Much of the early work of fragment screening involves avoiding artifacts. For high-concentration assays, compound purity is absolutely essential. However, this is not always easily assessed, as demonstrated in a recent paper in J. Med. Chem. by Alessio Ciulli, Helen Walden, and co-workers at the University of Dundee (see here for Derek Lowe’s discussion).
The researchers conducted a screen against Ube2T, a ubiquitin-conjugating enzyme involved in DNA repair and thus of interest as an anti-cancer target. About 1200 fragments were screened using both differential scanning fluorimetry (DSF) and biolayer interferometry (BLI). Most of the hits were quite weak (millimolar), but one showed low micromolar activity. Although this fragment was a destabilizer in the DSF assay, other destabilizers have turned out to be useful starting points.
Two-dimensional (HSQC) protein-detected NMR experiments suggested that the fragment binds near the catalytic cysteine residue, possibly with some protein rearrangement. The binding was reversible, as expected by the chemical structure of the fragment. The fragment was also active in a functional assay. Finally, isothermal titration calorimetry (ITC) revealed an impressively tight dissociation constant of 17.7 µM for the 16-atom fragment. All of these orthogonal assays suggested the researchers had a winning fragment on their hands, so they started acquiring and making analogs to further optimize the affinity. Then things went awry.
Of 14 molecules tested, some quite similar to the initial fragment, only two showed any activity, and these were way down. Concerned, the researchers examined the fragment itself by 1H and 13C NMR as well as high-resolution mass spectrometry, all of which revealed that the compound had the desired structure and appeared to be quite pure (not necessarily a given!) So what the heck was going on?
The mystery was finally resolved, after considerable effort, by a co-crystal structure of the fragment with the protein. Unlike previous structures of Ube2T, this one revealed an unusual domain-swapped architecture, in which a domain of one Ube2T protein interacts with a different molecule of Ube2T rather than with the rest of its own protein. More alarmingly, there was no electron density for the expected fragment, but there was a small, strong area of density connected to the catalytic cysteine residue. The researchers speculated that this could be a zinc ion, and sure enough, zinc chloride itself turned out to have essentially the same affinity for the protein as judged by ITC. Adding the zinc chelator EDTA to the fragment abolished activity, and a colorimetric probe revealed the presence of zinc in the original fragment as well as – to a lesser extent – the two active analogs.
Metal contamination is actually not uncommon – we mentioned a case where residual silver accounted for the apparent activity of many HTS hits. Enzymes with an active-site cysteine are particularly susceptible.
This type of artifact is particularly insidious because it is so difficult to discover. In this case, it was uninterpretable SAR that made the researchers suspicious, and crystallography that revealed the culprit. But SAR can be wonky, and crystallography often fails. What else can be done? Elemental analysis could have helped, but people usually only turn to this if they’re already suspicious.
Of the various fragment-finding methods, I think the only two besides crystallography that could have given warning are native mass spectrometry (MS) and ligand-detected NMR. The former is relatively specialized and doesn’t work for all targets, but it would be interesting to know whether standard NMR techniques such as STD, WaterLOGSY, or CPMG would have revealed that the initial fragment was not binding. Of course, there can be all sorts of reasons for negative results. Publications like this one are useful reminders that simply ignoring such data is unwise.