Fragments vs 53BP1

As we’ve noted (repeatedly), epigenetics is big. However, much of the focus has been on bromodomains, which recognize acetylated lysine residues. In a paper published earlier this year in ACS Chem. Biol., Lindsey James, Stephen Frye and collaborators at the University of North Carolina, the University of Texas, the Mayo Clinic, and the University of Toronto describe their efforts on a protein that recognizes methylated lysine residues (a Kme reader).

The protein 53BP1 is involved in DNA repair and could have anticancer potential. It recognizes a dimethylated lysine sidechain within a histone protein, so the researchers screened a set of molecules containing amines to mimic this moiety. They used an AlphaScreen assay, with each compound at 100 µM. This does not appear to have been a library of fragments (and unfortunately the number of compounds screened was not stated), but the most notable hit was the fragment-like UNC2170.

Although the affinity was modest, it was quite selective for 53BP1, showing no activity up to 500 µM against 9 other Kme readers. Since AlphaScreen assays can be prone to false positives (the original PAINS compounds were identified in this assay), the researchers tested their compound using ITC, which gave a dissociation constant of 22 µM, in good agreement with the AlphaScreen assay, though with unusual stoichiometry (more on that later).

Thus encouraged, the researchers set off to optimize their hit. Initially they tried modifications around the amine, but even changes as subtle as adding or removing a methyl group killed activity. Attempts to rigidify the propyl linker were also unsuccessful, and shortening it or lengthening it failed too. Replacing the amide with a sulfonamide or amine abolished activity. Most substitutions around the phenyl ring also gave dead compounds, though the bromine atom could be replaced with similarly hydrophobic moieties such as iodine, isopropyl, or trifluoromethyl. Many other analogs were made too, all to no avail. Though the text is measured, the frustration is palpable.

Ultimately the researchers were able to solve the crystal structure of the compound bound to 53BP1, which produced a surprise: one molecule of UNC2170 binds to two molecules of protein, making interactions with each. This explains the stoichiometry seen in the ITC data. It also explains the intolerance to substitutions, as “the ligand is encircled by both proteins,” with no room for modifications.

Happily, UNC2170 is highly cell permeable and non-toxic, and does show some modest activity in cell-based assays. Hopefully the researchers will ultimately find more potent compounds, though this may require a different approach. Indeed, another Kme reader also proved to be quite challenging, but was amenable to fragments. It would be fun to see whether an explicit fragment screen produces more tractable starting points against 53BP1.