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Rotten Rottlerin

Here’s one of the problems with the various schemes to do “deep learning” using the published scientific literature: a fair proportion of that literature is junk. This is not news, of course, and to some extent I feel that that’s always been the case. The pressures to publish, and the corresponding rise of publishing fraud, aren’t helping much, though. And there’s a lot of plain old sloppiness out there, too.
For a small example, consider the polyphenol compound rottlerin. It’s isolated from a plant in India and the surrounding area (and named after the botanist who first described the genus in the European literature). Rottlerin itself was isolated in the 1800s as a tan, crystalline substance, and in the early 1990s it was identified as a PKC-delta inhibitor, for which purpose it has been used in uncounted studies in the literature.
Problem is, though, like a lot of such compounds, it’s not anywhere near as selective as advertised. And it has all sorts of other activities in cells, too, as one can well imagine, looking at its structure. In fact, as that link shows, the original PKC activity reported for the compound may well have been due to an impurity – other papers have found no real PKC inhibition at all, much less anything selective (not that the original selectivity was anything to jump around about, either). So rottlerin, as a tool compound, is useless. It does too many thing to untangle, and the the thing that it’s most well-known for doing it probably doesn’t do at all.
So no one uses it any more, right? Har de har. That link will take you to the latest list of rottlerin papers, and on it you’ll find this paper from this year, using it as a PKC-delta control. And this 2015 paper. And this one. And this one. You can pick out more of them, all the way down the list.
Now, some of the papers you pull up are more defensible, since they’re more like “Here’s something rottlerin does phenotypically to cells, and we’re going to unravel what it is”. Fair enough. But why is anyone using it as a PKC-delta inhibitor, a good dozen years (at least) since that activity was called into question? I wouldn’t trust it as a pan-PKC inhibitor either, not when it does so many other things. Rottlerin is useful if you’re studying rottlerin, but getting it to illuminate something else for you is going to be a tricky business. Watching people just blithely treat it as if it were some automatic selective switch you can drop into an assay is disturbing.
That broad phenomenon has come up in discussions around here before, though. There’s a persistent lack of examination in the biological world when it comes to small-molecule tool compounds. Too many people seem willing to believe that there are all sorts of those perfectly selective compounds out there, ready to reach into cells and do exactly what it says in some catalog or some old paper. There are some, but nowhere near as many as you might imagine, or as many as people will sell you. Tocris is more up front about what the compound does, but here’s EMD/Millipore/Calbiochem, selling rottlerin as ” cell-permeable and reversible protein kinase C inhibitor that exhibits greater selectivity for PKC-delta (IC₅₀ = 3-6 µM) and PKC-theta”. So how can I blame some biologists for not trusting that sort of thing?
Update: from the comments, here’s a complaint about the use of this very compound from 2007. It spells it out in the title (“An Inappropriate and Ineffective Inhibitor”), and it appeared in what a colleague of mine once called “An obscure journal called Cell”). But rotterlin itself marches on, and neither editors nor reviewers seem to care, in many cases.

28 comments on “Rotten Rottlerin”

  1. Hap says:

    A polyphenol and a Michael acceptor? That doesn’t seem selective at first glance. It’d be helpful to know if it undergoes Michael additions under biological conditions, or if it does any funny redox stuff. Did anyone look?

  2. Anonymous says:

    And this has been complained about before….
    http://www.cell.com/trends/pharmacological-sciences/abstract/S0165-6147%2807%2900185-X (paywall)
    Only eight years ago – who could have possibly read that?

  3. DrSnowboard says:

    And there I was thinking Rottlerin was an April Fools compound…

  4. Ed-181 says:

    The top listing on Google a search for “rottlerin” is, not surprisingly, a Wikipedia page. That page has a section titled “Ineffective PKCδ selective inhibitor”. Anyone using rottlerin as a selective PKCδ inhibitor didn’t do even the most basic of research beforehand.

  5. Barry says:

    Lilly did (with a great deal of labor) carve a (promiscuous, micromolar) PI3K inhibitor LY29402 out of the related quercetin when that was found to be an inhibitor. It is still cited as a PI3K inhibitor (although quercetin is not?)

  6. luysii says:

    The nanoDomain is a classic example of this. It’s a small domain inside neurons next to the cell membrane promulgated by a Nobelist (Neher). The term is all over the literature and people speak of calcium concentrations within nanoDomains. Unfortunately at the microMolar concentrations of calcium thought to be found within neurons, the nanoDomain is so small that it contains very few to zero calcium ions.
    For details please see — https://luysii.wordpress.com/2013/10/09/is-concentration-meaningful-in-a-nanodomain-a-nobel-is-no-guarantee-against-chemical-idiocy/

  7. Sideline Chemist says:

    The fact that Sigma-Aldrich is offering rottlerin as part of their “Library of Pharmacologically Active Compounds, a biologically annotated collection of high-quality, ready-to-screen compounds” is particularly humorous. Sigma-Aldrich obviously has a low bar definition of “high-quality”.

  8. Non says:

    As a non-chemist, I’ve found reading your blog to be quite an interesting and insightful view of the drug industry.
    Although I don’t usually understand the vocabulary in these blogs, I can still well imagine how these things must go. The struggles in the field of organic chemistry are quite relatable to my own field.
    Thanks for the blog post.

  9. Shirley Hugest says:

    Rottlerin has nothing on Lupinone and Buldogone, particularly the caged version of the latter:
    CMAJ. 1992 Apr 1; 146(7): 1191–1197.
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1488342/
    MAIN RESULTS: Two putative lycanthropogens were isolated from the blood samples. Structural elucidation and synthesis permitted animal and clinical trials; in each of these, behavioural dysfunction was observed. Antilycanthropogen strategies included application of the principle of caged compounds and generation of a therapeutic immunoglobulin. The effects of a newly developed antihirsutic agent seemed promising. An interaction of the lycanthropogen-secretion system and ethanol was noted, which may explain behavioural aspects of alcoholism.
    CONCLUSIONS: The incidence of lycomania in North America is underestimated. Soon-to-be-available pharmacotherapies should promote its early detection and treatment. Full control may depend upon advances in gene therapy.

  10. anonymous says:

    You mean I can’t just sit here in my house while my computer predicts a cure for the measles?!?

  11. industrybiologist says:

    Quiet! I make a living running assays like this!

  12. johnnyboy says:

    Sadly, this is just an example of the type of indiscriminate, poorly thought-out, badly executed research that goes on in academia. I’ve worked for a couple of years now as a sort of ‘consultant’ to various life science and medical research groups, in what you could call a top tier university. The amount of baseless, badly carried out, and ultimately useless research I have seen is mind-boggling. Most of academic research is carried out by PhD students, who don’t have the perspective and experience to really understand what they are doing and why; they just want to get on with it and get done. They should be getting proper guidance and supervision from their PI, but from what I’ve seen the level of guidance is very variable, and often woefully inadequate. PIs have grant money to spend, and while they have it they throw it around this way and that, often on things that do not make sense or have not been thought through. Unfortunately as this is academia, there is no one there to tell them so – to paraphrase John Oliver talking about the Starbucks CEO, clearly nobody has told these guys “No” in the last twenty years.
    Maybe this is how academic research is supposed to work. Maybe you’re supposed to throw millions of dollars at the wall until something sticks. But for someone goal-directed like me, the amount of wasted time, effort and money often leaves me speechless, and not terribly hopeful about the future.

  13. steve says:

    Given all the problems with pharmacological inhibitors one wonders why people don’t just do the experiments with siRNA instead. Yes, there can be off-target effects with siRNA as well but at least you can try 2-3 different sequences to prove specificity.

  14. anonomoujs says:

    #5 More recently LY294002 (the correct number) has also been reported to interact with BET bromodomains at similar concentrations to its PI3K activity. While it almost certainly has other activities, it is possible that much of its phenotypic activity is due to targets other than PI3K.

  15. Cellbio says:

    A point I have made here before, worth repeating I believe, is that the pharmacology of a compound is just that, the pharmacology of a compound. Results in complex biological systems at one high concentration may say something about a protein target, or they may not. The sloppiness of thought derives from the adherence to a schema that holds the central question is about a target of knowledge which leads to rapid and easy interpretation of results despite extremely limited measures of other phenomena, or ignorance of existing data.
    While this may be more rampant in academia, that may only be because there are fewer chemists, screeners enzymologists, etc working in collaboration with the biologists. The biologists I have worked with in industry get stuck on this as well.

  16. anon-ymous(e) says:

    Derek: Is this a typo ….did you mean Resveratrol ???

  17. George Kaplan says:

    Re: EMD/etc’s advertising rotterlin as ” cell-permeable and reversible protein kinase C inhibitor that exhibits greater selectivity for PKC-delta (IC₅₀ = 3-6 µM) and PKC-theta:”
    I think you could argue this text makes a specific fitness claim and violates the implied warranty of merchantability, and importantly fitness for the intended purpose. UCC 2-315.

  18. anon the II says:

    Another example:
    Pitstop 2 (google it) is reported to be a selective Clathrin inhibitor and is commercially available as such. I don’t know the level of validation that it’s undergone, but it clearly falls in the PAINS category. I’d be leery of anything done with it.
    Pitstop II is a video game.

  19. Anonymous says:

    As 13 mentioned, rottlerin isn’t enough to make conclusions and biologists follow-up with siRNA or other methods of confirming a mechanism.
    My lab uses rottlerin to “inhibit” PKC-theta. Do we think this is selective? No, but it is easy to use and if you put enough of it on cells then it prevents phosphorylation of PKC-theta at several sites that are necessary for its downstream activation of NFkB.
    Rottlerin is quick and dirty. If it doesn’t have an effect on our system, then that is enough evidence to assume (not conclude) that it is PKC-theta independent. If it does have an effect, then it may be worth following-up with more careful studies.

  20. Anonymous says:

    @19:
    I think the whole point is that you can’t say that at all with Rottlerin. It looks pretty cysteine reactive regardless of its polyphenol promiscuity, I am sure there are a multitude of effects that could be altering PKS-theta phosphorylation independent of direct inhibition.
    Look how many targets have come up with EGCG and the like as a ‘direct inhibitor’ and yet any real interaction is tenuous at best and can’t be delineated from these off-target effects.
    As many people say, Derek included, a tool/probe compound should be even more specific than a leading drug candidate and that just isn’t the case for compounds like this.

  21. lynn says:

    Some of us biologists have been preaching this for a long time. Just because your enzyme is inhibited in vitro, you have to prove its selectivity, especially in vivo [or in bacterio as the case may be]. I have seen this error both in drug discovery and probe identification in papers (and grants) written by chemists and enzymologists. I start from the assumption that any small molecule can have multiple effects/targets unless proven otherwise.

  22. anonymous says:

    @19: It’s well established now that rottlerin does NOT directly inhibit the PKC family.
    It does inhibit (albeit weakly) PLK1 and CHK1.
    Don’t make excuses for using a bad compound. If you want to look at PKC-theta inhibition, at least use something that is known to be a direct inhibitor. And yes, there are compounds commercially available that are validated to be direct inhibitors. They may not be selective, but at least they actually inhibit the target.

  23. anonymous says:

    re: @13 & 19. A quick caveat re: use of siRNA, even when properly controlled. Due to the scaffolding potential of proteins/kinases, biologies noted following kinase reduction/ablation with siRNA may not mirror the pharmacology which may arise with a (hopefully) selective small molecule kinase inhibitors.

  24. JAB says:

    Any compound with that many phenolics is bound to bind to a lot of targets. I’d run away from it pretty quickly…

  25. Anonymous says:

    @22
    I put inhibit in quotes for a reason and I did not say that rottlerin directly inhibits the kinase activity of PKC-theta. If rottlerin has an effect on our system – we call it a rottlerin-mediated effect. Then we may decide to look at PKC-theta more direclty.
    @23
    We are actually more interested in PKC-theta as a scaffold as you suggest. Kinase dead mutants of PKC-theta are still very biologically active.

  26. Paul Brookes says:

    One thing it does appear to do (based on some nice work from Sellke and Clements at Rhode Island Med Ctr.) is activate BK channels of the Slo1 isotype. The distinguishing feature of that work, is they controlled it using knockout mice, wherein the effect was absent. They’ve sought to repurpose the compound as a specific Slo1 activator, and it does appear to elicit cardio-protection in animal models of ischemic injury, but I suspect the same limitations that applied to the compound in its former PKC life will probably hold true here as well. If something’s non-specific then it’s likely to be non-specific for a lot of things.

  27. YG says:

    Agree with 23.
    It’s always a dilemma. Use as many as ‘reportedly’ selectively inhibitors, and a nice cocktails of siRNAs in parallel, and hope the results come together. Yet these are completely different routes to block a target. even when you see different results, can you confidently say which is true, which is not?
    Ironically, academia world does kill creativity, in a certain way.

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