Epigenetics involves turning genes on or off without changing their sequence. This often relies on modifications to proteins or DNA that are recognized by other proteins. As Teddy pithily observed, this is a big field. However, in the realm of fragments, most of the attention has been on bromodomains; other classes of proteins, such as methyltransferases, have been largely neglected. A new paper in J. Med. Chem. by Masoud Vedadi and Matthieu Schapira and collaborators at the University of Toronto and Bayer suggests fragments are promising for these targets as well.
The researchers were specifically interested in protein arginine methyltransferases (PRMTs), which transfer a methyl group to one of the terminal side chain nitrogen atoms on specific arginine residues. PRMT6 in particular targets histone proteins to modulate transcription and has been implicated in cancer as well as neurodegenerative diseases. A few potent inhibitors have previously been reported for PRMTs, and the team started by deconstructing them to hunt for active fragments.
Ligand deconstruction involves chopping a known ligand into fragments to see whether any of these pieces will still bind. In this particular case, EPZ020411 had previously been characterized crystallographically bound to PRMT6 with the basic amine-containing “tail” in the substrate arginine-binding groove. Testing this fragment 6 by itself revealed a low micromolar inhibitor with a ridiculously high ligand efficiency.
Thus encouraged, the researchers ran a functional screen of 2040 diverse fragments (about half from Maybridge) at 1 mM concentration and retested hits at 0.5 mM. About half the resulting hits were false positives or other uglies, leaving the researchers with 14 fragments with IC50 values from 0.3 – 400 µM. As might be expected given the cationic nature of the substrate, 12 of these have basic nitrogens.
Compound 7 was particularly interesting: at 300 nM this is one potent fragment! ITC revealed a dissociation constant of 970 nM, with a favorable enthalpy and unfavorable entropy of binding. It did hit other PRMTs, but was remarkably selective against a panel of 25 other human methyltransferases.
The researchers also determined the crystal structure of compound 7 bound to PRMT6, which revealed it binding, as expected, in the arginine site, making hydrogen bonds with a conserved catalytic glutamic acid. Weirdly though, it seems to be a noncompetitive inhibitor: increasing concentrations of substrate peptide or cofactor had no effect on inhibition. The team speculates that the noncompetitive behavior could be because the substrate makes strong interactions with the protein outside the arginine-binding site. Nonetheless, the fragment did inhibit PRMT6 activity in a cell assay with IC50 = 21 µM.
Overall then it seems that the PRMTs are amenable to FBLD. They are interesting drug targets, and at the very least having more probes will help to unravel the biology.