SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase - PubMed (original) (raw)

. 2001 Nov 20;98(24):13681-6.

doi: 10.1073/pnas.251194298.

D T Sasaki, B W Murray, E C O'Leary, S T Sakata, W Xu, J C Leisten, A Motiwala, S Pierce, Y Satoh, S S Bhagwat, A M Manning, D W Anderson

Affiliations

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase

B L Bennett et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

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Figures

Figure 1

Figure 1

Characterization of SP600125. (a) Chemical structure. (b) Structure-activity relationship study of anthrapyrazolones (IC50 values).

Figure 2

Figure 2

Inhibitory profile of SP600125. (a) SP600125 is an ATP-competitive inhibitor of JNK2 with a _K_i value of 0.19 ± 0.06 μM. At fixed concentrations of JNK2 (9 nM) and c-Jun (2 μM), both ATP and SP600125 concentrations were varied; (□) 0 nM, (○) 50 nM, (▵) 100 nM, and (◊) 300 nM SP600125. The _K_i value was derived from a nonlinear least-squares fit of the data to the kinetic equation for competitive inhibition. (b) SP600125 is a selective inhibitor of JNK. The inhibition profiles of SP600125 vs. related MAP kinases (ERK2 and p38–2) and an unrelated serine threonine kinase (PKA) were performed at _K_m levels of substrates. (c) Kinase selectivity data for SP600125.

Figure 3

Figure 3

Activity in T cells. (a) Jurkat T cells were resuspended in growth medium and stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), anti-CD3 (0.5 μg/ml), and anti-CD28 (2 μg/ml) for 30 min. SP600125 was added as a 10-min pretreatment at the concentrations indicated. The same cell lysates were examined by using different Abs targeting mitogen-activated protein kinase and NF-κB signaling pathways. Levels of nonphosphorylated IKK-1 were used as an indication of equal loading. (b) Inhibition of CD4+ cell activation and differentiation. Th0 cells isolated from either human cord or peripheral blood were cultured with anti-CD3/antiCD28, IL-12, anti-IL-4 in the absence or presence of SP600125 (30 μM). Culture aliquots were analyzed at different times for the expression of cell surface markers CD4, CD45RO, CXCR4, and CCR5. (c) CD4+ cells were differentiated into either Th1 or Th2 cultures over 5 days by using polarizing conditions before stimulating with anti-CD3/anti-CD28 in the presence of increasing concentrations of SP600125 for 15 h in triplicate. Culture supernatants were analyzed by simultaneous analyte reagent technology multiplex cytokine bead array. Data shown represent one of the duplicate experiments. Results are expressed as the mean of triplicate samples.

Figure 4

Figure 4

Inhibitory activity in primary human monocytes. (a) Human PBMCs isolated from peripheral blood were stimulated with LPS (100 ng/ml) for 4 h in the presence of increasing concentrations of SP600125. Cell lysates were prepared and analyzed by using multiplex mRNA detection (Systems Integration Drug Discovery Company). Results are expressed as the mean of triplicate determinations. (b) Primary human monocytes were isolated by positive selection (CD14) and stimulated with LPS (100 ng/ml) in the presence of increasing concentrations of SP600125. After 15 h, culture supernatants were analyzed for expression of IL-1β, -8, and TNF-α with an ELISA. Values are mean and SD for quadruplicate samples.

Figure 5

Figure 5

In vivo activity of SP600125. (a) CD-1 mice were dosed with SP600125 i.v. 15 min before injection with LPS or per os 30 min before injection with LPS. At 90 min, a blood sample was recovered, and the serum was obtained. Samples were analyzed for mouse TNF-α by using an ELISA (BioSource). Results are expressed as the mean and standard error with 4 animals per compound treatment group and 6 animals in vehicle control group. Asterisk (*) indicates P ≤ 0.05. DEX, dexamethasone 21-acetate. (b) Apoptosis in murine thymocytes. Thymocytes were isolated from C57BL/6 mice 48 h after injection with anti-CD3 (50 μg) i.p. and analyzed for CD4 and CD8 by flow cytometry. Control animals received no anti-CD3 or vehicle. SP600125 was administered at 0, 12, 24, and 36 h, 15 mg/kg s.c. Anti-CD3 administration was at a single time immediately after the first dosing of SP600125. Data for double-positive (CD4+ CD8+ ) cells is shown expressed as the mean percentage of total thymocytes (n = 4 per group).

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