Native electrospray ionization mass spectrometry (ESI-MS) is
one of the less-commonly used fragment finding methods. The technique relies on
gently ionizing a protein-fragment complex without causing denaturation; bound fragments
reveal themselves as shifts in mass. The technique is truly label-free, and can
use very small amounts of protein and fragments. In practice the technique can
work really well, reasonably well, or quite poorly. Two new papers shed light
on factors that influence success.
The first paper, by Kevin Pagel (Freie Universität Berlin),
Benno Kuropka (Bayer), and collaborators, examines four different
cancer-related proteins. Let me say up-front that that the paper is remiss in
not disclosing the chemical structures of any of the fragments, so in a very real
sense this work is not reproducible. It is a shame the editors of ChemMedChem were not more demanding.
That said, there is some useful information here.
Most of the focus is on the protein MTH1, screened at 10 µM
concentration with 100 µM of each fragment. This was not a naïve screen; the
fragments were previously identified from a thermal shift assay (TSA): 24
stabilized the protein, 4 destabilized it, and 5 had no effect. Remarkably, all
of the fragments showed complexes in ESI-MS ranging between 6 – 66%, even those
that had no effect in the TSA! Choosing an (admittedly arbitrary) 20% cutoff
weeded out most of the false positives: 16 of the 24 stabilizers passed, while
none of the destabilizers or neutral molecules did.
The best hit by ESI-MS also gave the strongest thermal shift,
and a titration curve revealed an impressive dissociation constant of 1.7 µM.
However, even at high concentrations of fragment the amount of bound complex
did not exceed 70%, meaning that interpretation of single-dose experiments (for
example, from a primary screen) could be problematic.
The results were similar for the protein KDM5B: 8 of 9
stabilizing fragments were hits by ESI-MS, as were two of 7 destabilizing
fragments. Note that fragments that destabilize proteins can still be tight
binders, as illustrated here.
For two additional proteins, however, ESI-MS was
disappointing. For BRPF1, ESI-MS didn’t find any of the 11 hits from TSA, while
for UHRF1 it found only a single hit – though this hit was not one of the 10
stabilizers identified by TSA. One could argue that the TSA hits were false
positives were it not for the fact that, in the case of BRPF1, 6 of them were
confirmed by crystallography.
The second paper, in Angew.
Chem., comes from Chris Abell and coworkers at the University of Cambridge,
and focuses on the protein EthR, a potential target for tuberculosis that we’ve
previously discussed.
EthR binds to DNA, so rather than look for direct binding of
fragments to EthR the researchers instead looked for fragments that could
disrupt the EthR-DNA complex. A small library of 73 fragments was tested (at
0.5 mM each, in 2% DMSO), yielding 8 hits. The same library was screened under
the same conditions using differential scanning fluorimetry (DSF), yielding 7 hits, 4
of which had also been identified using ESI-MS. All 11 of these molecules were
then tested under the same conditions in an SPR assay to see if they could
disrupt the interaction between EthR and chip-bound DNA. The 7 best SPR hits
were all fragments that had been identified by ESI-MS. Moreover, two fragments
– one identified solely by ESI-MS and one identified by both ESI-MS and DSF –
were characterized bound to EthR crystallographically, and these represent new
chemotypes for this target.
So what are we to make of all this? In common with other
techniques, ESI-MS works well for some targets and less well for others. The
problem is that it is not clear what distinguishes the two classes of targets.
If you have access to the equipment and expertise you might consider adding
ESI-MS to your screening cascade. But if you can only afford to buy one instrument
for fragment screening, you’d probably be better off investing in NMR or SPR.
2 comments:
You tell us there is no one method that is a clear winner for all targets; so I was with you until you got to your "solution". The best solution may not be to lock yourself into one technology because you can only afford one but to pick & choose among the many fragment screening service providers.
Nice post, and thank you for the mention!
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