    What is the difference between a probe and a lead anyway?

  21. Hap says:

    Sorry. I was frustrated. For the most part, I figure that people looking to show evidence that a specific enzyme needed to use something to specifically inhibit or alter the enzyme they’re interested in. If they can do that without probes (siRNA, etc.) then there’s no reason to develop probes in the course of one’s work. Most things, though, probably do still need small molecule inhibitors to tinker with an enzyme, and if the users are going to be able to make conclusions about the biology they are perturbing, they have to be specific inhibitors. If they’re going to make the conclusions they’re trying to make about the molecular biology they’re investigating, they probably have to have a probe, and so people would be making (or finding, more likely) probes in the course of other work.
    I assume a lead is something capable of structural modifications that has a desired biological activity. Lots of times, since people look at single targets (or one target out of a similar set of enzymes), a lead might be similar to a probe, but not so selective (selectivity for the target is one of the things I figured would be optimized in the process of going from lead to drug).
    It’s reasonable to say that probes are hard, and if there’s a better way to investigate and disentangle biology, then it might be quicker, or cheaper. It just seems like people claim lots of things for the biology of molecules that they haven’t bothered to substantiate, because they haven’t bothered to learn about it, or don’t care because they already got a big-name publication, and that’s what matters. People think they’re paying for reliable research based on what people say in their publications and find out that it bears little resemblance to what’s actually going on, and it is frustrating.
    Paying to create lots of future unemployed people (because you’re training people for “jobs that don’t exist”) in order to generate research that makes things more expensive (because people have to redo your research to find out what they thought they knew, or to spend money acting on the basis of things that aren’t what they thought they were) seems like a bad use of research money. The abuse of probes seems like an egregious example. If you can’t make a selective probe for something, or don’t have one to use, then the claims you make for your work need to be consistent with that fact, or use other methods that can get you data consistent with those claims. It might also make NIH more willing to pay for probe work – if they realize that probes don’t grow on trees and are useful in giving people reliable biological knowledge, they might be more willing to fund their development.

  22. Anonymous says:

    @ 21
    You say “The same qualities that make a “high impact” probe are the qualities that you want in a drug”. I am surprised of this comment.
    To my best knowledge, and as others have said in this forum, drugs need not to be selective at all.
    And potency… well, since CombiChem we have had a lot of discussion on potency and drug quality…
    So, IMHO, probes and drugs are quite different beasts…

  23. Spiny Norman says:

    My personal fave: claims that inhibition of a response by BAPTA vs. EGTA demonstrates a requirement for spatially-local Ca2+ release. Have any of these authors actually looked at the structure of BAPTA?

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