Integrating Fragment Assembly and Biophysical Methods in the Chemical Advancement of Small-Molecule Antagonists of IL-2:  An Approach for Inhibiting Protein−Protein Interactions

Abstract

Fragment assembly has shown promise for discovering small-molecule antagonists for difficult targets, including protein-protein interactions. Here, we describe a process for identifying a 60 nM inhibitor of the interleukin-2 (IL-2)/IL-2 receptor (IL-2Ralpha) interaction. By use of fragment-based approaches, a compound with millimolar affinity was evolved to a hit series with low micromolar activity, and these compounds were optimized into a lead series with nanomolar affinity. Fragment assembly was useful not only for hit identification, but also for lead optimization. Throughout the discovery process, biophysical methods and structural biology demonstrated that compounds bound reversibly to IL-2 at the IL-2 receptor binding site.

Hot-spot mimicry of a cytokine receptor by a small molecule

“…Activity of SP4206 against WT IL-2 and each IL-2 variant was measured by the inhibition of the IL-2͞IL-2R␣ interaction as a function of compound concentration in an ELISA format as described in ref. 9. Approximately 10-20 nM biotinylated IL-2R␣ was immobilized in the wells of a streptavidin (Pierce, Rockford, IL)-coated 96-well plate (Maxisorp; Nunc, Rochester, NY).…”

“…However, mutational studies suggest that protein-protein interactions are driven by a small set of the contact residues, termed ''hot spots,'' whose footprints are not significantly larger than those covered by small molecules (4)(5)(6). Moreover, there have been several reports of small molecules that disrupt discontinuous protein-protein interactions with reasonable potencies (K i values Ͻ1 M), and their binding sites have been confirmed by highresolution structural analysis (7)(8)(9)(10)(11)(12).…”

“…Recently, SP4206, a small molecule (Structure 1) was discovered that binds with high affinity (K d Ϸ 70 nM) to IL-2 ( Fig. 1A) and blocks binding to its natural receptor, IL-2R␣ (8,9). SP4206 was assembled from smaller fragments by using a structureguided approach with a binding and functional assay (8,9).…”

“…1A) and blocks binding to its natural receptor, IL-2R␣ (8,9). SP4206 was assembled from smaller fragments by using a structureguided approach with a binding and functional assay (8,9). Subsequently, the structure of IL-2 bound to the IL-2R␣ (K d Ϸ 10 nM) was described (Fig.…”

17 The protein epitope mimetic approach to protein-protein interaction inhibitors

“…From this starting point, new molecules that bind to IL-2 were optimized, with dissociation constant in the mid-nanomolar range. These molecules were assembled in a fragment-based approach, guided by X-ray structures and medicinal chemistry (Arkin et al 2003;Braisted et al 2003;Raimundo et al 2004). Although the small molecules were assembled before the structure of the IL-2-IL-2Rα complex had been reported (Rickert et al 2005), they bind close to the center of the receptor contact region on IL-2.…”