Fragments finger the BPTF PHD Finger

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.