Fragments vs choline kinase alpha

Fragments and kinases have a long and successful association, as demonstrated by nearly half of FBLD-derived clinical candidates. Most of the attention has been on protein kinases, which transfer phosphate groups to other proteins. But there are more kinases out there, and in a recent paper in J. Med. Chem., Stephan Zech and colleagues at Ariad describe how they developed inhibitors against choline kinase α (ChoKα), a potential anticancer target involved in phospholipid synthesis.

A screen of known kinase inhibitors came up largely empty, so the researchers used STD NMR to screen a library of 1152 diverse, rule-of-three compliant fragments in pools of five, each at 3 mM. This yielded 55 hits, which were then tested in a fluorine-detected NMR (FAXS) assay to see whether they could displace molecules that bind in either the ATP or substrate binding sites. These experiments suggested that 13 fragments bind in the choline binding site while 21 bind in the ATP site; the remaining 21 either bind elsewhere or are artifacts.

Most of the fragments showed minimal activity in functional assays, but compound 11 was an exception. SPR confirmed binding, though it also seemed to bind to an unrelated protein and displayed super-stoichiometric behavior at high concentrations. Nonetheless, it could successfully be soaked into crystals of ChoKα, and the resulting structure revealed that it binds deep in the choline-binding pocket, with the terminal methyl group in a small pocket at the bottom. This is also consistent with STD epitope mapping of a related fragment, which showed that the azepane ring was closely associated with the protein.

An initial search for commercial analogs followed by several rounds of medicinal chemistry led to compound 43, with low micromolar potency, and several more rounds of optimization led to compound 65, with nanomolar binding (by SPR) and inhibition. A crystal structure of this molecule bound to the enzyme revealed that the fragment binding mode is roughly conserved, while the added basic moiety binds close to several acidic residues near the surface of the binding pocket. A very closely related molecule showed low micromolar activity in several cell-based assays (data for compound 65 is not reported). 

This is an interesting paper for several reasons. First, it reports the successful use of fragment screening against an unusual target. Second, although multiple fragments were found to bind in the ATP-binding site (a productive starting point for many conventional kinases), these fragments could not be optimized. On the other hand, a fragment that binds in the choline-binding site could rapidly be improved to nanomolar inhibitors. Third, although fragment 11 did show some red flags, it was ultimately optimizable – a reminder that some misbehavior in a fragment should not necessarily disqualify it. Finally, the iterative and structure-based nature of the medicinal chemistry – which is well beyond what this brief blog post can cover – makes a nice case study in fragment growing. Of course, the final molecule still has high hurdles to surmount, and it will be fun to see the story progress.