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Discovery of Small Molecule Inhibitory Compounds Targeting Toll-like Receptors 7 and 9 : Experimental Validation and Insights

  • 최양선 (Yang Seon Choi, 아주대학교 일반대학원)

초록/요약

Inflammatory diseases, including conditions like systemic lupus erythematosus and rheumatoid arthritis, are primarily driven by the abnormal release of proinflammatory cytokines from immune cells. Key to the onset and progression of these diseases are Toll-like receptors 7 (TLR7) and 9 (TLR9), which are found within the endosomes of dendritic cells and macrophages. Consequently, developing drugs that target these dysregulated endosomal TLRs is crucial for reducing systemic inflammation. Our research utilized computer-aided drug discovery to identify TLR inhibitory compound 10 (TIC10), a new low-molecular-weight compound, and its more potent derivative, TIC10g. Both compounds demonstrated the ability to inhibit both TLR7 and TLR9 signaling pathways. Specifically, TIC10g showed a stronger, concentration-dependent suppression of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) secretion mediated by TLR7 and TLR9 in a mouse macrophage cell line and mouse bone marrow dendritic cells, compared to TIC10. While TIC10g had a minor effect on TLR3 and TLR8 activation, it did not affect cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), highlighting its selectivity for TLR7 and TLR9. Further mechanistic studies suggested that TIC10g disrupted TLR9 activation by CpG DNA and suppressed downstream pathways through direct binding to TLR9. Western blot analysis confirmed that TIC10g reduced the phosphorylation of p65 (a subunit of NF-κB) and various mitogen-activated protein kinases (MAPKs), such as extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings collectively indicate that TIC10g acts as a novel, specific dual inhibitor of endosomal TLR7 and TLR9, thereby interfering with MAPK- and NF-κB -driven proinflammatory gene expression.

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목차

CHAPTER 1 : Introduction 1
1.1. The recognizer Toll-Like Receptor(TLR) of nucleic acid molecules 2
1.2. Correlation between TLRs and autoimmune diseases 2
1.3. Modulators of nucleic acid TLRs 3
CHAPTER 2 : Delelopment of TIC10 and its Derivates TIC10g 4
2.1. In-Silico drug screening method & materials 5
2.1.1 Compound library preparation for virtual screening 5
2.1.2 Homology modeling and establishing of the receptor structures 6
2.1.3 Phamacophore model generation and ligand-based screening 6
2.1.4 Structure-based virtual screening and derivates generation from initial lead 7
2.2 In-Vitro screening method & materials 8
2.2.1 Cell culture 8
2.2.2 Cell viability assay 9
2.2.3 Enzyme-linked immunosorbent assay (ELISA) and cytokine measurement 9
2.3. Screening result 10
2.3.1 Computationally designated lead-compound TIC series 10
2.3.2 TIC10 presents the dual-inhibition to both TLR9 and TLR 7 12
2.4. Advanced inhibition potency result of TIC10g 16
CHAPTER 3 : Mechanism Study of TIC10g 19
3.1. Validation and MOA(Mode of Action) method and materials of TIC10g 20
3.1.1 Molecular dynamics simulations 20
3.1.2 Signaling analysis by westernblot 20
3.1.3 Mouse bone marrow-derived dendritic cell (BMDCs) extraction and maturation 21
3.1.4 mRNA expression level measurement 21
3.1.5 Binding affinity test in surface plasmon resonance (SPR) 22
3.1.6 Competitive ELISA 22
3.1.7 Flow cytometry and count of active BMDC population 22
3.1.8 Schild analysis 23
3.1.9 Statistical analysis 23
3.2. Result of TIC10g Mechanism Study 24
3.2.1 TIC10g demonstrates pan-inhibition through endosomal TLRs but not non- nucleic TLRs 24
3.2.2 TIC10g interrupts CpG-DNA and modulates TLR9-dependent NK-κB/MAPK and IRF7 activation 24
3.2.3 TIC10g inhibits CpG-stimulated expression and maturation in mouse BMDCs 26
3.2.4 Discovery of inhibitory mechanism of TIC10g 28
3.2.5 Molecular dynamics study revealed the binding property of TIC10g to TLR7, 9 32
CHAPTER 4: Discussion & Conclusion 36
4.1 Discussion 37
4.2 Conclusion 39
References 40
Appendix 44

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