Fragments vs hIL-1β: Growing into a cryptic pocket to inhibit a protein-protein interaction

Protein-protein interactions have a well-deserved reputation for being difficult to drug with small molecules. This is particularly true for cytokine-receptor pairs, which are involved in a host of extracellular signaling functions. Human interleukin-1β (hIL-1β) plays a key role in inflammation by binding to its receptor IL-1R1. Biologics such as anakinra and canakinumab have been approved as drugs, but apart from some very low affinity fragments no small molecule inhibitors are known. In a new (open access) Nat. Commun. paper, Frédéric Bornancin, and collaborators at Novartis and University of Leicester report the first.

 

The researchers started by screening the 3452-compound LEF4000 library, which we described here, using ¹⁹F-NMR. After confirmation using protein-observed 2D NMR just a single super-sized fragment hit remained, consistent with the difficulty of the target. The individual enantiomers of this racemic compound were studied, and only (S)-1 was found to be active. Further characterization revealed that, despite weak affinity, this compound had both slow association and dissociation rates. More on that below.

 

Fragment growing in multiple directions led to mid-micromolar compounds such as 11 and 12. Combining elements from these molecules ultimately led to compound (S)-2, with low micromolar affinity as assessed by SPR. 

 

 

Compound (S)-2 specifically blocked the binding of hIL-1β with its receptor IL-1R1, but did not inhibit the binding of the related cytokine hIL-1α to IL-1R1. Even better, the compound blocked IL-1R-mediated signaling in cells at low micromolar concentrations in two different assays. The similar activity in biochemical and cell assays is likely due to the fact that the compound only needs to act at the cell surface, so permeability is not an issue, in contrast to our post last week.

 

A crystal structure of (S)-2 bound to hIL-1β revealed important interactions between the protein and both the phenol and lactam nitrogen, two contacts that were maintained during fragment optimization. The structure explains why only the (S)-enantiomer is active, as maintaining these contacts would cause clashes for the other enantiomer.

 

The structure also explains the mechanism of inhibition. (S)-2 binds to a cryptic pocket that forms in a region of hIL-1β important for interacting with IL-1R1, and formation of the pocket involves a loop movement that would be incompatible with the protein-protein interaction. The researchers argue convincingly that that the compound stabilizes the cryptic pocket, which naturally exists as a minor population within solution. This also explains the slow kinetics, which would be expected if the compound essentially has to wait until the cryptic pocket opens before it can bind.

 

There is still a long way to go to a drug. Not only is the affinity of (S)-2 modest, the two carboxylic acid moieties and the phenol are likely to impede oral bioavailability. Nonetheless, this is a lovely paper, and the researchers point out that cryptic pockets frequently involve “large movements of secondary structural elements” that could block biological function. Indeed, this is the case for approved drugs such as sotorasib. Don’t give up just because your protein of interest appears like a featureless billiard ball: there may well be opportunities hidden just beneath the surface.