ABSTRACT
The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.
Subject(s)
Interleukin-6 , Molecular Docking Simulation , Protein Binding , Receptors, Interleukin-6 , Interleukin-6/metabolism , Interleukin-6/genetics , Humans , Receptors, Interleukin-6/metabolism , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/chemistry , Cytokine Receptor gp130/metabolism , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/chemistry , Mutagenesis, Site-Directed , Binding Sites , STAT3 Transcription Factor/metabolism , Phosphorylation , MutationABSTRACT
Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.
Subject(s)
Cytokine Release Syndrome , Interleukin-6 , Humans , Cytokine Release Syndrome/drug therapy , Interleukin-6/metabolism , Interleukin-6/antagonists & inhibitors , Crystallography, X-Ray , Receptors, Interleukin-6/antagonists & inhibitors , Receptors, Interleukin-6/metabolism , Interleukin-1/metabolism , Interleukin-1/antagonists & inhibitors , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin 1 Receptor Antagonist Protein/chemistry , Interleukin 1 Receptor Antagonist Protein/metabolism , Drug Design , Cytokine Receptor gp130/metabolism , Cytokine Receptor gp130/antagonists & inhibitors , Cytokine Receptor gp130/chemistry , Protein Binding , Signal Transduction/drug effects , Receptors, Interleukin-1 Type I/antagonists & inhibitors , Receptors, Interleukin-1 Type I/metabolismABSTRACT
Cytokines play a major role in the pathogenesis and progression of psoriasis. Interleukin (IL)-30 is a multifunctional cytokine. It binds to glycoprotein 130 (GP130) and inhibits the GP130 signaling pathways of psoriasis associated cytokines such as IL-6, IL-11, and IL-27. The study intended to assess associations of IL-30 and GP130 with the risk of psoriasis and Psoriasis Area Severity Index (PASI) score. Therefore, we measured the serum levels of IL-30 and GP130 in psoriasis patients and in a control group. An enzyme linked immunosorbent assay (ELISA) technique was used to measure IL-30 and GP130 levels in the serum of 43 patients and 43 normal controls. Statistical analysis of IL-30 and GP130 serum levels among patients and control groups and their correlation with PASI scores were performed. IL-30 serum levels showed a significant increase in patients with psoriasis compared with controls (p < 0.001) and a positive correlation with PASI scores. While serum levels of GP130 were not different in psoriatic patients and in the control group. Furthermore, the receiver operating characteristic (ROC) curve showed that IL-30 had diagnostic ability for prediction of psoriasis in comparison to controls, at cut of point of >14.34 showed a sensitivity of 97.7%, 100% specificity. In conclusion, IL-30 was elevated in psoriasis patients than controls, therefore, it can be considered a sensitive biomarker for diagnosis of psoriasis.