When you take an NSAID (naproxen, ibuprofen, aspirin, etc.), how does it work? This is one of those questions that improves on further inspection – or deteriorates, according to your point of view, because it just keeps on getting more complicated. For decades, there was no good answer at all, but then there was “It reduces signaling in the inflammation pathways”, followed by “OK, it seems to do that by decreasing this prostaglandin signaling molecule” and “Ah, it does that by inhibiting cyclooxygenase, a key enzyme in the pathway to make those”. Then we moved to “Hold it, there’s more than one cyclooxygenase”, which is how we ended up with the COX-2 inhibitors like Vioxx, which led to “You know, the functions of these two are more complicated than we even thought”.
And on top of all these, there are clearly effects of NSAID drugs that have nothing to do with the COX enzymes at all. Blood clotting you can pretty much chalk up to the COX isoforms, but there are more things going on, and they’re very poorly defined. Which brings up a more general question: how do you ever know about all the things that a small-molecule drug might be doing in a living system? The answer is, you don’t. That’s not generally appreciated outside the biomedical professions, but it’s the truth. We don’t have some way to track things around and watch them interact at a molecule level through out a cell or an organism.
Well, maybe. Mapping compound-protein interactions is a big part of chemical biology, and this new paper from the Woo lab at Harvard shows an attempt to track small molecules in just this way. It uses photoaffinity labeling, via the now-nearly-standard combination tag of a diazirine and an acetylene, which lets you form a covalent bond to (most) of the thing the molecule of interest is spending time next to, and on the back end of the process, it uses isotope-labeled tags on the acetylene-click-conjugation step to give the mass spec detection a much higher signal/noise. Just digging through the whole cellular proteome without the kind of validation that isotope tagging gives you is a tall order, but the deliberately-weird-mass-combination effect of the stable isotope tags moves things up out of the noise.
Three NSAIDs are given the treatment: naproxen, celecoxib (Celebrex), and indomethacin. It’s a good spread of activity: naproxen hits both COX-1 and COX-2 pretty much equally, celecoxib is of course a COX-2 selective compound, and indomethacin is in a structurally distinct class that’s already known to have some other modes of action. (Update: here’s work from another group on an alkyne-tagged aspirin derivative, which also interacts with a long list of cellular proteins). All the photolabel-derivatized compounds were still active against COX enzymes (albeit with somewhat lower affinity), and all of them could be displaced by the parent compounds in competition assays. All of them photo-labeled COX-2 in an in vitro experiment, as they certainly should, and this experiment was used to validate the mass spec analysis methods for the whole-cell experiments. (For example, each of these compounds produces six or seven different photoadducts, depending on which nearby amino acid gets snagged by the reactive carbene that forms from the diazirene, and the group was able to see and account for all of these without difficulty).
So what happens in cells? They tried the dosing/photolabeling experiment in Jurkat cells, along with experiments with photoactive negative control (non-NSAID) compounds. About 700 proteins were labeled in all, with quite a bit of overlap between the three NSAIDs (40% of them are hit by all three), and that figure alone should make a person stop and think a bit: as simple and widely used a molecule as naproxen binds to hundreds of proteins well enough to photolabel them. No, we don’t know the details of what’s going on in there, do we? Admittedly, that’s at a high concentration (250 micromolar), but (1) patients take whopping doses of this stuff in the real world, and (2) the majority of these interactions also showed up at a 50 micromolar dose as well.
This same set of experiments was also run in K562 cells, a leukemia line. In this case, there were 513 enriched protein targets and only 206 of them overlapped with the set from the Jurkat cells. And that’s also something to think about – not only do small, well-known compounds like these interact with a long list of proteins, that list can change dramatically in different cell backgrounds. Only about 30% of the total list of proteins have ever been report to interact with any small molecules at all.
The distribution of these doesn’t show any particular compartmentalization effects in the cells; it pretty much tracks protein abundance across the cytoplasm, various organelles, nucleus and so on. A closer look at the proteins and their interaction sites can be found in the paper, but one thing worth mentioning is a “hot spot” in histone H2A, which is certainly not a target that anyone had been thinking about for NSAIDs, to my knowledge. Assigning all the specific sites of labeling across the proteome is still beyond current technology, although you can think of some experiments that would help narrow things down a lot (different protein digestion before the mass spec, and so on). But even at this level – which is more detailed than we’ve ever had for such compounds – there’s a lot to deal with here.
So if you needed a reminder about just what a complicated mixing bowl we’re throwing our compounds into, here you go. The hope is that such techniques and the ungodly huge piles of data that they generate will help up build up a clearer picture of drug action. But what a picture that’s going to be!