I remember reading Barry Sharpless’ big “click” chemistry paper in 2001, where he proposed the term for reactions that take place rapidly, selectively, and without any outside reagents, and proposed such techniques for the rapid assembly of diverse molecules. In the years since, the term has drifted away a bit at times to mean “any reaction that forms a triazole from an acetylene and an azide”, but the concept is still a good one. And no one could have predicted where it would lead, almost twenty years later.
For example, it is (from what I can see) perhaps the central enabling technology of chemical biology. The ability to put a small unreactive tag on a biomolecule or small organic species and then label it at will, in vitro or in vivo, with whatever you like as a coupling partner is like having magic powers. It’s led to a vast amount of new knowledge about the protein landscape in living systems and their handling of small molecules. The versatility of the coupling reaction allows it to fit perfectly with other powerful techniques such as fluorescence microscopy, mass spectrometry, and photoaffinity labeling, and the amount of information generated through such methods is literally more than we can take in. Several of the people who’ve developed such experiments have been mentioned as candidates for a Nobel prize, along with Sharpless himself (for his second one!), and I would have no problem with that whatsoever. (There’s no space in this post to even go into the applications of click reactions in other fields; it’s a long and interesting list all its own and it keeps growing every year).
But as for chemical biology, this paper will serve to illustrate the sorts of things that you can do. It’s looking at bromodomain ligands, a field that everyone seemed to pile into at once a few years ago when small-molecule chemical matter was found to bind to the lysine-binding pockets on the proteins themselves. The lysines that they bind to are the ones that dangle off of histone proteins in the nucleus, and the bromodomain proteins themselves are involved heavily in the intricate dance of chromatin packing, DNA unwinding, and the incredibly complex process by which genes are selectively transcribed. (As an aside, this recent paper in Science has an extraordinary new look at the details of chromatin packing, thanks to a new electron microscopy technique).
Unraveling just which bromodomain-containing proteins are doing what, and when, and for what reasons and just exactly where is a tall order, but those are just the sorts of details that have to be tracked down to understand them. (Not to mention that such an understanding would greatly assist trying to turn any of the small-molecule inhibitors into drugs – it would greatly decrease the finger-crossing and breath-holding that accompanies any transition into human trials). The paper referenced in the paragraph above shows how to go about doing that.
The authors (from a multicenter collaboration in France and Australia) took several of the reported bromodomain compounds and added short chemical tags to them to participate in click reactions – either the classic azide/alkyne, or another later combination, tetrazine/trans-cyclooctene (an exceptionally fast reaction developed by the Fox lab at Delaware). Cell assays showed that these modified ligands seemed to behave just the same as the parent compounds, which is a key step any time you do one of these add-a-tag experiments. After using a variation of the crosslinking assays for chromatin structure, they could localize the compounds to particular DNA sequences by attaching a reporter group to them via the “click” handles.
This showed some interesting patterns that hadn’t been worked out before. As it turns out, the bromodomain protein under study (BRD4) behaves quite differently around different types of genes. It was already known that adding a BRD4 inhibitor immediately downregulates some genes, while other BRD4-associated genes don’t show this effect. This experiement showed that of the ligand accumulates at the downregulated ones, and the hypothesis is that the BRD4 protein interacts with them through its first bromodomain, leaving its second one free to pick up ligand. Meanwhile, BRD4 binding at genes where an inhibitor doesn’t have much effect seems to take place by a bromodomain-independent mechanism, probably a set of protein-protein interactions that not only don’t involve the lysines on histone proteins, but seem to make the lysine-binding sites less accessible to ligands.
Meanwhile, at enhancer sites in the genome, BRD4 inhibitors seem to generally downregulate things. Looking at these, some BRD4 protein-associated enhancer regions also showed noticeably greater accumulation of labeled drug than others. In this case, the binding does always seem to be dependent on the bromodomain pockets, and the differences may well be that in the high-ligand-amount sites the second bromodomain is left open (as above) to pick up compounds, while at the other sites both bromodomains might be occupied by lysines at the same time. (Alternatively, it could also be that binding to one of them is taking place in a way that makes the other one inaccessible).
Two different bromodomain inhibitors (IBET-151 and JQ-1) showed exactly the same pattern of accumulation in these experiments, which argues strongly that the two of them are mechanistically pretty much identical. They were already known to show very similar effects in cellular assays, but it wasn’t clear if they were getting to those endpoints via the exact same route, or if somewhat different compound mechanisms were just ending up with the same readout.
Another thing you can do with labeled drugs of this sort is to quantify their locations inside different cells, at different points in the cell cycle, and in different tissues. You can do some of these experiments on a less detailed level with radiolabeled compounds, but fluorescence microscopy gives you a real advantage here, not to mention being much easier to handle from the facilities and regulatory standpoints. It turns out that localization of the bromodomain inhibitors to the nucleus isn’t affected by the phase of the cell cycle or how proliferative the cells are in general. In mixing experiments, flow cytometry could be used to pull out the ones that had been exposed to higher levels of compound (another advantage of fluorescence). You could try doing that with a drug that’s already had a fluorescent tag on it from the start, but it’s much harder to do that and not affect its properties. A small click handle has a better chance of slipping by (as it does here), and lets you add the fluorescent group at a time of your choosing.
These studies were used to shed some light on an effect that had been noticed before, that bromodomain inhibitors, when used in models of acute myeloid leukemia (AML), seemed to do a much better job clearing out the cancerous cells in circulating blood and in the spleen than the ones in the bone marrow. Using the fluorescence accumulation in the nuclei as a marker showed that cells from the spleen and bone marrow can take up the compound equally, but the ones in the spleen just seem to be getting exposed to a lot more drug, which makes sense from a pharmacokinetic standpoint, since the spleen (technically speaking!) is perfused to hell and gone and access to the bone marrow is more limited. (The figures I’ve seen would indicate that, at least under normal resting conditions, blood flow through the spleen is at least ten times greater, mL/min per weight of tissue).
In general, this type of compound labeling, followed up by either fluorescence or mass spec detection (depending on the label you’d like to use) lets you track levels of drug between different cells types, different tissues, and down to intracellular compartments in vivo or even particular locations in the genome, if you’re targeting that machinery (as these compounds do). That’s a level of detail that just didn’t exist not so long ago, and a lot of it is made possible by these bio-orthogonal coupling reactions. Couple that with advances in handling genomic data and in protein identification (such as modern mass spec proteomics) and you can routinely run experiments that would once have gotten you laughed out of the room for even bringing them up. The process continues, too – we may well look back on these as the early days and feel pity for our younger selves for having to work under such primitive conditions. But for now, we can enjoy what we have and make the most of it!