Development of New IL-1R Antagonists with Improved Anti-inflammatory Efficacy.

Background

Anakinra, a recombinant human interleukin-1 receptor antagonist (hIL-1Ra), is a widely used anti-inflammatory biologic for conditions like rheumatoid arthritis and gout. However, its limited potency and dose-dependent side effects restrict broader therapeutic application, highlighting a need for more potent and stable IL-1R antagonists.

Methods

To develop improved IL-1R antagonists, we rationally designed six hIL-1Ra variants using structure-guided mutagenesis. Molecular dynamics simulations and thermodynamic integration predicted enhanced binding stability, with an average binding free energy improvement of -7.8 ± 0.9 kcal/mol compared to wild-type hIL-1Ra (hIL-1Ra WT). We assessed variant functions in microglia-derived HMC-3 cells by measuring IL-1β and IL-6 mRNA suppression and evaluated their ability to attenuate IL-1β-induced NMDAR hyperactivation in cultured cortical neurons using electrophysiological recordings. In vivo validation was performed using Nlrp3 D301N knock-in mice, a model of chronic neuroinflammation.

Results

All six hIL-1Ra variants demonstrated enhanced anti-inflammatory activity, suppressing IL-1β and IL-6 expression by 25-53% in HMC-3 cells. The E127Q variant exhibited the greatest efficacy. In primary cultured neurons, hIL-1Ra E127Q more effectively inhibited IL-1β-induced NMDAR-mediated postsynaptic responses at lower concentrations than hIL-1Ra WT. Furthermore, acute administration of hIL-1Ra E127Q, but not hIL-1Ra WT, reversed elevated NMDAR activity in the medial prefrontal cortex of Nlrp3 D301N knock-in mice.

Conclusion

This study successfully developed next-generation hIL-1Ra variants with superior receptor binding and anti-inflammatory activity. E127Q emerged as a promising therapeutic candidate, effectively attenuating inflammatory signaling and neuroinflammatory responses both in vitro and in vivo. These findings underscore the significant therapeutic potential of engineered IL-1R antagonists for treating inflammation-driven neurological and systemic disorders, paving the way for improved anti-inflammatory therapies.