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Discovery and pharmacological investigation of MyD88 inhibitory peptides in autoimmune diseases

초록/요약

Myeloid differentiation primary-response 88 (MyD88) serves as a critical adaptor protein in innate immune signaling pathways, mediating downstream responses initiated by Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) through its Toll/interleukin-1 receptor (TIR) domain. Structurally, the MyD88 TIR domain facilitates homotypic interactions with receptor complexes, such as the TLR4-Mal-TIR assembly, enabling recruitment of downstream kinases like IRAK to activate NF-κB and MAPK pathways. Given its central role in propagating inflammatory signals, MyD88 has emerged as a therapeutic target for autoimmune diseases characterized by dysregulated immune activation. In this study, a rational peptide design strategy was employed to develop MyD88 inhibitory peptides (MyDIPs) targeting key interaction interfaces within the TIR domain. Computational modeling of the MyD88 TIR structure informed the synthesis of MyDIPs, peptides mimicking critical binding motifs. In the first round of screening, MyDIP2 was found to be inhibiting TLR4-induced TNF-α and IL-6 and thus was selected for further modifications. The MyDIP2 peptide was modified through alanine scanning method and consequently, four derivative peptides were designed. Through in vitro analysis of TNF-α inhibition and cytotoxicity, MyDIP2-4 was selected as the best peptide among the derivatives. Further in vitro evaluations in human THP-1 monocytes and murine RAW264.7 macrophages demonstrated that MyDIP2–4 potently suppressed MyD88-dependent signaling across both TLR and IL-1R pathways. For instance, pretreatment with MyDIP2–4 (IC50 = 2.6 µM) reduced LPS-induced TNF-α and IL-6 secretion, while also inhibiting IL-1β-mediated NF-κB activation. Surface plasmon resonance (SPR) assays confirmed high-affinity binding between MyDIP2–4 and recombinant MyD88-TIR (KD = 37 nM). This specificity underscores the peptide’s mechanism of competitively disrupting MyD88 homodimerization and receptor complex assembly. In vivo efficacy was validated in an imiquimod (IMQ)-induced psoriasis model, where topical MyDIP2–4 application significantly attenuated disease progression. Treated mice exhibited reduction in Psoriasis Area and Severity Index (PASI) scores compared to controls, alongside normalized epidermal thickness. Histopathological analysis revealed reduced epidermal thickness, while immunohistochemistry showed a dose dependent reduction in IL-17 expressing cells within lesional skin. These effects correlate with MyD88’s role in driving IL-23/IL-17 axis activation, a hallmark of psoriatic inflammation. The therapeutic potential of targeting MyD88’s TIR domain extends beyond psoriasis. Structural conservation across TLR/IL-1R family members positions MyDIP2–4 as a prototype for treating conditions like rheumatoid arthritis or sepsis, where MyD88 hyperactivity exacerbates tissue damage. Furthermore, the peptide’s modular design allows for optimization of delivery systems—such as nanoparticle encapsulation—to enhance bioavailability. This study highlights the feasibility of structure-guided peptide inhibitors to precisely modulate adaptor protein function, offering a novel strategy to counteract autoimmune pathogenesis while preserving essential immune surveillance.

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목차

Chapter 1 1
1. Introduction 2
1.1. Structure of Toll-like receptors 3
1.2. MyD88-mediated pathogenesis in autoimmune disorders. 7
1.3. MyD88 inhibitors against autoimmune diseases 9
1.4. MyD88 in psoriasis 12
Chapter 2 15
2. Materials and methods 16
2.1. Peptide design and synthesis 16
2.2. Materials and Reagent Preparation 18
2.3. Cell lines and Culture Conditions 19
2.4. Assessment of Peptide Cytotoxicity 19
2.5. Cytokine Quantification Experiments 20
2.6. Protein Quantification and Western Blot Analysis 20
2.7. RNA Extraction, Reverse Transcription, and Quantitative PCR 21
2.8. Surface Plasmon Resonance (SPR) Analysis 22
2.9. In Vivo Psoriasis Mouse Model: Induction, Treatment, and Histological Analysis 23
2.10. Statistical Analysis 23
Chapter 3 25
3. Results 26
3.1. Identification of MyDIP2 as an Inhibitor of MyD88-Mediated Signaling 26
3.2. Broad Inhibition of TLR Ligand-Induced Responses by MyDIP2 26
3.3. Peptide Optimization and Enhanced Inhibition by MyDIP2-4 29
3.4. Determination of IC₅₀ and Broader TLR Inhibition by MyDIP2-4 29
3.5. Inhibition of TLR and IL-1R Signaling in Human Macrophages 31
3.6. Biophysical Characterization of MyDIP2-4 and MyD88 Interaction by SPR 33
3.7. In-Depth Analysis of Peptide-MyD88 Interactions: Molecular Stability, Dynamics, and Binding Energetics 33
3.8. Thermodynamic Analysis of Binding Energetics 35
3.9. Evaluation of the therapeutic efficacy of MyDIP2-4 in imiquimod-induced psoriasis 37
Chapter 4 40
4. Discussion 41
Conclusion 47
Chapter 5 48
5. References 49

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