Fluorine NMR meets make-on-demand libraries

Last month Practical Fragments wrote about make-on-demand libraries, such as those offered by Enamine. We’ve also written about fluorine-detected NMR screening of fragments, which is both rapid and particularly sensitive for low affinity binders. A new paper in ChemMedChem by Patrick Penner, Anna Vulpetti, and colleagues at Novartis combines these two concepts.

 

The researchers had previously built a library of 5472 fluorinated fragments. These were compared with the 77 billion molecules then in Enamine REAL Space, which are based on a smaller set of building blocks that can be combined using validated chemistries. More than a third of the Novartis library members could be found in REAL Space, and nearly 80% had close analogs, supporting the notion that one could do rapid follow-up studies of fragment hits without requiring resource-intensive in-house chemistry.

 

To test whether this would work in practice, the researchers turned to embryonic ectoderm development protein (EED), an oncology target that Novartis has been pursuing for some time; here’s a 2017 post. An undisclosed number of known fragment ligands were screened against Enamine REAL Space using three computational methods: a search of the 180,000+ Enamine building blocks themselves, SpaceMACS to find close analogs, and FastROCS to find more distant analogs.

 

Of 150 compounds ordered across the three categories, 125 arrived: 66 building blocks, 30 close analogs, and 29 distant analogs. All of these were first tested by SPR, and nine (mostly close analogs) showed at least double-digit micromolar binding.

 

To assess whether ¹⁹F NMR could identify weaker binders, the remaining 116 compounds were screened in mixtures of nine each. This led to 43 additional hits, of which several were characterized in more detail by competing them in a dose-response format against a reporter molecule to calculate dissociation constants. One of these came in at sub-micromolar affinity, though it was a close analog of a known binder; the others were high micromolar.

 

Two of the more novel (and less potent) molecules were used as starting points to select new molecules from Enamine REAL space, and 74 of the 81 selected were delivered and tested by SPR or DSF. Two of these were more potent than the starting molecules, one of them by more than an order of magnitude.

 

In the end, although the results are modest, the paper provides a detailed framework for applying fluorine NMR to make-on-demand libraries. And with only four authors, it seems to be a low-effort approach. Although this work was done at Novartis, it should be suitable for smaller companies or academic labs that have access to an NMR.