Here’s a paper that will give many medicinal chemists a shiver of recognition. A group from Dundee reports a screening hit against the enzyme Ube2T, a ubiquitin-conjugating (E2) enzyme. For those outside the field, ubiquitin is indeed ubiquitous. It’s a short bit of protein that gets hung onto other proteins (or removed, if it’s there already), a process that mostly happens on exposed lysine amino acids. Ubiquitination has evolved into a massive signaling system in living cells, with whole biochemical pathways set up to sense whether a protein has been so tagged, where the ubiquitin has been added, and how many times. There are three stages involved in just the adding-ubiquitin stage of things, catalyzed by E1, E2, and E3 enzymes, and all of these are under investigation, as are the de-ubiquitinating pathways on the other side of the process.
Polyubiquitinated proteins are almost always marked for the trash heap – it’s a biochemical marker for them to disappear into the proteasome to be broken up for amino acid scrap and recycled. Right now, a very hot topic indeed in chemical biology and early-stage drug research is the attempt to hijack and control that garbage-hauling machinery. We’d like to be able to target a given protein for the shredder before its time (especially proteins that we haven’t found any other way to shut down), or keep one around when it would otherwise disappear. Doing this at will and selectively is not quite a real thing yet, although some very interesting examples have appeared, but a lot of effort is going into making it one.
The reason Ube2T shows up as a target is because it’s overexpressed in many tumor lines, and is part of a DNA repair pathway. Interfering with DNA repair in general is a bad idea, since that would probably lead to tumor formation, but doing it temporarily would also make the fast-replicating tumor cells more vulnerable to radiation and some types of chemotherapy, and several of these pathways have been pursued. Targeting an E2 ubiquitin-conjugating enzyme is a tough way to do it (few inhibitors are known for that class), but the Dundee group had already reported discovery of an allosteric pocket on the Ube2T enzyme, and had run a fragment screen to find chemical matter for it. So there’s your disease, your target, your screen, and your small-molecule hits – the medicinal chemist’s equivalent of “All I ask is a tall ship and a star to steer her by”. Time to go earn that salary!
That’s exactly what everyone was trying to do in this latest paper. Their lead compound from the fragment screen was a hit in several orthogonal assays, showing reversible dose-dependent effects on Ube2T. NMR, DSF, ITC – these three-letter acronyms will gladden the heart of any early-stage drug discovery person, because normally you’d expect any compound that shows activity in all three of these to be the real thing. The group set up crystallization experiments with the compound and the protein (in just that try-all-sorts-of-things-at-once style that I referred to yesterday!), and while those were going, they bought or synthesized a series of analogs.
There the worries began. Ideally, you’d want to see the beginnings of a real SAR (structure-activity relationship) – this change did nothing, this one made it worse, this one made it better. But everything they did to the compound wiped out the binding. That sort of flat SAR is not a good sign – there are real singleton compounds out there, for which every change is a bad one. But this is also the sign of a false positive, where something different is going on than what you think.
And so it proved, sadly. They did get crystals suitable for X-ray, which showed extensive structural rearrangement of the protein. But what it didn’t show was any of the small molecule hit in there. Looking at the catalytic cysteine, though, there was a strong electron density peak, a solo atom that diffracted plenty of X-rays. . .and it turned out to be a zinc atom. The team went back and ran their nice-looking ITC experiments in the presence of the classic metal chelator, EDTA, and found that it totally abolished any activity. The starting compound was contaminated with zinc, and that’s where all the activity was coming from.
I have been down this exact same road, exactly twenty years ago. Fortunately, we didn’t get as far as letting an X-ray crystal structure tell us the bad news. We had a hit for a phosphatase enzyme (an unlikely event), but none of the analogs that we bought or synthesize showed any activity at all. I ordered up another sample of the original powder, and at the same time had someone in the group synthesize a fresh batch of it. The freshly made stuff had no activity at all – and the library sample turned out, by elemental analysis, to have a goodly amount of zinc in it. That was, of course, the source of all activity in the enzyme assay.
Always check the purity of your screening hits, folks. In this case, the NMR and mass spec came out fine for the Dundee group, which is the sort of thing that will lead you down the path. More recently, I had another situation like this, in which a compound checked out by LC/MS and NMR, but had flat SAR and batch-to-batch variability. Turned out that if you ran a bit of the sample through a silica gel plug, it looked the same analytically, but now was totally dead in the assay. A tiny amount of purple stuff was stuck to the very top layer of that silica – and that junk (which was some sort of oxidative polymer) was what was lighting up the assay. Always check the purity, and then check it again.