Fragments vs the PWWP1 domain of NSD3: a chemical probe

Epigenetics is a topic we’ve covered frequently at Practical Fragments. Much attention has been focused on bromodomains, which recognize acetylated lysine residues. However, lysine side chains are also methylated to affect gene expression. The PWWP1 domain of the protein NSD3 (NSD3-PWWP1) recognizes these modified lysines. This protein is amplified in several tumour types, and so makes an intriguing cancer target. At the CHI DDC conference last year Jark Böttcher presented how Boehringer Ingelheim and a large multinational group of collaborators developed a chemical probe for NSD3. The story now appears in Nat. Chem. Biol. (and see here for a fun animated short set to music).

The researchers started by screening a library of 1899 fragments against NSD3-PWWP1. STD NMR (at 0.25 mM of each fragment, in pools of four) as well as differential scanning fluorimetry (at 0.5 mM of each fragment) resulted in 285 and 20 hits, respectively. Two-dimensional NMR was used to confirm hits. Interestingly, only three fragments were identified from both STD-NMR and DSF, and these did not confirm – a cautionary reminder that screening orthogonal methods is not necessarily the best path.

Fortunately, 15 fragments not only confirmed, but also caused the same changes to the 2D-NMR spectra as a histone-derived peptide containing a dimethyl-lysine residue, suggesting that the fragments bind at the recognition site for modified lysines. Those fragments with dissociation constants better than 2 mM were pursued crystallographically, and some of the successes included compound 4. This molecule was used in a virtual SAR-by-catalog screen of internal compounds. Of the 601 fragments experimentally tested, compound 8 was the most potent. Crystallography confirmed that the compound binds in the expected site, and further structure-based design ultimately led to BI-9321.

BI-9321 was put through a battery of tests. Affinity was confirmed in biochemical, SPR, and ITC assays, and crystallography revealed the binding mode to be similar to the initial fragment. BI-9321 was selective for NSD3-PWWP1 when tested against 14 other PWWP domains, and showed no activity against 35 protein methyltransferases, 31 kinases, and 48 bromodomains. Solubility, in vitro metabolic stability, permeability, and plasma protein binding all look good.

Multiple assays also demonstrated selective target engagement in cells at a concentration of around 1 µM. BI-9321 showed downregulation of MYC mRNA levels, though the effect was both modest and transient. Antiproliferative activity was also observed in cells, and the effects were synergistic with a bromodomain inhibitor. Moreover, these effects were only seen in NSD3-dependent cells, suggesting that the activity is on-target and that the compound is not generally cytotoxic.

All of this makes BI-9321 an attractive chemical probe, at least for cell-based assays. More work will need to be done to improve potency and further understand the biology. Laudably, to this end, the researchers have made the molecule publicly available.