From noncovalent to covalent fragment for NSD1

As evident at the CHI Drug Discovery Chemistry meeting a couple weeks ago, covalent fragment-based lead discovery is becoming increasingly popular. Normally this entails screening a library of electrophile-containing fragments. However, it is also possible to start with a noncovalent fragment and add the “warhead” later. This is the approach taken by Jolanta Grembecka, Tomasz Cierpicki, and collaborators at University of Michigan, Memorial Sloan Kettering Cancer Center, and Columbia University in a paper just published in Nat. Chem. Biol.

 

The researchers were interested in NSD1, one of three related histone methyltransferases whose potential role in cancer is uncertain – in part due to the lack of good chemical probes. They started with a two-dimensional (¹H-¹⁵N HSQC) NMR screen of 1600 fragments in pools of 20, with each fragment present at 250 µM. BT1 was one of the hits, and synthesis of several analogs led to BT2, which showed low micromolar affinity as assessed by ITC as well as an IC₅₀ of 66 µM in a functional assay. Crystallography was unsuccessful, but NMR experiments suggested considerable changes in an autoinhibitory loop that blocks the substrate-binding region of the so-called SET domain.

For whatever reason, the researchers tested thiocyanate analog BT3 and found that this binds covalently as a disulfide to a cysteine in the autoinhibitory loop. They were able to get a crystal structure of BT3 bound to the protein, which revealed significant rearrangements that allow BT3 to bind deep in the SET domain, where it makes multiple polar and hydrophobic contacts. Prudently they chose to replace the unstable thiocyanate warhead, and while acrylamide derivatives were inactive, aziridine BT5 modified the protein as assessed by mass spectrometry.

 

BT5 inhibited NSD1 with an IC₅₀ of 5.8 µM after a four hour incubation and was somewhat selective against the related proteins NSD2 and NSD3 as assessed both by mass spectrometry and activity assays. It also showed good selectivity at 50 µM against 20 other epigenetic enzymes and 291 kinases. Interestingly though, at this concentration BTK was inhibited by 41% and EGFR was inhibited by 49%; both these kinases are targeted by approved covalent drugs.

 

Next, the researchers conducted multiple cell assays. A cellular thermal shift assay (CETSA) revealed that BT5 stabilized NSD1 but not NSD2 or NSD3. Growth of an NSD1-dependent cancer cell line was inhibited with GC₅₀ = 1.3 µM after 3 days, NSD1-mediated histone methylation was suppressed, and several target genes showed reduced expression. BT5 also inhibited growth of non-NSD1-dependent cell lines, though at 6-8 higher concentrations, and did not alter methylation or target gene expression. Finally, the compound impaired colony formation of cells from a primary patient-derived sample with an NUP98-NSD1 translocation.

 

This is a nice, carefully conducted study. Refreshingly, the researchers do not attempt to oversell their results, and acknowledge that “further optimization is needed to develop NSD1 chemical probes.” But they’re off to a good start, and it will be fun to see what they – and others – will come up with.