Fragments vs RIP2: from flat fragment to shapely selectivity

Last week we highlighted the utility of shapely fragments. However, as the latest review of fragment-to-lead success stories again shows, starting with a “flat” fragment does not condemn a lead to flatland. This is illustrated in a recent J. Med. Chem. publication by Adam Charnley and colleagues at GlaxoSmithKline.

The researchers were interested in receptor interacting protein 2 kinase (RIP2), which is implicated in various inflammatory diseases. A fluorescence polarization screen of 1000 fragments at 400 µM yielded 49 hits with inhibition constants ranging from 5-500 µM. Thirty of these confirmed in a thermal shift assay, and 20 were characterized crystallographically bound to the enzyme. Hit-to-lead chemistry was pursued for five series; the most successful started with compound 1a.

The crystal structure revealed that the carboxamide of compound 1a makes interactions with the hinge region of the kinase, with the phenyl group in the back pocket. A search of related molecules available in-house led to compound 2a, with a satisfying boost in potency. Interestingly, the crystal structure of this molecule bound to RIP2 revealed that the binding mode of the pyrazole moiety had flipped to keep the phenyl ring in the back pocket (compound 1a in cyan, 2a in gray). Enlarging the phenyl group to better fill the pocket led to compound 2k.

This molecule had relatively poor selectivity against several other kinases, but introducing a ring as in compound 8 improved the situation. Crystallography suggested that installing a bridged ring would pick up further interactions with the protein, and although the resulting molecule did not have better affinity, selectivity improved. Finally, a hydroxyl group was introduced (compound 11) to try to pick up interactions with a non-conserved serine residue. This addition did not improve biochemical activity, and in fact a crystal structure revealed that the hydroxyl group was pointing towards solvent, but the activity in human whole blood improved. Importantly, compound 11 was remarkably selective for RIP2: just 1 of 366 other kinases tested at 1 µM showed >70% inhibition.

This is a lovely fragment-to-lead success story that reiterates several important lessons. First, a generic (in this case commercial) and nonselective fragment can lead to novel, selective series. Second, as has been seen multiple times, fragment binding modes can flip unexpectedly, especially during early optimization. Finally, despite the relative flatness of fragment 1a (Fsp³ = 0, though the two aromatic rings are slightly twisted), it could be optimized to a more shapely lead, and the increased complexity is likely responsible for the impressive selectivity. Left unreported is the stability and pharmacokinetics of compound 11: the hydroxyl and all those sp³-hybridized carbons are likely metabolic hotspots. As is so often the case in lead discovery, what solves one problem can too often create another.