항 류마티스 금제제의 분자적 작용기전

1. 항 류마티스 금제제의 분자적 작용기전 주 대 명 가톨릭대학교 의과대학 생화학교실

2. Cytokine signaling pathway involved in inflammatory arthritis (Choy et al., 2001)

3. NSAID Corticosteroids Auranofin Parenteral gold Hydroxychloroquine Minocycline Sulfasalazine Azathioprine Antirheumatic drugs “Second-line” drugs (“Disease-modifying” or “Slow-acting”) “First-line” drugs Penicillamine Methotrexate Cyclosporin Chlorambucil Cyclophosphamide

4. 항염작용을 나타내는 금제제. (1) gold sodium thiomalate, (2) gold thioglucose, (3) gold sodium thiosulfate, (4) gold sodium thiopropanosulfonate, (5) auranofin

5. Auranofin inhibits TNF gene expression by blocking NF-B activation

6. Proteins regulated by NF-B Proinflammatory cytokines TNF, IL-1, IL-2, IL-6, GM-CSF, M-CSF, G-CSF Chemokines IL-8, MIP-1 Gro-, -, and - Eotaxin Inflammatory enzymes iNOS, COX-2 5-Lipoxygenase cPLA2 Adhesion molecules ICAM-1, VCAM-1 E-selectin Receptors IL-2R ( chain) TCR ( chain)

7. NF-kB activation pathway LPS Virus TNF P NF-kB P P IKK P IkB IkB IkB IkB p50 p50 p50 p50 p65 p65 p65 p65 p50 p65 kB TNF

8. IkB kinase (IKK) • 500-900 kDa multi-subunit complex • Phosphorylates Ser 32 and 36 of IkBa • Inducible by NF-kB inducers • Contains IKKa,IKKb, andIKKg

9. IKK assay • Preparation of cell lysate • Immunoprecipitation with anti-IKK antibody • Incubation with [-32P]ATP and GST-IkB • Electrophoresis and autoradiography

10. Auranofin inhibits signal-induced activation of IKK

11. NF-kB activation pathway LPS Virus TNF P NF-kB P P IKK P IkB IkB IkB IkB p50 p50 p50 p50 p65 p65 p65 p65 p50 p65 kB TNF

12. Inhibition of in vitro IKK activity by gold compounds

13. Metal compounds that inhibit IKK activity in vitro ID50 (M) Metal compounds ZnSO4 Aurothiomalate HgCl2 Aurothioglucose AuCl3 CuSO4 CoCl2 Na2Cr2O7 8.7 10.9 12.3 19.6 24.1 26.9 55.7 69.2 CaCl2, FeSO4, FeCl3, NiCl2, (NH4)6Mo7O24, CdCl2, cis-Pt(NH3)2Cl2 (cisplatin), and lead acetate were not effective.

14. Zn2+ and Cu2+ inhibit TNF synthesis by blocking IKK activation

15. Reducing and thiol-reactive agents modulate IKK activity in vitro

16. Proposed pathway for signal transduction by reactive cysteine modification (Finkel, 2000)

17. Oxidizing agents inhibit TNF-induced NF-B reporter gene expression

18. Oxidizing agents inhibit signal-induced IKK activation and in vitro IKK activity

19. LPS TNF PKC IKK P P IkB IkB p50 p50 p65 p65 Gold Oxidative Stress

20. The three components of IKK. (Karin, 2000)

21. Inhibition of homodimeric IKK and IKK by various thiol-modifying agents

22. Schematic representation of proposed model of IKK regulation by phosphorylation. (Karin, 2000)

23. Activation loops of IKK, IKK, JNK, and p38

24. KD LZ HLH  IKK 165 178 191 IKK wtDLG Y A KDVDQG SL CT SFVG T LQ Y L APE IKK CASLAT S IKK SE ELCT E 166179 192 IKK wtDLG Y A KELDQG SL CT SFVG T LQ Y L APE IKK CA SLAT S IKK SE ELCT E IB Kinase(IKK) mutants

25. (A) IKK WT IKK CA Gold inhibits IKK through modification of Cys-179 in its activation loop Auranofin 03 10 30 03 10 30 (M) KA GST- IB IB IKK (B) IKK WT IKK CA Auranofin 03 10 30 03 10 30 (M) GST- IB KA IB IKK

26. H2O2 Diamide Cys-179 of IKK is necessary for the effect of H2O2 and diamide: kinase assay 0.3 1 3 0.3 1 (mM) TNF  + + + + + + KA GST- IB IB IKK IKK-HA  + + + + + + WT CA IKK-Flag  + + + + + + WT CA

27. Constitutively active IKK and IKK are inhibited by gold

28. Conclusion • Gold and oxidizing agents inhibit NF-B and IKK activation by modifying Cys-179 of IKK. • Modification of Cys-179 of IKK by gold directly inhibits activated IKK.

31. The activation segment in active forms of different kinases

32. The activation segment in inactive forms of different kinases