Plant homeodomain (PHD) fingers,
despite their name, are found in nearly 300 human proteins. They are small (50-80
amino acid) domains that typically recognize post-translational modifications
such as trimethylated lysine residues in histones. The PHD finger in BPTF is
implicated in certain types of acute myeloid leukemia. However, because of the
large number of PHD fingers as well as their small binding sites, few attempts
have been made to develop corresponding chemical probes. (Indeed, the only mention
of them on Practical Fragments was in 2014.) In a just-published ACS
Med. Chem. Lett. paper, William Pomerantz and collaborators at University
of Minnesota and St. Jude Children’s Research Hospital report the first steps.
The researchers started by
screening a library of 1056 fragments (from Life Chemicals) against the BPTF
PHD finger using ligand-observed (1H CPMG) NMR. Fragments were at
100 µM in pools of up to five. This gave a preliminary hit rate of 5.7%, but
only ten compounds (<1%) reproduced when compounds were repurchased and retested
individually.
These ten fragments were next
tested by SPR (at 400 µM), which confirmed six of them. Also, all ten CPMG hits
were tested in an AlphaScreen assay in which they competed with a known peptide
binder. This confirmed nine, including the six that confirmed by SPR.
Interestingly, the most potent fragment
in the AlphaScreen assay was the starting point for the KRAS inhibitor we
highlighted last year. However, this fragment did not show binding to the BPTF
PHD finger by SPR, and the researchers had identified the 2-aminothophene substructure
as a hit against an unrelated protein. Whether this fragment is privileged or pathological
may be context dependent.
This and the top three fragments
that confirmed in all assays were used as starting points for SAR by catalog,
and a handful of analogs were purchased. The researchers also resynthesized two
of the compounds. Oddly, resynthesized F2 turned out to be three-fold more
active in the AlphaScreen assay than the commercial material. One analog, compound
F2.7, showed mid-micromolar activity.
Docking and two-dimensional
protein-observed (1H,15N HSQC) NMR experiments suggest
that most of the fragments bind in the “aromatic cage” which normally recognizes
methylated lysine residues, but F2 may bind in an adjacent region. Both subpockets
were also identified as being ligandable using the program FTMap.
This paper is a nice example of using
orthogonal methods to find and carefully validate fragments against an underexplored
class of targets. The researchers conclude by stating that “these hits are
suitable for further SAR optimization and development into future methyl lysine
reader chemical probes.” I look forward to seeing more publications.
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