Identification of Small-Molecule Inhibitors of Human Siglec-1?Sialic Acid Interaction via Structure-Based Virtual Screening

다국어 초록 (Multilingual Abstract)

Xenotransplantation is a potential solution to the shortage of human organ donors, yet acute and chronic rejection remain major barriers. This study examined the role of human Sialoadhesin (Siglec-1/CD169), a sialic acid-binding receptor, in recognizing porcine cells due to glycan incompatibility, and assessed candidate molecules targeting hSiglec-1 for their ability to suppress macrophage-driven inflammation and phagocytosis. An AI-based protein structure prediction model was used to screen 1,615 candidate molecules for hSiglec-1 binding. THP-1 monocytes were differentiated into high Siglec-1-expressing M1 macrophages via PMA, LPS, and IFN-γ stimulation. Cells were treated with anti-hSiglec-1 antibody or candidate molecules, then co-cultured with porcine endothelial cells (PECs) or triple-knockout porcine red blood cells (pTKO RBCs) to evaluate cytokine production and phagocytic activity. hSiglec-1 was identified as a key mediator of M1 macrophage responses to PECs. Three candidate molecules markedly reduced pro- inflammatory cytokines (TNF-α, IL-6, IL-12) and coagulation factors (Tissue factor, Thrombospondin-1, PAR-1). Two also significantly suppressed phagocytosis of pTKO RBCs. In this study, we nominated hSiglec-1 as a promising therapeutic target for mitigating macrophage-mediated inflammation and phagocytosis in xenotransplantation. Leveraging molecules with hSiglec-1-binding potential may offer a practical strategy to improve graft survival and reduce rejection risk.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1. Current status of organ transplantation 1
• 1.2. Graft rejection barrier in Xenotransplantation 1
• 1.3. Macrophage-driven inflammation, coagulation and phagocytosis in xenotransplantation 2
• 1.4. Lectin-glycan mediates xenograft rejection 3
• 1.5. Artificial Intelligence (A.I)-driven the virtual drug screening development 4
• 1.6. Human Sialoadhesin (hSiglec-1/hCD169) – A potential therapeutic target in xenotransplantation 6
• 2. MATERIALS AND METHODS 8
• 2.1. Molecular Docking: Computational Tools and Online Resources 8
• 2.2. Molecular Dynamics Simulation 8
• 2.3. Animal 9
• 2.4. Cell culture 9
• 2.5. Generation of THP-1-derived M1 macrophages express high levels of Siglec-1 9
• 2.6. Cytotoxicity Measurement by MTT Assay 9
• 2.7. Drugs’ concentration optimization 10
• 2.8. Coculture experiment 10
• 2.9. RNA archival and quantitative real-time polymerase chain reaction (qPCR) for the gene expression 11
• 2.10. ELISA 11
• 2.11. Phagocytosis assay 11
• 2.12. Red blood cell counting. 12
• 2.13. Cell marker measurement 13
• 3. RESULTS 15
• 3.1 Characterization of High Siglec-1-Expressed Human M1 Macrophages 15
• 3.2 Molecular Docking 18
• 3.3 Molecular Dynamics Simulation. 23
• 3.4. Cytotoxic Analysis of LBS-004, LBS-005, and LBS-006 28
• 3.5. Cytotoxic Alleviation of Screened Molecules in The Xenogenic Model 31
• 3.6. Siglec-1-Driven Inflammatory and Coagulatory Responses between hM1 Macrophages and Pig Endothelial Cells 34
• 3.7. LBS-004, LBS-005, and LBS-006 Alleviate Inflammation and Coagulation in Xenogenic Model. 37
• 3.8. LBS-004, LBS-005, LBS-006 Reduce hM1 Inflammatory Cytokines in a Sialoadhesin–Dependent Manner 40
• 3.9. LBS-004, LBS-005, and LBS-006 Mitigate Phagocytosis Activity of Macrophages Against Triple Knockout Pig Red Blood Cells 43
• 4. DISCUSSION 46
• 5. REFERENCES 50