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

Interleukin-1β (IL-1β) is a pivotal pro-inflammatory cytokine crucial to innate and adaptive immunity. Produced by myeloid cells in response to stimuli, mature IL-1β binds to its receptor (IL-1R) initiating inflammatory responses. Its central role in inflammation links it to various disorders including rheumatoid arthritis, gout, and neuroinflammation. While antibody-based therapies targeting IL-1β have shown promise, the need for an orally bioavailable, low-molecular-weight antagonist remains. Such a molecule would broaden the therapeutic applications of IL-1β-targeted therapies, particularly in conditions like neuroinflammation where the blood-brain barrier poses a challenge for antibody-based drugs. Although some peptide-based antagonists have been described, their in vivo efficacy is uncertain. Existing low-molecular-weight binders identified through fragment screening lack reported affinity or functional activity data. This study aimed to identify and characterize a novel low-molecular-weight IL-1β antagonist with significant potential for therapeutic translation.

Extensive research highlights the critical role of IL-1β in inflammatory diseases. The IL-1 family, including IL-1α, IL-1β, IL-1Ra, and others, regulates immunity through complex interactions. The proteolytic processing of IL-1β precursor proteins, typically by inflammasomes, is essential for its release and activity. IL-1β's involvement in atherosclerosis and cancer progression has also been established, with studies like the CANTOS trial demonstrating the efficacy of canakinumab, an anti-IL-1β antibody, in reducing cardiovascular risk. However, the need for an orally available small molecule antagonist remains unmet, limiting the therapeutic potential of IL-1β inhibition, particularly in diseases affecting the central nervous system. Previous attempts to develop peptide antagonists, while showing some promise, have not demonstrated consistent in vivo efficacy.

The study employed a fragment-based screening approach using a library of 3452 fluorinated compounds. Initial screening utilized ¹H-NMR to identify potential binders. Selected hits were validated using deuterated ¹H-¹⁵N-HMQC NMR experiments. The binding site of a prototypical hit, compound 1, was mapped using NMR, revealing interactions with loops β4–5 and β7–8 of hIL-1β, regions also involved in the IL-1β/IL-1RI complex interface. Compound 1 was then optimized using a combination of NMR and ¹⁹F-reporter based displacement assays to improve potency, leading to the identification of compound (S)-2. Structure-activity relationship (SAR) studies guided modifications, focusing on the indoline and phenol moieties of the molecule. The binding kinetics of (S)-2 were characterized using surface plasmon resonance (SPR). Cellular activity of (S)-2 was assessed using IL-6 release assays in primary human dermal fibroblasts and a reporter gene assay in HEK293 cells. The crystal structure of the hIL-1β/(S)-2 complex was determined using X-ray crystallography. Chemical exchange saturation transfer (CEST) NMR experiments were conducted to investigate the conformational equilibrium of hIL-1β and its relationship to (S)-2 binding. Protein expression and purification methods involved E. coli expression systems with subsequent chromatographic purification techniques.

A low-molecular-weight hIL-1β antagonist, compound (S)-2, was identified and characterized. (S)-2 exhibits single-digit micromolar affinity for hIL-1β (SPR Kd = 1.1 µM) and effectively inhibits the hIL-1β/IL-1RI interaction (FRET IC50 = 0.40 µM). NMR studies mapped the binding site to a previously unknown cryptic pocket formed by residues in loops β4–5 and β7–8 of hIL-1β. X-ray crystallography confirmed this binding site and revealed a conformational change in loop β4–5 upon ligand binding. This conformational change prevents proper interaction with IL-1R1, explaining the antagonistic activity. (S)-2 effectively inhibited hIL-1β-induced IL-6 release in primary human fibroblasts and IL-1β-mediated signaling in HEK293 cells, demonstrating its cellular activity. CEST NMR experiments revealed that the binding site is part of a minor, conformationally excited state of hIL-1β, suggesting that targeting this state is crucial for effective antagonism. The high stereoselectivity of (S)-2 binding was explained by the X-ray structure, demonstrating the importance of specific interactions with the cryptic pocket. The selectivity for hIL-1β over hIL-1α was confirmed in cellular assays.

The discovery of compound (S)-2 represents a significant advancement in the development of orally available, low-molecular-weight IL-1β antagonists. The identification of a novel cryptic binding site on IL-1β expands the druggable space of this important therapeutic target. The allosteric mechanism of action, involving a conformational change that disrupts the interaction with IL-1R1, highlights the potential of targeting dynamic protein states for drug development. The successful inhibition of IL-1β activity in primary human cells validates the therapeutic potential of (S)-2. The study's findings support further investigation of compound (S)-2 and its analogs as potential therapeutics for various IL-1β-mediated inflammatory diseases. Comparison with a known IL-36γ antagonist suggests a potentially conserved mechanism for inhibiting cytokine-receptor interaction.

This research successfully identified and characterized a novel low-molecular-weight, selective, and biologically active antagonist of human IL-1β, compound (S)-2. This molecule binds to a previously unknown cryptic pocket on IL-1β, disrupting its interaction with the IL-1R1 receptor via an allosteric mechanism. The compound’s efficacy in cellular assays indicates promising therapeutic potential. Future work should focus on optimizing (S)-2's pharmacokinetic properties for in vivo studies and exploring its therapeutic application in various IL-1β-related diseases.

The study primarily focused on in vitro characterization. Further in vivo studies are needed to evaluate the efficacy, safety, and pharmacokinetic properties of (S)-2. The current study did not investigate the long-term effects of (S)-2 treatment. Although the study demonstrated high selectivity for hIL-1β over hIL-1α, further investigation may be needed to assess selectivity against other members of the IL-1 family.