Challenge
Mettl3/Mettl14 is a large (90 kDa) heterodimeric “writer” complex that uses S-adenosyl methionine (SAM) as a cofactor.  Identifying low affinity, validated fragment hits in the large amount of electron density derived from X-ray diffraction data was extremely difficult.

Solution
X-ray crystallography provides the necessary high resolution structural data needed to efficiently elaborate hits from fragment or HTS campaigns. However, even highly validated fragment hits typically exhibit low affinity (100s to 1000s of µM). As a result, the occupancy of ligand binding sites in the crystal may be substantially less than 100%, which degrades the quality of the electron density of the fragment. Moreover, fragments are small and overall somewhat symmetric, further reducing identifiable features of their electron density.

We set up a solution competition binding experiment using NMR as a readout to focus on fragments that bind at the SAM site. We quickly found a fragment containing a CF₃ moiety that bound at the SAM site. We could then use ¹⁹F NMR studies as a clean, sensitive and fast approach to reveal other ligands that bound at the same site. Once we were certain of the binding site, we were readily able to identify electron density at the SAM site and confidently assign it to the ligand.

Achievements
We developed a highly efficient structure pipeline capable of determining up to 10 sub 2Å structures per month which has been the basis for elaborating fragment hits to cellular activity and beyond. Multiple chemotypes are promising, allowing for a robust hit-to-lead campaign.