Finding fragments that bind to a
target is important but so is measuring their affinities. NMR methods can find even weak fragments, but accurately assessing affinities
takes time. In a recent (open-access) J. Am. Chem. Soc. paper, Felix
Torres, Roland Riek, and collaborators at the Institute for Molecular and
Physical Science and NexMR provide a new, fast method.
The approach is based on photochemically
induced dynamic nuclear polarization (photo-CIDNP), which we wrote about here;
Felix also spoke about it at the FBDD-DU meeting in June. As the name
implies, the technique involves illuminating NMR samples to electronically
excite ligands, thus increasing the signal to noise ratio of the NMR signal by as much as 100-fold. Previous
work focused on using the method to identify binders, even with cheap, benchtop
NMR instruments.
The new paper describes how to quantitatively
measure dissociation constants using photo-CIDNP. The theory gets a bit hairy,
but the basic idea is that the more photochemically excited ligand that binds
to the protein, the more the signal decreases. A series of samples are prepared
with increasing concentrations of ligand and either no protein or a fixed concentration
of protein. After measuring the NMR signals, the data are plugged into equations
to derive the KD values in a method called CIDNP-KD.
As the researchers have
previously noted, not every ligand can be photosensitized. However, dissociation
constants can still be measured for these using competition experiments with previously
characterized reporter ligands that can polarized, akin to using to NMR competition
studies with 19F reporter ligands (see here).
So how well does the technique
work? The researchers first turned to the PDZ2 domain of a phosphatase called
hPTP1E, which is involved in cell proliferation. They measured the affinities
of a series of peptides having 4 to 8 amino acid residues and compared these values
to those obtained using two dimensional [1H,15N]-HSQC
chemical shift perturbation, the gold standard NMR technique. Affinities ranged
from low micromolar to low millimolar, and there was reasonable agreement
(generally within about two-fold) between both techniques. Most of the peptides
contained tryptophan, which is suitable for photo-CIDNP, but CIDNP-KD
also worked in competition mode when non-tryptophan containing peptides were
competed against peptides containing tryptophan. And the technique was fast,
with each datapoint taking only 30 seconds for photo-CIDNP compared to as long
as 80 minutes for HSQC NMR.
Next the researchers turned to fragments.
They had previously conducted a screen against the oncology target PIN1 and
identified a number of fragment hits, two of which had been characterized in
detail. The affinities of these were measured by CIDNP-KD, and the
low millimolar values agreed with those from HSQC NMR.
Another neat application described
in the paper is “CIDNP-based epitope mapping,” which is based on the fact that
an excited proton on a ligand that is in close proximity to the protein will
relax more rapidly than one that is distant from the protein. This phenomenon
is similar to STD epitope mapping, and the two methods yielded similar
information for the two PIN1 ligands: one region of each molecule was buried in
the protein, consistent with crystal structures.
One drawback of the technique is
that, because measurements require fast protein-ligand exchange, CIDNP-KD
is limited to relatively weak binders (KD > 10 µM), but this is
usually not a problem in the early stages of a fragment program. A full affinity
measurement takes about 15 minutes, which compares very favorably to two hours
using [1H,15N]-HSQC and without the need for isotopically
labeled protein. It would be interesting to run head-to-head comparisons with two
ligand-based NMR techniques we wrote about last year, imaging STD NMR and R2KD,
to see how they compare in terms of speed, accuracy, and generality. Please let
us know if you’ve done so.
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