Combining fragments and HTS hits to target PHGDH

Boehringer Ingelheim has been on something of a tear reporting new chemical probes for difficult targets – see here for their NSD3 inhibitor and here for their RAS inhibitor. This is part of an ambitious effort to develop probes for the entire human proteome by 2035. In a new paper published in J. Med. Chem., Harald Weinstabl and collaborators at BI and Shanghai ChemPartner describe the discovery of BI-4924, a potent inhibitor of phosphoglycerate dehydrogenase (PHGDH).

The enzyme is the rate-limiting step in serine synthesis, and has been implicated in multiple types of cancers. However, metabolic enzymes such as PHDGH are particularly challenging drug targets for several reasons: cofactors such as NADH are present at high concentrations in cells, the substrate binding pocket is both shallow and polar, and one often needs near complete inhibition to see an effect. Thus, the researchers chose multiple approaches.

An STD NMR fragment screen was conducted against the apo form of the protein (250 µM fragment and 20 µM protein) to find compounds that would bind in the NAD⁺-binding site. Of 1860 fragments screened, 60 hits were identified, and 19 of these gave measurable dissociation constants in an SPR assay and were selective against two other proteins. Compound 9 was found crystallographically to bind in the adenine pocket of the NAD⁺-binding site. Fragment growing was challenging due to the “kinked shape” of this pocket: elaborated molecules tended to point out of the pocket into solvent. Careful design led to modest improvements in potency (compound 11), and adding a negatively charged moiety led to potent molecules such as compound 43. To avoid problems with permeability, the researchers tried various uncharged bioisosteres, but these were not tolerated. Interestingly, crystallography revealed that the carboxylic acid does not seem to make specific interactions with the protein; its necessity may be due to long-range electrostatic interactions with multiple nearby basic residues.

In parallel, a biochemical HTS screen of more than a million molecules yielded 27,000 hits, which were whittled down to 11,250 that confirmed and didn’t interfere with the assay. Removing PAINS and large, lipophilic molecules narrowed the set to 4750 compounds. Further rigorous assessment included biophysical methods, as recently recommended. Aware of the potential for metal contaminants to give false positives, the researchers examined select samples with inductively coupled plasma mass spectrometry and found that some contained mercury or copper, which inhibited the enzyme. Ultimately 77 hits were validated with dissociation constants better than 300 µM, including compound 8, which crystallography revealed binds in a similar manner to fragment 9.

Combining information from both campaigns and growing to engage an aspartic acid side chain ultimately led to BI-4924. This compound is soluble, stable, and selective against other dehydrogenase enzymes. Unfortunately, the carboxylic acid moiety does indeed impart low permeability, and perhaps because of this the molecule has only low micromolar activity in cells. However, the ethyl ester (BI-4916) transiently accumulates in cells and modulates serine levels.

Unfortunately, the researchers appear to have been scooped; as they politely note, “subsequently, these findings were independently confirmed….” As it stands BI-4916 is too unstable for use in vivo. Still, it could be useful for further unraveling the biology around serine biosynthesis and its role in cancer cells, and the paper itself stands as a nice example of structure-based lead design combining information from multiple sources.