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.
1 comment:
Interesting paper. The completion could potentially be improved. Looking at their data I might make a case for the compounds being Partially Uncompetitive rather than non-competitive as it seems to my eye that the IC50 decreases with substrate concentration (and saturates at a bout a 3 fold increase in potency when substrate binding sites are saturated) but the compound is able to bind in the absence of substrate. This mechanism might make sense for a compound which binds adjacent from to SAM and also sits within, but not covering the peptide binding pocket as it could potentially make further interactions with SAM and the peptide.
What irks me about this is that the models to properly determine mechanism are available in Graphpad Prism, which the authors have used to make their figures.
However I have no doubt the data are real, and it's an interesting find worthy of publication.
Post a Comment