NMR is among the more sensitive fragment-finding techniques: the starting point for clinical compound ASTX660 had low millimolar affinity at best. Now, three papers by A. Joshua Wand and colleagues at University of Pennsylvania have taken sensitivity to a new level, enabling the detection of fragments that bind hundreds of times less tightly. (Derek Lowe recently wrote about one of them, and I highlighted a talk last year.)
All three papers focus on a method called reverse micelle encapsulation, in which an aqueous solution of protein and ligand is encapsulated in nanoscale reverse micelles measuring less than 100 Å in diameter. At this size, each micelle will contain at most just a single protein and a few thousand water molecules. Because of the small volume, the protein concentration – and that of any fragments – will be extraordinarily high. The micelles have polar groups pointed inwards towards their watery interior, and their hydrophobic tails point out towards solvent, typically pentane. The overall water content of the sample is typically around 2%.
Various NMR techniques can be used to study the proteins. Although the reverse micelles are larger than the proteins themselves and thus would be expected to tumble more slowly, the low viscosity of the pentane solvent makes up for this, providing high-quality spectra.
The primary paper, in ACS Chem. Biol., focuses specifically on fragments. To establish that the technique can detect weak interactions, the researchers show that they can measure the 26 mM dissociation constant of adenosine monophosphate to the enzyme dihydrofolate reductase.
Next, they turned to the protein interleukin-1β (IL-1β), an inflammatory target with no reported small-molecule binders. One challenge of the method is that hydrophobic fragments could partition into the micelles or even diffuse into the pentane, thus reducing their concentration. To avoid this, the researchers assembled a library of 233 very polar, water-soluble fragments with cLogP values < 0.5. A 2-dimensional NMR screen (15N-TROSY) using standard conditions (100 µM protein and 800 µM fragment) yielded no hits.
In contrast, NMR screening using reverse micelles with the protein at an effective concentration of 5 mM and fragments at 40 mM yielded 31 hits. Chemical shift perturbations (CSPs) were used to determine where they were binding. Ten of the fragments didn’t show clear binding to specific sites on the protein, but the remaining 21 did, with all but one binding to multiple sites. Of these, 13 also showed non-specific interactions with other regions of the protein. Altogether, the fragment binding sites covered 67% of the protein surface, with the receptor-binding interface particularly well-represented.
Concentration-dependent CSPs were used to determine dissociation constants, which ranged from 50 mM to over 1 M. An SAR-by-catalog exercise was able to improve the affinity of one fragment from 200 mM to 50 mM at one site, though it also binds three other sites with slightly weaker affinity.
The second paper, also in ACS Chem. Biol., uses IL-1β but focuses on the interaction of even smaller molecules such as pyrimidine, methylammonium, acetonitrile, ethanol, N-methylacetamide, and imidazole. Not surprisingly, the dissociation constants are even weaker, averaging 1.5 – 2.5 M.
Finally, a Methods in Enzymology paper goes into depth on how to actually run the experiments, including details on choosing detergents and making the micelles. At high fragment concentrations, for example, pH needs to be carefully controlled.
Five years ago we asked “how weak is too weak” for a fragment. In terms of practicality, I’d say that these fragments qualify. Indeed, the ligand efficiency for the best fragment mentioned above is just 0.15 kcal mol-1 atom-1.
But the findings do raise the almost philosophical question of what exactly constitutes a small molecule binding site. Astex researchers reported several years ago that most proteins have more than one, and their more recent work with MiniFrags suggest on average 10 sites at high enough concentrations. Similar results were also reported earlier this month from Monash. Whether or not the fragments from such screens turn out to be immediately useful, they could certainly advance our understanding of molecular recognition.