Historically the most popular method for finding fragments has been ligand-detected NMR. Preliminary results of our current poll (to the right) suggest crystallography has pulled ahead. (Please do vote if you haven’t already done so.) However, NMR has many uses beyond finding fragments, as illustrated in a recent J. Med. Chem. paper by Sacha Larda, Steven LaPlante, and colleagues at INRS-Centre Armand-Frappier Santé Biotechnologie, NMX, and Harvard.
Among the many artifacts that can occur in screening for small molecules, one of the most insidious is aggregation. A distrubing number of small molecules form aggregates in water, and these aggregates give false positives in multiple assays. Unfortunately, determining whether aggregation is occurring is not always straightforward. The new paper provides a simple NMR-based tool to do just that.
All molecules tumble in solution, but small fragment- or drug-sized molecules tumble more rapidly than large molecules such as proteins. The “relaxation” of proton resonances is faster in slower tumbling molecules, and in the NMR experiment called spin-spin relaxation Carr-Purcell-Meiboom-Gill (T2-CPMG) various delays are introduced and slower tumbling molecules show loss of resonances. Indeed, this technique has frequently been used in fragment screening: if a fragment binds to a protein, it will tumble more slowly, resulting in loss of signal.
The researchers recognized that an aggregate could behave like a large molecule, and they confirmed this to be the case for known aggregators, while non-aggregators did not. The experiment is relatively rapid (~30 seconds), and has been used to profile a 5000-compound library to remove aggregators.
One of the frustrations of aggregators is that it is currently impossible to predict whether a molecule will aggregate, and indeed, the researchers show several examples of closely related compounds in which one is an aggregator while the other is not. Even worse, the phenomenon can be buffer-dependent: the researchers show a fragment that aggregates in one buffer but not in another, even under the same pH.
Many fragment screens are done with pools of compounds, and the researchers find that molecules can show a “bad apple effect”, whereby previously well-behaved molecules appear to be recruited to aggregates.
The limit of detection for T2-CPMG is said to be single-digit micromolar concentration of small molecule, though the researchers note that double- or triple-digit micromolar concentrations are more practical, which is more typical of fragment screens anyway. And some compounds may show rapid relaxation due to non-pathological mechanisms, such as tautomerization or various conformational changes.
Still, this approach seems like a powerful means to rapidly assess hits, and pre-screening a library makes sense. Another NMR technique using interligand nuclear Overhauser effect (ILOE) has also been used to test for aggregation, though not to my knowledge so systematically. For the NMR folks out there, which methods do you think are best to weed out aggregators?