Fragments win in a virtual screen against Notum

Wnt proteins are implicated in a variety of diseases, from Alzheimer’s to colorectal cancer. The enzyme Notum shuts down signaling by removing a palmitoyl group from Wnt. Last year Practical Fragments highlighted several series of Notum inhibitors identified from biochemical and crystallographic fragment screens. The researchers behind those efforts, including Paul Fish and Fredrik Svensson (University College London), have now published a successful virtual screen against the enzyme in J. Med. Chem.

 

Starting with 1.5 million compounds available from ChemDiv, the researchers chose 534,804 based on a variety of computational filters including molecular weight (200-500 Da), number of hydrogen bond donors (<=2) and ClogD (-4 to 5). A virtual screen of these (using Glide) produced 1330 high-scoring hits, of which 1088 were chosen for purchase. Of these, 952 were available, a much higher percentage than the ZINC15-reliant paper we wrote about earlier this year.

 

All 952 compounds were tested in a biochemical assay, and the 44 that gave >50% inhibition at 1 µM were then tested in dose-response format. This yielded 31 compounds with IC₅₀ values < 500 nM. These could be subdivided into four structurally related clusters and eight singletons. Further triaging removed compounds likely to cause assay interference as well as those similar to known Notum inhibitors. This left two clusters and two singletons.

 

Compound 1f was the most potent member of a series of 9 related (and possibly covalent) inhibitors. Although these strongly inhibited the enzyme in the biochemical assay, they were essentially inactive in a cell-based assay. They were also highly insoluble and showed low cell permeability, and were thus dropped.

 

Compound 2a was one of two related molecules that were also quite potent when initially tested. Unfortunately, when the molecules were resynthesized they turned out to be significantly weaker and were also not very soluble, so this series was also halted.

 

The singleton compound 3 turned out to be a covalent inhibitor; the catalytic serine formed an ester with the molecule. The mechanism is more fully described in this open-access J. Med. Chem. paper.

 

That leaves the second singleton. Compound 4d was not just active in the biochemical assay, it also showed sub-micromolar cell activity. SAR, guided by crystallography, ultimately led to low nanomolar inhibitors. The pK_a of compound 4d was measured to be 7.9, which is less acidic than many previously reported Notum inhibitors and thus more likely to be cell permeable. This turned out to be the case experimentally, and the compound was also stable in mouse liver microsomes. Pharmacokinetics in mice were promising for several compounds, but unfortunately brain penetration – which the researchers were hoping for – was negligible. (This could be an advantage for peripheral diseases.)

 

This is a nice example of lead discovery in academia. Like last week’s post, it also illustrates that fragments themselves can be quite potent. Indeed, although the researchers were looking for molecules up to 500 Da in their virtual screen, all of the best hits were fragment-sized. Another illustration that small is beautiful.