I’ve been meaning to write about this paper (open access) on some problems with chemical probes, and now’s a good time. There was a well-known article a few years ago about “The Promise and Peril of Chemical Probes”, and this is a deliberate follow-up, starting with its title. Even if you don’t give much of a hoot about chemical probes, the point it makes is worth keeping in mind.
All chemical probes have some off-target effects. Some of the things sold as such in the catalogs (and referenced in the literature, to this day) have so many of these that they’re worthless. In fact, they’re worse than worthless; they cause active confusion and harm. One way to validate your experiments with the more useful ones is to use a very closely related compound that doesn’t have the desired probe activity, in the hopes that it will still maintain the off-target binding activities. It would be nice if every chemical probe had a selection of these available, but that is not the world we find ourselves in.
And what this new paper says is that even some of the ones that people have been trusting are. . .not so great. For example, A-196 is a pretty well-validated probe for the SUV420H1 and SUV420H2 enzymes, which are specific lysine demethylases. A similar compound, A-197, is sold as a negative control for experimental validation. But if you profile the two compounds against a wide panel of receptors, enzymes, transport proteins and so on, you find that A-196 has six off-target activities on that list, and A-197 not only loses activity against SUV420H1, but it loses activity against five of those other targets as well.
The same goes for another epigenetic probe, NVS-MLLT-1. It has six off-target activities in the profile screen, but the compound that’s suggested as its negative control, NVS-MLLT-C, loses activity at five of them, too. To be sure, all of these six targets are being hit at low micromolar range or a bit below (depending on your assay technique), whereas the interaction with the probe target (interaction of histones with the the YEATS/MLLT proteins) is about tenfold more potent. But the recommendation is to use this one at 5 to 10 micromolar concentrations in cell assays, so depending on the tone of the off-target systems, you could still get misled.
Many of these negative control compounds change out a charged atom (such as a nitrogen) for something uncharged, like a straight carbon or a basic-nitrogen-to-amide switch. And the problem, which is going to be very hard to completely escape, is that these sorts of atoms are likely going to be involved in binding to those other targets as well, so you can’t get as clean a slice in the activity profile as you’d want. This paper only looks at four compounds (and their suggested controls), but tries to extend the reach computationally. The authors pulled data from the PDB on identical ligands bound to different protein sites, and then looked at the predicted effect of adding a methyl group to such a compound by systematically replacing every plausible hydrogen with a methyl.
An example of what happens is with the JAK2 inhibitor TG101209, which also binds BRD4. There are 25 possible methylation sites, and five of these would be predicted to knock out the JAK2 binding. But four of those would also be predicted to knock out BRD4 binding as well. So there’s a significant risk that plausible negative control compounds around TG101209 would be less useful than you would want, whether you’re approaching this from the JAK2-binding side of the story or the BRD4-binding side. The authors extended this to 41 other such pairs, using protein binding domains that were as dissimilar as possible, and found an average 50% chance of trouble with this methyl-substitution idea. That leads one to think that there’s generally a substantial chance of a given negative control compound modification wiping out on other unrelated targets as well.
What the paper ends up recommending is developing at least two chemically distinct probes for every target, and while that is going to involve a lot of work, I think that they’re right: that’s the only way that we’re going to be (reasonably) sure that we’re looking at what we think we are. They note the example of LLY507, a SMYD2 inhibitor probe that kills off glioblastoma cells. But before you run out and publish a paper on the importance of SMYD2 in glioblastoma (it’s another lysine methyltransferase for histones), or worse yet try to use such an inhibitor as a drug for the disease, you might want to know that a structurally different SMYD2 inhibitor, BAY-598, doesn’t show that cytotoxicity at all. So a selection of probes would be rather handy, and comparison of the negative controls for both such compounds would be, too. We have enough noise in the literature already, right?