Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1β

The study addresses whether human IL-1β, a key pro-inflammatory cytokine implicated in numerous inflammatory diseases and cancer progression, can be selectively antagonized by a low-molecular-weight small molecule that disrupts its interaction with the IL-1R1 receptor. Antibody therapies (e.g., canakinumab) validate the target clinically but require parenteral administration and have limited tissue penetration (e.g., CNS). Prior efforts with peptide antagonists and fragment binders lacked in vivo efficacy or functional activity data. The purpose of this work is to discover and characterize a selective, biologically active small-molecule antagonist of hIL-1β, define its structural binding mode, and demonstrate functional antagonism in cellular systems, thereby opening avenues for orally available therapeutics targeting IL-1β-driven pathologies.

- hIL-1β is central to inflammation and is linked to disorders such as rheumatoid arthritis, gout, periodic fevers, neuroinflammation, atherosclerosis, and cancer. Antibody blockade (e.g., canakinumab) has shown clinical benefits, including reduced cardiovascular risk (CANTOS). - Peptidic IL-1R1 antagonists (up to 21 amino acids) were reported but with no clear in vivo efficacy. - Crystallography-based fragment screening previously identified binders to hIL-1β, but without reported affinity or functional activities. - Small-molecule antagonism of related cytokines (e.g., IL-36γ antagonist A-552) suggests that allosteric, non-overlapping sites can modulate cytokine-receptor engagement; however, binding sites differ sufficiently to confer isoform specificity. - Conceptual advances in drug discovery emphasize cryptic pockets and protein conformational dynamics as opportunities to expand druggability for challenging protein-protein interactions.

- Protein production: Mature hIL-1β expressed in E. coli and purified via ion-exchange and size-exclusion chromatography. Additional His-tagged constructs produced and purified by immobilized metal affinity chromatography and SEC. - Fragment-based screening: 3,452 fluorinated fragments screened in mixtures using 1H-NMR relaxation-based methods on Bruker spectrometers; hits validated with 1H-15N HMQC NMR. - Hit validation and binding-site mapping: Enantiomer separation of hit compound 1; binding assessed by NMR with chemical shift perturbation mapping to identify affected residues and locate the binding site (loops β4–5 and β7–8). - SAR and optimization: Semi-quantitative 1H-15N HMQC readouts and quantitative 19F reporter displacement assays guided SAR. Modifications on indoline (ring A) and phenol (ring C), plus growth vectors from ring C and indoline C-3 quaternary center, explored to enhance affinity and functional activity. - Biophysical characterization: - NMR (various 1H/13C/15N experiments) to monitor binding regimes and kinetics; CEST NMR to interrogate conformational exchange and excited states. - Surface plasmon resonance (SPR) to determine Kd and kinetics (kon, koff) using a 1:1 Langmuir model. - Structural biology: X-ray crystallography of hIL-1β in complex with (S)-2; structure determination via molecular replacement and refinement; analysis of conformational changes (notably loop β4–5 displacement) and ligand interactions. - Receptor interaction assay: TR-FRET assay quantifying inhibition of hIL-1β binding to IL-1R1; selectivity assessed versus IL-1α. - Cellular assays: - Primary human dermal fibroblasts IL-6 release assay in response to hIL-1β or hIL-1α; pharmacological controls canakinumab (anti-IL-1β) and anakinra (IL-1R antagonist). - HEK293 reporter gene assay (NF-κB/AP-1-driven SEAP) to measure IL-1 signaling; tested inhibition by (S)-2 versus controls; assessed selectivity for IL-1β over IL-1α. - Data analysis: NMR processing with TOPSPIN/CCPN tools and custom scripts (FRPire); SPR analyzed with Sierra Analyzer; IC50 fitting with Prism/Helios; crystallographic data processed with autoPROC/Phaser/autoBUSTER/Coot/Phenix.

- Discovery of a selective small-molecule antagonist (S)-2 that binds to hIL-1β and blocks its interaction with IL-1R1. - Affinity and kinetics: (S)-2 binds hIL-1β with Kd ≈ 1.1 µM by SPR and displays slow association kinetics (kon ≈ 1.2×10^6 M−1 s−1; koff ≈ 0.12 s−1). - Functional inhibition of receptor engagement: In a TR-FRET assay, (S)-2 inhibits the hIL-1β/IL-1R1 interaction with an IC50 ≈ 4.0 ± 1.1 µM; no inhibition observed for IL-1α up to >200 µM, indicating selectivity. - Cellular activity: - HEK293 reporter gene assay: IC50 ≈ 5.3 ± 2.0 µM for hIL-1β-induced signaling; inactive when signaling is driven by hIL-1α. - Primary human dermal fibroblasts: Inhibits IL-1β–induced IL-6 release with IC50 ≈ 7.9 ± 2.0 µM; consistent with an extracellular mechanism of action and aligned with TR-FRET potency. - Structural mechanism: X-ray structure reveals (S)-2 binds to a cryptic, allosteric pocket formed by displacement of loop β4–5 (≈1 Å) and involving loop β7–8 and β-strand 5 residues; this antagonist-bound conformation is incompatible with engagement of IL-1R1 domain 3 (site B), explaining antagonism. - Stereoselectivity: Activity resides in the (S)-enantiomer; the (R)-enantiomer shows no measurable binding or functional activity. - Conformational dynamics: CEST NMR identifies a minor, conformationally excited state of hIL-1β (≈30% population, kex ≈ 22 s−1) comprising residues that form the cryptic pocket; ligand binding stabilizes a single state, linking access to the pocket with the excited-state ensemble.

The work demonstrates that hIL-1β, a classical cytokine with a challenging protein-protein interaction surface, possesses a cryptic allosteric pocket that can be targeted by small molecules to antagonize receptor engagement selectively. By exploiting fragment-based discovery and structure-guided SAR, the team optimized an initial low-affinity hit into (S)-2, a single-digit micromolar antagonist that disrupts IL-1R1 binding and downstream signaling in cells, while sparing IL-1α. Structural analysis elucidates how ligand-induced displacement of loop β4–5 perturbs site B interactions with IL-1R1 domain 3, providing a clear mechanistic rationale for antagonism. NMR evidence connects this binding mode to an excited conformational state of the cytokine, underscoring the role of protein dynamics in enabling access to otherwise hidden sites. Collectively, the findings validate hIL-1β as amenable to small-molecule allosteric antagonism and highlight cryptic pockets as actionable features for drug discovery against cytokine-receptor interfaces.

This study identifies and characterizes a selective, low-molecular-weight hIL-1β antagonist, (S)-2, that binds an allosteric cryptic pocket to prevent IL-1R1 engagement and block downstream signaling in cellular assays. The integrated biophysical, structural, and cellular data establish a mechanistic link between a conformationally excited state of hIL-1β and ligand access to a druggable site. The work expands the druggability landscape of cytokines and supports the feasibility of small-molecule antagonism for IL-1β. Future research should focus on enhancing potency and pharmacokinetic properties, evaluating in vivo efficacy and safety, and exploring brain-penetrant or orally bioavailable analogs to broaden therapeutic applications, including neuroinflammatory conditions.

- Potency remains in the micromolar range; further optimization is needed for therapeutic development. - No in vivo efficacy or pharmacokinetic/pharmacodynamic data are reported. - Selectivity was primarily assessed against IL-1α; broader cytokine profiling and off-target assessment are not detailed. - The antagonist engages a cryptic pocket linked to conformational dynamics; potential context-dependent variability in pocket accessibility in vivo is unaddressed. - Some inconsistencies between summarized and tabulated values (e.g., reported SPR Kd) highlight assay and data-integration complexities.