항 류마티스 금제제의 분자적 작용기전
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Transcript of 항 류마티스 금제제의 분자적 작용기전
항 류마티스 금제제의 분자적 작용기전
주 대 명가톨릭대학교 의과대학 생화학교실
Cytokine signaling pathway involved in inflammatory arthritis (Choy et al., 2001)
Antirheumatic drugs
NSAID Corticosteroids
AuranofinParenteral goldHydroxychloroquineMinocyclineSulfasalazineAzathioprine
Chlorambucil Cyclophosphamide
“Second-line” drugs(“Disease-modifying” or “Slow-
acting”)“First-line” drugs
Penicillamine Methotrexate Cyclosporin
항염작용을 나타내는 금제제 . (1) gold sodium thiomalate, (2) gold thioglucose, (3) gold sodium thiosulfate, (4) gold sodium thiopropanosulfonate, (5) auranofin
Auranofin inhibits TNF gene expression by blocking NF-B activation
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)
NF-B activation pathway
IB
p65p50
IkB
p50
p50
p50p65 p65 p65
IB IBP P
P PIKK
B
p50 p65
TNF
NF-B
LPSTNF Virus
IB kinase (IKK)
• 500-900 kDa multi-subunit complex• Phosphorylates Ser 32 and 36 of IB• Inducible by NF-B inducers• Contains IKKIKKandIKK
IKK assay
• Preparation of cell lysate• Immunoprecipitation with anti-IKK antibody• Incubation with [-32P]ATP and GST-IkB• Electrophoresis and autoradiography
Auranofin inhibits signal-induced activation of IKK
NF-B activation pathway
IB
p65p50
IkB
p50
p50
p50p65 p65 p65
IB IBP P
P PIKK
B
p50 p65
TNF
NF-B
LPSTNF Virus
Inhibition of in vitro IKK activity by gold
compounds
Metal compounds that inhibit IKK activity in vitro
Metal compounds ID50 (M)ZnSO4
AurothiomalateHgCl2AurothioglucoseAuCl3CuSO4
CoCl2Na2Cr2O7
8.710.912.319.624.126.955.769.2
CaCl2, FeSO4, FeCl3, NiCl2, (NH4)6Mo7O24, CdCl2, cis-Pt(NH3)2Cl2 (cisplatin), and lead acetate were not effective.
Zn2+ and Cu2+ inhibit TNF synthesis by blocking IKK activation
Reducing and thiol-reactive agents modulate IKK activity in vitro
Proposed pathway for signal transduction by reactive cysteine modification (Finkel, 2000)
Oxidizing agents inhibit TNF-induced NF-B reporter gene expression
Oxidizing agents inhibit signal-induced IKK activation and in vitro IKK activity
IB
p65p50
IKK
LPS
TNF PKC
GoldOxidative Stress
IB
p65p50
P P
The three components of IKK. (Karin, 2000)
Inhibition of homodimeric IKK and IKK by various thiol-modifying agents
Schematic representation of proposed model of IKK regulation by phosphorylation. (Karin, 2000)
Activation loops of IKK, IKK, JNK, and p38
IKK KD LZ HLH
165 178 191
IKK wt DLG Y A KDVDQG SL CT S FVG T LQ Y L APEIKK CA SL AT SIKK SE EL CT E
166 179 192
IKK wt DLG Y A KELDQG SL CT S FVG T LQ Y L APEIKK CA SL AT SIKK SE EL CT E
IB Kinase(IKK) mutants
Gold inhibits IKK through modification of Cys-179 in its activation loop
AuranofinIKK WT
0 3 10 30 0 3 10 30 (M)
GST-IB
KA
IB IKK
IKK CA
IKK WT
0 3 10 30 0 3 10 30 (M)GST-IBKA
IB IKK
IKK CAAuranofin
0
50
100
150
0 3 10 30Auranofin (uM)
%Con
trol
WTCA
(A)
(B)
TNF + + + + + +
H2O2 Diamide
0.3 1 3 0.3 1 (mM)
GST-IB
KA
IB IKK
IKK-Flag + + + + + +
WT
CA
IKK-HA + + + + + +
WT
CA
Cys-179 of IKK is necessary for the effect of H2O2 and diamide:kinase assay
Constitutively active IKK and IKK are inhibited by gold
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.
The activation segment in active forms of different kinases
The activation segment in inactive forms of different kinases