Cyclin-dependent kinase (CDKs) have been drug targets for quite a while now. There are 20 different ones, and they help to regulate a whole list of important functions, particularly involving the cell cycle (which has made them of great interest in oncology research). There are three approved drugs in the area so far: Kisqali (ribociclib), Ibrance (palbociclib), and Verzenio (abemaciclib). Here’s a 2014 post on that second one and its history. All of these are CDK4/6 inhibitors, approved (thus far) as part of treatment regimes for breast cancer. These CDK subtypes are involved in the “checkpoint” after the G1 phase of the cell cycle (which requires phosphorylation of the Rb protein), and inhibiting them keeps this from taking place and interrupts cell division.
That’s the CDK area that we know the most about, and have the most tools to address (CDK1 and CDK2 also have a good amount of work done on them). They’re a pretty complex group, though, and many of their functions are still being worked out. The usual complications are there: the same enzyme subtype can be performing several quite different functions on different substrates in the cell, for example. Alternatively, sometimes you find things that look different at first but can be traced back to new functions of the same substrate protein. The CDK family is evolutionarily rather conserved (you can swap the human forms into yeast and they seem to do just fine, for example), so they’re not something to be messed around with lightly.
But about those tools. . .here’s a new paper that takes a look at the reported compounds, and finds some confusion. The authors (a Czech team) note that there are around 100 inhibitor compounds out there with reported activity, but (as is so often the case) taking those reports at face value is not recommended. They’ve evaluated a list of compounds that needed to be better characterized against CDKs 1, 2, 4 ,5, 7, and 9 in enzyme assays, and in cell assays as well.
As it turns out, there are a lot of mischaracterized compounds out there. CGP74514A, for example, is usually described as a CDK1 inhibitor, but it’s more potent against CDK2 (and shows activity against plenty of other kinases besides). Benfluorene and elbfluorene are available from several prominent vendors as selective CDK1 inhibitors, too, but this group was unable to confirm any CDK activity against any subtype, and they don’t seem to do much of anything to HCT116 cells, either (!) The most suitable CDK1 compound, if you’re in need of one, appears to be RO3306.
The pattern continues on the other subtype compounds. NU6140 is sold cheerfully by many vendors as a CDK2 inhibitor, but this work shows that it’s more properly described as an Aurora kinase inhibitor, which will make your CDK2-based conclusions hard to stand by. And commercially available CVT313, which the catalogs say is a “selective and potent” CDK2 compound, is actually a dual targeting CDK2/5 inhibitor. And you can buy GW8510 as a CDK2 inhibitor as well, but you’d better not: it’s actually a CDK 1/2/5 compound, hits other things besides, and if you’d looked at chemicalprobes.org you could know these things before getting tangled up in trouble. Sigma-Aldrich might want to consider updating its catalog. In the end, the authors find that none of the commercial “selective CDK2 inhibitors” are suitable for cellular experiments.
Now, if you want to inhibit the CDK4/6 types, you’re in luck. As mentioned above, these have marketed drugs against them, and the companies involved have put in the work to show that they do what it says on the label. You can buy other compounds in this area, but it’s not a good idea: NSC625987, for example, is reported as a CDK4 inhibitor, but the Czech group report that they can’t get it to show any activity at all (up to 20 micromolar) against any of their CDK subtypes, and they note that this lack of activity has been reported before. But that’s not slowing down Tocris, EMDMillipore, Sigma-Aldrich, Santa Cruz, Axon, Fisher, Apex, Probechem and several others. They all have it listed as a selective CDK4 compound, and let the buyer beware.
The paper has plenty of further information, but the authors have compiled their results into a publicly accessible database to help out researchers in the field. If you are doing CDK-dependent assay work, you should absolutely have a look – there might be some unpleasant surprises waiting for you, but the time to find out is now. Overall, the lessons are clear: just because that’s what the vendor says it does, doesn’t make it so. Just because the literature says that’s what a compound does, doesn’t make it so. And just because others have used this as a control compound doesn’t necessarily mean that you should, too. Check it out.
You may think that “due diligence” is a phrase from the business world, but that is far from true. Every time you pick up on an assay, a probe compound, a cell line, what have you from the literature or a catalog, it is incumbent on you to do the homework and make sure that you’re using what you think you’re using. Make sure it’s what it says on the label. Then even if it is, make sure to the best of your ability that it’s going to do what you brought it in to do. Otherwise, you are inviting the laziest, sloppiest links in the whole chain to do their worst to you and your research. Why would you allow that to happen?