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