Inter-ligand STD NMR: Better than ILOE?

Although our poll in 2019 suggested that crystallography has surpassed NMR in FBLD, not all proteins can be crystallized. Ligand-detected methods such as saturation transfer difference (STD) NMR can be particularly useful for quickly identifying individual fragment binders and getting some sense of how they bind. A new variation published (open access) in Pharmaceuticals by Jesus Angulo and collaborators at University of East Anglia and Universidad de Sevilla provides information on the relative binding modes of two ligands.

 

Long-time readers may remember the inter-ligand NOE method (ILOE) we wrote about in 2010, in which proximity of two ligands is assessed by measuring NOE signals between them. However, despite being described more than 20 years ago, the technique seems to be rarely used, with fewer than a dozen papers in Pubmed, perhaps because ILOE requires large amounts of both protein and NMR time.

 

The new method is called inter-ligand STD NMR (IL-STD NMR), and it was discovered serendipitously while studying the binding of the drug naproxen to bovine serum albumin (BSA). As Teddy discussed several years ago, STD NMR normally involves irradiating specific protons in a protein (for example, the hydrogen atoms on buried methyl groups) and then measuring the “transfer” of this magnetization to bound ligands. When the researchers instead irradiated protons on one end of naproxen, they found that while the STD effect fell along the length of the molecule as expected based on distance, the signal suddenly increased at the other end of the molecule. Naproxen is known to bind to three sites on BSA, and this increased signal was attributed to the proximity of two adjacently bound naproxen molecules.

 

Inspired by this observation, the researchers developed IL-STD NMR. The experiment requires two samples, with two NMR experiments on each. One sample contains the protein and ligand of interest, while the other sample also contains a “reporter ligand” with a known binding mode. For each sample, one NMR experiment is run with selective irradiation of protons on the protein, while the other is run using irradiation of the reporter ligand. Comparison of the spectra reveals which regions of the ligand of interest are near the reporter ligand. The researchers demonstrated that the method works using a model system they had previously studied, the cholera toxin subunit B (CTB), which binds two ligands at nearby sites.

 

Importantly, the time and amount of protein is considerably less than required for ILOE: in this case 2 hours of NMR time and 0.3 mg of protein compared with 88 hours (!) and 1.8 mg. Moreover, the experiment could be run on a 500 MHz NMR, which is a relatively common instrument.

 

IL-STD NMR does have limitations. The researchers note that it is important to avoid irradiating the ligand of interest while irradiating the reporter ligand. Also, the approach obviously only applies to proteins with two nearby pockets (or one larger pocket). Still, it does look interesting, and I could imagine it being used as part of a screening cascade to find candidate fragments for merging or linking. What do the NMR aficionados think?