Fragments vs CDC25B phosphatase – from behind

Protein phosphatases, which remove phosphate groups from proteins, fall into the category of low-hanging but firmly attached fruit: many make great targets, but getting lead-like inhibitors is tough. Indeed, the enzymes seem to be particularly susceptible to PAINS (see for example here and here). A major challenge is the phosphate-binding site, which has a predilection for highly negatively charged (and non-druglike) moieties. In a paper just published in ACS Chem. Biol., Tomasz Cierpicki and his group at the University of Michigan neatly sidestep this issue.

The researchers were interested in the dual-specificity protein phosphatase CDC25B, which is important in cell cycle regulation and thus a potential anti-cancer target. They started with a ¹H–¹⁵N HSC NMR screen of 1500 fragments in pools of 20, with each fragment present at 0.25 mM. This yielded a single hit: 2-fluoro-4-hydroxybenzonitrile.

Because the researchers were using protein-observed NMR and had previously assigned the backbone resonances, they were able to use chemical shift perturbations to identify the binding site. Surprisingly, this turned out to be not the active site at all, but rather a region about 15 Å away. They were able to confirm this site using X-ray crystallography, which further revealed that the fragment binds in a small pocket near where the substrate protein CDK2 binds.

The researchers noticed a nearby sulfate ion (from the crystallization buffer) and, after first doing a brief SAR by catalog survey, they tried to link this to their hit. Although this certainly didn’t improve physicochemical properties, it did result in tighter binding, and crystallography confirmed that the new molecule bound as designed. This molecule also inhibited the phosphatase, albeit modestly (IC₅₀ 1-2 mM). The result suggests that blocking this protein-protein interaction is effective at blocking activity.

It remains to be seen how much affinity there is to be had at this site. Still, I do have a soft spot for phosphatase inhibitors that bind outside the active site. At the very least this paper provides a new direction for an old – and very difficult – class of targets.