Beware Greeks bearing gifts

The publication that I wanted to discuss was bought to my attention whilst reading Derek Lowe’s superb blog “in the Pipeline”. I would wholeheartedly recommend readers of this particular website to read the original blog discussion of the publication there. The link to in the Pipeline article is here and the link to the original article is as follows

In the publication a group at Dundee University were exploring a fragment based approach, against an enzyme which has a role in ubiquitin conjugating called Ube2T. The team had been successful in identifying a fragment hit in an earlier publication. In this first publication they had utilised differential scanning fluorimetry (otherwise known as a thermal shift assay) and followed up with biolayer interferometry, as an orthogonal technique to confirm primary hits from the DSF primary screen. To further generate confidence, ligand observed NMR was carried out.

From this work a specific fragment was chosen as the top hit and was then measured with 15N- labelled NMR and ITC measurements against which at this stage showed positive binding. At this point analogues were ordered around this fragment and this is where the project hit some rough water. None of the analogues seem to show any significant SAR, and the limited number of analogues that did bind were much weaker in potency than the original hit

In parallel, co-crystallization of the fragment against Ube2T was attempted. While this was unsuccessful in showing specific binding, it did highlight a rearrangement of the protein, and the presence of a metal ion close to the catalytic residue, this metal ion was then identified as Zn2+

As no zinc was present in the co-crystallization buffer system, the team investigated the original fragment hit compound, running a zincon colorimetric assay on the sample which was gave a positive result for the presence of zinc. The team re-ran the original ITC experiment with the fragment hit in the presence of EDTA (which should chelate all zinc present), this showed that all binding was lost. In summary, the fragment hit must have contained Zn2+ ions which was the cause of the activity.

Gareth 1

Figure A showing ITC binding experiment of Ube2T with the original fragment compound with and without EDTA present. ZnCl2 against Ube2T is also shown as a comparison.

Taken from: F. E. Morreale et al., Mind the Metal: A Fragment Library-Derived Zinc Impurity Binds the E2 Ubiquitin-Conjugating Enzyme Ube2T and Induces Structural Rearrangements. Journal of Medicinal Chemistry60, 8183–8191 (2017).

The authors did point out that this hit was from a commercial library and one of the actions they undertook was to ask for the QC data from the supplier, at the point when the related analogues of the fragment compound did not show any activity. This obviously did not highlight the presence of the Zn2+

I did wonder when reading this publication if a different screening cascade may have identified this type of false positive, before having to get to the step of a very labour intensive co-crystallization process. For example a cell based assay might by more resilient to the presence of metal ions. However with a fragment based project, this may not be possible due to the poor affinity of fragments and also obtaining an effective concentration within the reduced DMSO tolerance of a cell based format compared to a biochemical assay. The only recommendation for this specific case, would be carry out further purification of samples, and then re-assay. It would also appear that the data from commercial suppliers is lacking and maybe that can be changed.

The authors should be commended for this work and putting it in the public domain. Drug discovery is a hard, long and quite often an unsuccessful process, and anything we can do to reduce the time following red herrings the better.

Blog written by Gareth Williams



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