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.
10 comments:
Hi Dan, the issue seems primarily about purity of samples and I'd have thought that MS would have revealed the presence of zinc. Perhaps a 'metal check' by MS for all test samples could be recommended in screening campaigns? It's worth remembering that paramagnetic cations can often be 'seen' by NMR.
Just to note that Zn++ is not paramagnetic but other metals used in synthetic chemistry such as copper and iron do have paramagnetic ions.
Hi Pete,
Thanks for your comments. You are correct in theory that zinc could be detected by MS, but with an atomic weight of 65 and a doubly positive charge the m/z is well below the lower limit used in most analyses.
In SPR, Rmax is proportional to Mw. Is that not the case for BLI? As it is Zn that binds, and not the fragment, a surprisingly low Rmax would be observed. Especially as saturation is achieved cf the high affinity.
This story ring a bell! From experience, I would have either synthesize a new batch of the hit either at the beginning or after the lack of SAR. Having a defined process for hit validation is critical in drug discovery. But if you have the budget for obtaining crystal structure...
Fairly certain we'd have seen the Zn by native MS. This is quite a small protein. And despite the lower mass accuracy with native MS the mass difference is signficant even without using the highest spec. instrument.
The BLI traces shown in Supplementary Figure 1 were clearly suspicious. There was no saturation + exceptionally high Rmax, it was more indicative of protein unfolding rather than proper binding. In addition to that it was destabilising in DSF; two strong reasons to discard such hit. But this doesn't look well in PhD theses or grant applications...
Pro tip: Add 1 mM EDTA to your screening buffer
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