Tiny fragments at high concentrations give massive hit rates

Screening fragments crystallographically is becoming more common, especially as the process becomes increasingly automated. Not only does crystallography reveal detailed molecular contacts, it is unmatched in sensitivity. At the FBLD 2018 meeting last year we highlighted work out of Astex taking this approach to extremes, screening very small fragments at very high concentrations. Harren Jhoti and colleagues have now published details (open access) in Drug Discovery Today.

The researchers assembled a library of 81 diminutive fragments, or “MiniFrags”, each with just 5 to 7 non-hydrogen atoms. Indeed, the fragments adhere more closely to the “rule of 1” than the “rule of 3.” Because the fragments are so small, they are likely to have especially low affinities: a 5 atom fragment with an impressive ligand efficiency of 0.5 kcal mol^-1 per heavy atom would have a risibly weak dissociation constant of 14 mM. In order to detect such weak binders, the researchers screen at 1 M fragment concentrations, almost twice the molarity of sugar in soda! Achieving these concentrations is done by dissolving fragments directly in the crystallographic soaking solution and adjusting the pH when necessary. Although this might mean preparing custom fragment stocks for each protein, it avoids organic solvents such as DMSO, which can both damage crystals and compete for ligand binding sites.

As proof of concept, the researchers chose five internal targets they had previously screened crystallographically under more conventional conditions (50-100 mM of larger fragments). All targets diffracted to high resolution, at least 2 Å, and represented a range of protein classes from kinases to protein-protein interactions. The hit rates were enormous, from just under 40% to 60%, compared to an average of 12% using standard conditions.

Astex has previously described how crystallography often identifies secondary binding sites away from the active site, and this turned out to be the case with MiniFrags: an average of 10 ligand binding sites per protein. In some cases protein conformational changes occurred, which is surprising given the small size and (presumably) weak affinities of the MiniFrags.

All this is fascinating from a molecular recognition standpoint, but the question is whether it is useful for drug discovery. The researchers go into some detail around the kinase ERK2, which we previously wrote about here. MiniFrags identified 11 ligand-binding sites, several of which consist of subsites within the active site. Some of the MiniFrags show features previously seen in larger molecules, such as an aromatic ring or a positively charged group, but the MiniFrags also identified new pockets where ligands had not previously been observed. The researchers argue that these “warm spots” could be targeted during lead optimization.

One laudable feature of the paper is that the chemical structures of all library members are provided in the supplementary material. Although it would be easy to recreate by purchasing compounds individually, hopefully one or more library vendors will start selling the set. If MiniFrag screening is standardized across multiple labs, the resulting experimental data could provide useful inputs for further improving computational approaches, as well as providing more information for lead discovery.