15 February 2016

Selectivity in STD

Among NMR-based fragment screening methods, saturation transfer difference (STD) came in as most popular in a recent poll. The technique is very sensitive and thus able to identify weak fragments. Unfortunately, it’s a bit too sensitive; hit rates of >30% are not uncommon. Many of these hits interact non-specifically with the protein. These can be weeded out using orthogonal screening methods or competition assays, but it would be nice to make STD itself more discriminating. In a paper published late last year in J. Med. Chem., Olivier Cala and Isabelle Krimm describe how to do just this.

In an STD experiment, the protein target is irradiated and transfers some of its magnetization to bound ligands. When these dissociate they retain some of the magnetization, and so the NMR signals of the fragments decrease. The problem is that lots of fragments interact non-specifically with proteins. For example, if a lipophilic fragment dances across greasy patches on a protein surface to escape from water without making any specific interactions, it will still get magnetized. Can such signals be distinguished from fragments that bind in a single, well-defined manner?

Within a ligand that binds to a protein, a proton that binds closer to the protein will show a stronger STD effect than one that is exposed to solvent. This is in fact the basis for STD “epitope mapping”, which allows one to roughly model how a ligand binds to a protein – or at least which parts of a ligand are closest to the protein. In the new paper, the researchers argue that simply observing differences in the STD effect between different protons in a ligand can distinguish whether or not that ligand is binding in a single binding mode.

Several examples support this assertion. For one protein, all the fragments that showed significant epitope mapping could be competed with a known reference molecule, suggesting binding to a specific site; this was less often the case for fragments that did not show epitope mapping. In another example, the privileged fragment 7-azaindole was found to bind to two different proteins with different epitope maps, suggesting different (but specific) binding modes for each protein. The technique also seems fairly robust to the affinity of the fragments (KD 50 ┬ÁM to > 1 mM), the details of the NMR experiment (saturation time from 0.5 to 4 seconds), and ligand/protein ratios between 66 to 1 and 400 to 1.

As the researchers note, there are caveats. For example, if a fragment can bind in two different but nonetheless specific binding modes, it may show uniform STD effects and will be a false negative. Nonetheless, comparing STD effects across a ligand does seem a worthwhile exercise. Not only could it help prioritize fragments, it could also reveal which protons are further from the protein, and so suggest growth vectors.


Darren W. Begley said...

Is it not also possible for a fragment to bind in a single, specific mode, in which all protons are equally close to the surface of the protein? I have seen this experimentally, for fragments proven to bind specifically to (at least) one binding site on a protein (i.e. XRD). Also, think of the number of fragments with only 2 or 3 non-exchangeable protons, for which this can be the case. These are among the caveats for why the STD-NMR epitope mapping approach does not work in all cases (i.e. for all small molecule-macromolecule) pairs.

Anonymous said...

std is notoriously sensitive to cross relaxation effects..perhaps reverse noe pumping or epitope mapping by diffusion nmr would be better?