Identification of an Induced Orthosteric Pocket in IL-23: A New Avenue for Non-biological Therapeutic Targeting

Abstract

Interleukin 23 (IL23) is a key driver of autoimmune inflammatory pathology and has been successfully targeted by therapeutic antibodies for the treatment of psoriasis and ulcerative colitis. Identifying small-molecule inhibitors of IL23 signaling is of potential interest for drug development. We report the identification of an induced-fit orthosteric binding pocket on the IL23p19 subunit that may be suitable for small-molecule inhibition. X-ray crystallography was used to determine the structure of the IL23 heterodimer when bound to inhibitory peptide 23-446 and to confirm peptide binding to the IL23p19 subunit at the location of its interface with the IL23 receptor (IL23R). We then applied structure-based design to modify peptide 23-446. This process involved identifying key residues responsible for inhibitory activity and generating structure-activity relationship-optimized peptides with low nanomolar affinity for IL-23 and corresponding inhibitory potency against IL-23R binding. These optimized peptides show promise as potential therapeutic candidates in their own right and may also serve as valuable starting points for further discovery. The most potent of these peptides was used to develop a fluorescence polarization probe and to design a high-throughput screening assay, which was validated through a pilot screen using a small fragment-based compound library. This screening strategy has the potential to support the discovery of peptides or small molecules that bind to the orthosteric pocket, thereby blocking the IL-23R interaction and downstream signaling.

1

X-ray structure of peptide 23–446 (blue) bound to IL23 (p19 red + p40 yellow). The peptide-induced fit binding pocket is represented as a solid surface. IL23R (orange, from an overlay with PDB 6WDQ) has been added to illustrate the orthosteric blockade resulting from the bound peptide. The picture was generated using Molecular Operating Environment software. Coordinates and structure factors were deposited in the Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do) under accession code 8UUI. IL23, interleukin 23; IL23R, IL23 receptor.

2

Comparison of (A) apo (p19 in pink; PDB 5MXA) vs (B) 23–446 peptide bound (peptide in blue, p19 in red; PDB 8UUI) X-ray structures of IL23. Peptide (blue) binding induces flexible disorder of p19 residues 47–51 (magenta) and a significant shift in loop Thr110–Pro120 and helices 2, 3, and 4, resulting in the formation of an induced-fit pocket. The picture was generated using Pymol software. IL23, interleukin 23; PDB, Protein Data Bank.

3

Key interactions between peptide 23–446 (blue) and IL23p19 (red) (A) π-stacking between Phe163 and Phe14 and Tyr10 (23–446). (B) π–Cation interaction between Arg162 and Trp18 (23–446). (C) H-bond between Leu116 backbone and Asn17 backbone (23–446). (D) H-bond between Ser114 backbone and Tyr19 backbone (23–446); CH–π interaction between Pro113 and Trp20 (23–446). IL23, interleukin 23.

4

FP assay development. (A) Titration of different probes to IL23. (B) Dose–response curve of peptide 14 and peptide 1. (C) Illustration of FP competition assay setup. (D) Quality assessment of the FP HTS pilot screen. Three-times SD values are labeled as blue or red lines in the graph. (E) FP single-point pilot screen: top 5 hits (% of inhibition). To confirm their specific binding to IL-23, the 5 Hits binding to IL23 was confirmed by DSF. They were subsequently titrated to determine their binding affinity with MicroScale Thermophoresis (MST), including a counter-screen against IL-23R to ensure selectivity. Hit 3 did not show any binding by DSF and MST and is likely a false positive. Hit 1 exhibits intrinsic fluorescence, which accounts for the observed signal exceeding 100%. However, its binding to IL-23 was independently validated using DSF and MST. We confirmed that the compound’s fluorescence did not interfere with the performance or interpretation of these assays. Compounds were purchased at enamine, and their purity was confirmed by LC/MS. FP, fluorescence polarization; HTS, high-throughput screening; IL23, interleukin 23; and SD, standard deviation.