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).
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