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Computational Elucidation of the IL-17 receptor A/ACT1 Interaction Mechanism and Discovery of a Novel Antagonist Targeting IL-17 Signaling for Autoimmune and Inflammatory Disorders

초록/요약

The increasing prevalence of autoimmune and inflammatory diseases constitutes a significant worldwide public health concern, positioning the Interleukin-17 (IL-17) signaling pathway as a primary focus for therapeutic intervention. The central axis to the pathogenesis of inflammation driven by this pathway is the protein-protein interaction (PPI) between IL-17 receptor A (IL-17RA) and its downstream adapter protein, ACT1 (also known as TRAF3IP2). A fundamental obstacle in structure-based drug discovery (SBDD) aimed at targeting this pathway has been the absence of high-resolution structural data, detailing the therapeutically relevant IL-17RA-ACT1 complex. The absence of high-resolution structural characterization of the target substantially impedes the application of rational SBDD methodologies. This knowledge deficit was addressed through a comprehensive computational analysis, provided the first detailed mechanistic insight into the IL 17RA-ACT1 binding process. This computational approach revealed that the association between IL-17RA and ACT1 is not a simple, instantaneous event, but rather proceeds through a sophisticated three-phase mechanism involving initial fold recognition, conformational adaptation, and final SEFIR-SEFIR engagement. This detailed mechanism fundamentally alters the conventional understanding of how SEFIR domain interactions are mediated. By integrating techniques such as protein network analysis, molecular docking, and dynamics simulations demonstrated that IL-17RA functions as a rigid structural scaffold that accommodates the necessary conformational flexibility of ACT1 during the assembly of the complex. The analysis established exceptional binding stability for the complex, quantifying the free energy of binding (Δ Gbind) at -43.63 ± 0.48 kcal/mol. Furthermore, the study precisely identified specific hotspot residues that are critical for the initial recognition between the two proteins. The dynamic analysis highlighted a targetable conformational state—specifically, the dynamic opening of ACT1's SEFIR domain towards IL-17RA. These transient conformational states were previously undetectable using traditional structure-based methods, offering new opportunities for therapeutic intervention. This novel mechanistic and structural understanding is transformative, enabling the rational design of small molecules and biological agents capable of selectively disrupting pathogenic IL-17 signaling while preserving essential host defense mechanisms. The established computational framework provides a structural blueprint for developing targeted therapeutics against a range of IL-17-driven pathologies; including rheumatoid arthritis, psoriasis, and inflammatory bowel disease, which currently suffer from a lack of sufficiently targeted treatment options. Current therapies for Interleukin-17 (IL-17) driven diseases rely on large biological drugs, which are constrained by the need for injectable administration, high cost, and the risk of sustained, profound immune-suppression. The second project aimed to discover an orally bioavailable small molecule inhibitor targeting the upstream activation event of IL-17A ligand binding to the common IL-17 Receptor A (IL-17RA) subunit. A discovery strategy integrating ligand-based virtual screening with Generative Chemistry (Sequential Attachment-based Fragment Embedding, SAFE methodology) was employed, resulting in the identification and prioritization of the lead compound, ILR6. In vitro validation confirmed that ILR6 demonstrated potent broad-spectrum inhibitory activity against multiple IL-17 subtypes (IL-17A, E, and F), all of which utilize the IL 17RA subunit, with IC 50 values in the low micro molar range (10.99 μM for IL-17A, 6.56 μM for IL-17E, and 9.63 μM for IL-17F). Biophysical validation via Surface Plasmon Resonance (SPR) confirmed that ILR6 binds directly to IL-17RA with high affinity (KD = 4.01 μM), while exhibiting minimal binding to the IL-17A cytokine. Molecular Dynamics (MD) simulations (400 ns) and MM/PBSA analysis (ΔGbind = −22.65±3.83 kcal/mol) revealed the structural basis of antagonism. ILR6 strategically inserts into the interfacial valley near the D1/D2 elbow joint region of the IL-17RA extracellular domain (ECD). By acting as a molecular wedge, ILR6 prevents the cytokine from inducing the necessary D1-D2 conformational motions, which is the mechanical trigger for signal propagation. This binding stabilizes a non-productive conformation (RMSD divergence ≈ 1.9 Å), resulting in an allosteric arrest of the ECD that precludes the downstream SEFIR domains in the intracellular domain (ICD) from achieving the necessary alignment for ACT1 scaffold assembly. ILR6 thus provides a structurally validated lead compound with novel scaffold for developing selective, orally bioavailable antagonists of the IL-17 pathway.

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목차

Chapter 1 1
1. Introduction 2
1.1. Pro-inflammatory role of Interleukin 17 2
1.2. IL-17 Signaling pathology in inflammatory diseases 2
1.3. Molecular mechanism of IL-17 signaling initiation 3
Chapter 2 5
Computational elucidation of Interleukin-17 receptor A-E3 ubiquitin ligase TRAF3IP 2(ACT1) signaling nexus 6
2.1. Summary 6
2.2. Therapeutic rationale and research novelty 7
2.3. Protein Interaction Network 8
2.4. Physical Interactors of IL-17RA 8
2.5. Functional Profiling of IL-17RA and ACT1 9
2.6. Pathway Enrichment Analysis of IL-17 Signaling. 9
2.7. Protein-Protein Docking at the SEFIR Interface 10
2.8. Structural Dynamics 12
2.9. Inter-Residue Contact Analysis and Non-Covalent Interactions 13
2.10. Inter-Residue Network Centrality 14
2.11. 3D Interfacial Residue Analysis and Interaction Mapping 14
2.12. Relative Free Energy Calculation and Residue Decomposition 14
Chapter 3 16
3.1. Protein-Protein Interaction Network Analysis of IL-17RA 17
3.2. ACT1 as the Primary Direct Physical Interactor of IL-17RA 19
3.3. Co-biological Functions of IL-17RA and ACT1 20
3.4. Roles of IL-17RA and ACT1 in IL-17 Signaling Pathway 22
3.5. Refinement of Receptor and Adapter Protein Structures and Docking 23
3.6. Structural Dynamics Profile of the IL-17RA-ACT1 Complex 26
3.7. Conformational Energy Landscape and Stability of IL-17RA-ACT1 complex 29
3.8. Exploration of Dynamic Conformational Space via Principal Component 31
3.9. Dynamic Evolution of Inter-Residue Contacts 33
3.10. Inter-Residue Interaction Network and Critical Anchor Residues 35
3.11. Binding Site Search on Induced Conformations 36
3.12. Mapping of Druggable Small Molecule Binding Sites 39
3.13. IL-17RA-ACT1 Interfacial Residue Analysis and Polar Contact 40
3.14. Calculation of Total Binding Free Energy and Energy Term Correlations of IL-17RA- ACT1 system 42
3.15. Residue Decomposition Analysis of the IL-17RA-ACT1 Interface 44
Chapter 4 47
Generative Chemistry and CADD protocol- enabled Discovery and Experimental Validation of novel IL-17 Receptor Antagonist 48
4.1. Summary 48
4.2. Therapeutic rationale and research novelty 49
4.3. Database Preparation and Molecular Similarity Screening 50
4.4. De Novo Design, Optimization, and Diversity Enrichment via Generative 50
4.5. Homology Modeling and Refinement of the IL-17A-IL17RA Structure 52
4.6. Molecular Docking and Binding Mode Exploration 53
4.7. In Silico ADMET Evaluation of Hit Molecules 55
4.8. Synthetic Accessibility Assessment and Final Hit Prioritization 55
4.9. Cell Line and Culture Conditions for In Vitro Assay. 56
4.10. Assessment of Cell Viability using the Water-Soluble Tetrazolium Assay 56
4.11. Secreted Alkaline Phosphatase (SEAP) Reporter Assay for IL-17 Inhibition 57
4.12. Surface Plasmon Resonance (SPR) Analysis for Binding Kinetics 58
4.13. Geometric Refinement of Lead Compound ILR6 and Final Structural Validation 58
4.14. Molecular Dynamics Simulation 59
4.15. Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) Free 60
4.16. Residue-Wise Decomposition Analysis of Free Energy and Entropic Contribution 61
Chapter 5 63
5.1. Ligand Fingerprint Screening and Screening Library Construction 64
5.2. De Novo Design and Diversification of Chemical Space 65
5.3. Structural Preparation of the IL-17A-IL17RA Complex 66
5.4. Molecular Docking and Identification of Hit Compounds 67
5.5. Hit synthesizability 68
5.6. ADMET properties 69
5.7. Initial Cytotoxicity Screening of Hit Compounds 70
5.8. Inhibition of IL-17A-Induced SEAP Activity 72
5.9. ILR6 Dose-Dependent Cytotoxicity and Potency 72
5.10. Biophysical Validation: Surface Plasmon Resonance (SPR) Analysis 73
5.11. Geometric Optimization and Frontier Orbital Analysis of ILR6 75
5.12. Conformational Analysis and Complex Stability via Molecular Dynamics 77
5.13. MM/PBSA Binding Free Energy Estimation 80
5.14. Residue Contributions to Binding Energy 81
5.15. Structural Insights and Mechanistic Basis of ILR6 Inhibition 83
Chapter 6 87
6. Discussion 88
Chapter 7 91
7. Conclusion 92
Chapter 8 94
8. References 95
Chapter 9 100
9. Appendices & supplemental material 101

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