Among the many biophysical
techniques presented at the recent Novalix meeting, hydrogen/deuterium exchange
mass spectrometry (HDX-MS) was mentioned only a few times. Practical
Fragments last covered it nearly four years ago in the context of a paper
that described theoretical challenges and improvements to the method for
assessing fragment binding modes. A new open-access Comm. Chem. paper
from Tiago Bandeiras, Alessio Bortoluzzi, and collaborators at iBET-Instituto
de Biologia Experimental e Tecnológica and Merck KGaA implements some of these
suggestions.
The researchers find only two
examples where HDX-MS had been applied to fragments, one of which we covered
here. The new paper focuses mostly on Cyclophilin D (CypD), a mitochondrial
protein implicated in several diseases. A screen we wrote about in 2020
described several fragment hits, some of which were crystallographically found
to bind in three overlapping sites designated the proline pocket, the aniline
pocket, and the pyrazolo pocket. The
researchers chose a binder from each pocket to study as well as two more fragments
whose binding sites were not known. All five fragments are extremely weak hits,
with at best 7 mM (yes, millimolar) affinity as assessed by SPR, making this a
particularly challenging test case.
As a reminder, HDX-MS examines
the exchange of deuterium from D2O to protein backbone amides. Nearby
ligands can slow this exchange, and mapping the locations and magnitude of
these changes reveals where the ligand binds. Optimization experiments were
initially run on a compound with a KD of 44 mM for the aniline
pocket. Three protein concentrations were tested. Since the highest (10 µM)
yielded the highest number of detected peptides, it was used for subsequent
experiments.
As suggested in the 2022 paper, compound
was added both to the initial protein solution as well as to the deuterium
exchange buffer. The fragment was tested at 2.5, 5, and 10 mM, and changes in
deuterium uptake were found in all cases, which is remarkable given that the
theoretical occupancies range from 5 to 18%. An experiment using
substoichiometric concentrations of a high affinity ligand confirmed that 18%
protein occupancy is sufficient to generate a reliable signal.
Still, given the low occupancy,
the researchers used statistical methods to ensure that changes in signals were
significant. Mapping those that were onto the structure of the protein
confirmed that the fragment bound to the aniline pocket.
A second fragment was tested by
HDX-MS, and the results confirmed that it binds in the pyrazolo pocket, as
previously shown by crystallography. However, for a third fragment that had
been shown to bind in the proline pocket, the results suggested instead that it
binds in the aniline pocket. The proline pocket exhibited very low levels of
deuterium exchange, but when the researchers increased the pH from 7.4 to 9
they did find evidence that the fragment binds here as well.
Next, the researchers turned to
the two fragments with unknown binding sites. HDX-MS revealed that these bind
to the aniline binding site, though with subtly different protection patterns
suggesting that they bind in slightly different regions of the pocket.
Finally, the researchers used
their optimized HDX-MS conditions on a previously identified fragment that
binds the kinase FAK with low millimolar affinity. This showed that it binds in
the so-called hinge region, in agreement with crystallography. This fragment
was also used as a negative control for CypD, showing that it does not bind.
This paper is a nice resource for those
hoping to apply HDX-MS to fragments. The fact that the binding sites of such
weak binders can be determined is quite remarkable. That said, the resolution
is not as good as crystallography or protein-detected NMR; for FAK in
particular, the ligand reduces deuterium update across a large fraction of the
protein surface. And the fact that a proline-pocket binder was initially mapped
to the aniline pocket also gives one pause. Perhaps the binding mode in
solution really is different from that found crystallographically. It would be
interesting to see whether NMR can resolve the conundrum.
Posts
