Low-molecular weight human interleukin-1β antagonist discovered

In a recent study published in Nature Communications, researchers described the detection of a human interleukin-1β (hIL-1β) antagonist with low molecular weight (LMW) that disrupts interactions with the interleukin-1 receptor, type I (IL-1R1) receptor.

Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1β
Study: Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1β. Image Credit: Jarun Ontakrai/Shutterstock.com


Oral antagonists of the pro-inflammatory cytokine IL-1 are currently unavailable. Despite its therapeutic advantages, LMW antagonists are not available to treat a wider range of illnesses.

Further research is required to develop LMW-type antagonists with efficacies comparable to those of human interleukin-1β-targeted antibodies to lower the burden of inflammatory diseases and improve the standard of care.

About the study

In the present study, researchers reported on the development of an LMW human interleukin-1β antagonist compound that inhibited the cytokine's binding to its receptor, interleukin 1 receptor type I, in cellular and biochemical experiments with half-maximal inhibitory concentration (IC50) values within single-digit μM ranges.

The researchers performed fragment-based screening to identify compounds that could bind with human interleukin-1β. The local fluorine environment (LEF4000) library of 3,452 compounds containing CF, CF2, or CF3 moieties was screened using conventional 19F-nuclear magnetic resonance (NMR) spectroscopy transverse relaxation studies. Hits were nominated based on the extent of relaxation increase in protein presence.

The hIL-1 binder 1 was discovered by a fragment screen. Nuclear magnetic resonance (NMR) was then used to map the binding location of 1. Chemical shift variations of amide 1H- and 15N-resonances were mapped to protein sequences to identify the ligand binding site. Compound 1 was optimized to obtain the optimized form of human interleukin-1β antagonist enantiomer (S)-2.

1H-13C-HMQC nuclear magnetic resonance studies were conducted to evaluate the binding affinities of compound 1 derivatives. A 19F-reporter-based displacement assay was designed to improve throughput, minimize protein intake, and provide an accurate read-out. A structure-activity relationship (SAR) study was carried out.

A FRET-based receptor displacement experiment was performed once drugs attained double-digit M binding affinity. The researchers supplemented the findings obtained by evaluating the binding affinities of chosen chemicals by surface plasmon resonance (SPR). Compound binding to human interleukin-1β and IL-1 binding to IL-1R1 were investigated.

Using an IL-6 release experiment in human primary dermal fibroblasts, the researchers evaluated whether occupancy of the above-mentioned binding site may influence hIL-1β-induced cellular activity. The researchers also employed a reporter gene assay in human embryonic kidney (HEK)293 cells to detect IL-1 signaling.

To validate the existence of this equilibrium and further characterize the residues participating in the related conformational exchange mechanism, the researchers used chemical exchange saturation transfer (CEST) NMR measurements.


In cellular biochemical and biophysical studies, enantiomer (S)-2, a low-affinity fragment-based screening hit 1, was shown to bind to and inhibit hIL-1β with single-digit micromolar activity. The chemical was found to be attached to a previously unknown cryptic pocket on hIL-1β, involving residues at the interface with the complex’s third Ig domain. This finding underlines the therapeutic potential of targeting hIL-1β with low-molecular-weight molecules.

Compound 1’s binding site is located near strand 5, whose hydrophobic residues were similarly altered by ligand interaction in the 13C and 1H HMQC spectra. Compound 1 was verified as a binder by 2D NMR, and additional analysis included compound 1 enantiomer separation, giving enantiomers I-1 and (S)-1, with only the enantiomer (S)-1 binding to the protein.

Enantiomer (S)-2 bound to human interleukin-1β and blocked interactions with its cognate receptor interleukin 1 receptor of type I with comparable efficacy in the FRET-based experiment. The chemical modification of the main fragment improved ligand-protein interactions in the bound form, it did not affect the ligand-independent process preceding drug binding.

The antagonist-induced structural alterations in hIL-1β prevented contact with the heterodimeric receptor on the surface of native cells. (S)-2 binding to human interleukin-1β did not alter the structural conformation of human IL-1β residues at site A, but when bound to ligands, loop 4-5 adopted a conformation incompatible with the cytokine’s receptor-bound form at B site. This is the structural foundation for (S)-2 and its analog's antagonistic action.

Enantiomer (S)-2 binding to human interleukin-1β was affected by a conformational equilibrium. Val47, a key residue in the cryptic pocket, was shown to be centrally positioned in unliganded hIL-1β and to move by 4.3 Å when displaced by (S)-2. The cryptic pocket mutation altered the balance of human interleukin-1β toward its minor form, resulting in the hIL-1β form capable of ligand binding.

The binding process is now regulated by relatively rapid exchange kinetics, with protein resonances moving ligand concentration-dependently. This conformational exchange mechanism involving Val47 occurred concurrently with drug binding, establishing a mechanistic connection between the cryptic pocket and the minor state of human interleukin-1β.

The engagement of the cytokine at two key binding sites (sites A and B) was necessary for human interleukin-1β-to-IL-1R1 binding. The antagonists' targeting of the cryptic pocket allows for cytokine specificity.


Overall, the study findings highlighted the identification of fragment 1 and its development into the optimized human interleukin-1β antagonist molecule (S)-2, demonstrating the potential of cryptic binding sites in drug discovery.

This result adds to the rising number of instances in which cryptic pockets play a role in interactions between small-molecule ligands and therapeutic targets, highlighting the importance of cryptic binding sites in current drug development.

Journal reference:
Pooja Toshniwal Paharia

Written by

Pooja Toshniwal Paharia

Pooja Toshniwal Paharia is an oral and maxillofacial physician and radiologist based in Pune, India. Her academic background is in Oral Medicine and Radiology. She has extensive experience in research and evidence-based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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