A bright idea for rapid affinity measurements

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 K_D values in a method called CIDNP-K_D.

 

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-K_D 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-K_D, 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-K_D is limited to relatively weak binders (K_D > 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.