The researchers started with a low micromolar inhibitor of the interaction between the von Hippel-Lindau protein and the alpha subunit of hypoxia-inducible factor 1 (pVHL:HIF-1α), an interaction important in cellular oxygen sensing. The team had previously deconstructed this molecule into component fragments, but they were unable to detect binding of the smallest fragments.
In the new study, the researchers again deconstructed the inhibitor into differently sized fragments and used three ligand-detected NMR techniques (STD, CPMG, and WaterLOGSY) to try to identify binders. As before, under standard conditions of 1 mM ligand and 10 µM protein, none of the smallest fragments were detected. However, by maintaining ligand concentration and increasing the protein concentration to 40 µM (to increase the fraction of bound ligand) or increasing concentrations of both protein (to 30 µM) and ligand (to 3 mM), the researchers were able to detect binding of fragments that adhere to the rule of three.
Of course, at these high concentrations, the potential for artifacts also increases, but the researchers were able to verify binding by isothermal titration calorimetry (ITC) and competition with a high-affinity peptide. They were also able to use STD data to show which regions of fragments bind to the protein, suggesting that the fragments bind similarly on their own as they do in the parent molecule. (Note that this is in contrast to a deconstruction study on a different PPI.) Even more impressively for a large (42 kDa) protein, the researchers were able to use 2-dimensional NMR (1H-15N HSQC) to confirm the binding sites.
Last year we highlighted a study that deconstructed an inhibitor of the p53/MDM2 interaction. In that case, the researchers were only able to find super-sized fragments, and they argued that for PPIs the rule of three should be relaxed. The current paper is a nice illustration that very small, weak fragments can in fact be detected for PPIs, though you may need to push your biophysical techniques to the limit.
But back to the original question of how weak is too weak. With Kd values from 2.7-4.9 mM, these are truly feeble fragments. Nonetheless, they could in theory have been viable starting points had they been found prospectively. That assumes, though, that these fragments would have been recognized as useful and properly prioritized. The ligand efficiencies (LE) of all the fragments, while not great, are not beyond the pale for PPIs. Previous research had suggested that much of the overall binding affinity in compound 1 comes from the hydroxyproline fragment (compound 6, which was originally derived from the natural substrate). Not discussed in the paper, but perhaps more significantly, the LLE (LipE) and LLEAT values are best for compound 6, which despite having the lowest affinity is the only compound that could be crystallographically characterized bound to the protein. In the Great Debate over metrics, this suggests that LLE and LLEAT may be more useful than simple LE for comparing very weak fragments.