p38 (IC50 = 50 nM); p38β2 (IC50 = 500 nM)
p38α (IC₅₀ = 0.0006 μM; Ki = 0.0005 μM) and p38β (IC₅₀ = 0.0015 μM); the compound showed >100-fold selectivity over p38γ/δ (IC₅₀ >0.1 μM) and >1000-fold selectivity over other MAPKs (ERK1/2, JNK1/2) and non-MAPK kinases (AKT, EGFR, RAF1) when tested at 10 μM [1]

SB203580 has an IC50 of 3-5 μm and inhibits the proliferation of murine CT6 T cells, BAF F7 B cells, or primary human T cells when IL-2 is present. Although the concentration needed is a little bit higher and the IC50 is above 10 μm, SB203580 also inhibits IL-2-induced p70S6 kinase activation. With an IC50 in the 3-10 μm range, SB203580 also inhibits the activity of PDK1 in a dose-dependent manner.[1] SB203580 has an IC50 of about 0.07 μm for blocking p38-MAPK stimulation of MAPKAPK2, whereas it has an IC50 of 3–10 μm for blocking total SAPK/JNK activity. Higher concentrations of SB203580 cause the ERK pathway to be activated, which then improves the transcriptional activity of NF-κB.[2] Human hepatocellular carcinoma (HCC) cells are induced to undergo autophagy by SB203580.[3]\n\n

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\n\nSB203580 Inhibits IL-2-induced Rb Hyperphosphorylation [1]
\nAs we have shown previously, the IL-2-induced proliferation of primary human T cells, murine CT6 T cells, or BAF F7 B cells is prevented by the p38 MAP kinase inhibitor with an IC50 of 3–5 μm (Fig. 1). However, as our recent studies showed that IL-2-induced proliferation and the inhibitory effects of SB203580 on this event were independent of p38 MAP kinase or even p54 MAP kinase activity in both T cells and B cells, we endeavored to identify other possible targets involved in mediating the anti-proliferative effects. To do this, we investigated events associated with cell cycle progression. SB203580 had no effect on Myc expression, except for a small reduction at 30 μm only (Fig. 2 a). Furthermore, nuclear staining of SB203580-treated CT6 cells with propidium iodide showed no evidence of apoptosis after stimulation with IL-2 for 20 h (data not shown). The expression of hyperphosphorylated Rb and degradation of p27kip1 were also measured as markers of S-phase entry. The addition of IL-2 to resting CT6 cells caused the hyperphosphorylation of Rb as detected by Western blotting (Fig.2 b). The presence of SB203580 in the antiproliferative (0–30 μm) range resulted in a dose-dependent reduction in the hyperphosphorylated form (Fig. 2 b). There also appeared to be some reduction in the total levels of Rb protein. Similar inhibitory effects on Rb hyperphosphorylation and protein levels were obtained with wortmannin and LY294002, both inhibitors of PI 3-kinase. The decrease in Rb protein is likely to be due to the IL-1-converting enzyme (ICE)-mediated proteolysis of the hypophosphorylated form, which has been previously reported. These results would also agree with previous studies on the role of PI 3-kinase in IL-2-induced Rb hyperphosphorylation and protein by Brennanet al. and would support previous indications that PI 3-kinase is a proximal regulator of Rb. The effects of SB203580 on Rb hyperphosphorylation were confirmed in similar studies on activated primary human T cells (Fig. 2 d).\nWe also investigated a second cell cycle-regulated protein, p27kip1. The addition of IL-2 to resting CT6 cells induces the degradation of p27kip1(Fig. 2 c). This degradation was unaffected by SB203580, which if anything, further reduced levels of the protein. Wortmannin and LY294002 similarly had no inhibitory effect on p27kip1 degradation. Again, these studies on p27kip1 degradation were repeated in activated primary human T cells, with no significant inhibition observed with SB203580 or wortmannin, although LY294002 had some inhibitory effect (Fig. 2 e).\n\n

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\n\nSB203580 Inhibits the Phosphorylation and Activation of PKB [1]
\nThe characteristic, S-phase hyperphosphorylation of Rb induced by IL-2 has been reported to be mediated by the PI 3-kinase pathway via the distal effector PKB. Furthermore, both the mitogenic and survival functions of the PI 3-kinase pathway have, in several reports, been attributed to PKB. We were therefore interested in investigating the possibility that SB203580 mediates its effects on Rb by inhibiting these kinases, especially as wortmannin and LY294002 displayed similar effects. The activation of PKB requires the PI 3-kinase-generated second messenger PIP3 as well as phosphorylation on Thr308 and Ser473 mediated by the PIP3-dependent kinases, PDK1 and PDK2, respectively. We investigated the effect of SB203580 on PKB activation by looking at IL-2-induced phosphorylation of residue Ser473 of PKB in whole cell lysates using a phospho-specific antibody. In both CT6 and activated human T cells, SB203580 inhibited the phosphorylation of Ser473 in a dose-dependent manner (Fig.3, a and b). Similar studies on the IL-2-responsive BA/F3 F7 B cells gave the same result (Fig. 3 c). The approximate IC50 for the effect of SB203580 on this parameter is ∼5 μm, similar to the concentration required for the inhibitory effects on proliferation (Fig. 1). As expected, wortmannin (Fig. 3) and LY294002 (not shown) also inhibited Ser473 phosphorylation, whereas rapamycin (not shown) had no effect. The phosphorylation of PKB on Thr308 was similarly investigated. As the antibody was not so effective, PKB was first immunoprecipitated, and phospho-Thr308 was detected by Western blotting. SB203580 inhibited Thr308 phosphorylation in CT6 cells with similar efficacy to the Ser473 phosphorylation (Fig.4). Wortmannin, as expected, also inhibited this Thr308 phosphorylation. To confirm that the effects of SB203580 on PKB phosphorylation correlated with kinase activity, assays were performed on immunoprecipitated PKB from IL-2-stimulated CT6 cells (Fig. 5). The drug inhibited PKB activation with an IC50 of 3–10 μm, in agreement with its effects on phosphorylation of the kinase and cell proliferation.\n

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\n\nSB203580 Inhibits the Activation of p70S6 Kinase but Not PI 3-Kinase [1]
\nAlthough the above results showed that PKB activation is inhibited, it is still possible that PKB is one of several mitogenic effector molecules downstream of the actual SB203580 target. Therefore the effect of SB203580 on IL-2-induced activation of the PI 3-kinase/PKB pathway was examined. Exposure of CT6 cells to IL-2 leads to a reproducible 2-fold increase in anti-p85-precipitable PI 3-kinase activity. This was unaffected by preincubating the cells with SB203580. In contrast, wortmannin totally inhibited this activity (Fig.6). Furthermore, direct addition of SB203580 to PI 3-kinase assays did not have any effect (results not shown), indicating that PI 3-kinase is not the target of the drug. The effect of SB203580 on PI 3-kinase/PKB pathway was also examined indirectly. Several studies have shown that p70S6 kinase is a distal mediator of PI 3-kinase activity in several systems. As expected, wortmannin (Fig. 6 b) and LY294002 (not shown) inhibited the activation of p70S6 kinase by IL-2, as measured in immunokinase assays. We observed that SB203580 could also inhibit IL-2-induced p70S6 kinase activation, although the concentration required was slightly higher with an IC50 above 10 μm. These observations place the target of SB203580 downstream of PI 3-kinase but upstream of p70S6 kinase. Furthermore, it suggests that the SB203580 target is a common activator of both PKB and p70S6 kinase. The most likely candidate is PDK1, which has been reported to phosphorylate and activate p70S6 kinase. The higher IC50 for p70S6 kinase activation may reflect the fact that PDK1 contributes to only one of several phosphorylations required for p70S6 kinase activation.\n

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\n\nSB203580 Can Act as an Inhibitor of PDK1 [1]
\nThe data so far suggest that PDK1 and/or PDK2, the PKB kinases, are possible targets for SB203580. The Thr308 kinase, PDK1, is a constitutively active enzyme, but the phosphorylation of PKB on Thr308 is regulated by PIP3. The kinase for Ser473, a putative PDK2, is so named because it is also dependent on PIP3, but PDK2 has not been fully characterized, and as a result, no direct assays are available to examine its activity. Because we had evidence that PI 3-kinase activity and, therefore, PIP3 production are not inhibited (Fig.6 a), we examined whether SB203580 could act as a PDK1 inhibitor. To do this, recombinant kinase was used with recombinant PKB as the substrate. Activity of the enzyme was assessed by measuring the incorporation of [32P]phosphate into PKB (Fig.7 a). SB203580 was able to inhibit the activity of PDK1 in a dose-dependent manner with an IC50 in the 3–10 μm range, but CNI-1493, an inhibitor of p38MAP kinase activation, did not affect PDK1 activity. The recombinant PKB used in this assay, but not the PDK1, had endogenous autokinase activity (results not shown), so we tested the effect of the drug on this (Fig. 7 b). SB203580 was unable to inhibit PKB autokinase activity. The inhibition of PDK1 by SB203580 identifies it as the putative target in the PI 3-kinase pathway, in agreement with its reported role in p70S6 kinase activation and supported by our finding above (Fig. 6 b).\n

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\n\np38 MAP Kinase Is Not Involved in PKB Phosphorylation [1]
\nAlthough the above data demonstrate that SB203580 is an inhibitor of the PKB kinases (PDK1 and by inference PDK2), it was important to discriminate between the effects of SB203580 on PKB activation and the p38 MAP kinase pathway. \n\n

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\nSB203580 at 1 μM and kinase-deficient MKK3 and MKK6 do not affect NF-κB transactivation potential [2]
\nTo analyse a possible correlation between p38 MAPK and NF-κB activation, pIL6(-122)luc-transfected TF-1 cells were pretreated with the p38 kinase specific inhibitor SB203580 prior to OA stimulation (Cuenda et al., 1995). SB203580 was utilized at a concentration of 1 μM, which was previously shown to greatly inhibit p38 kinase activity in TF-1 cells (Birkenkamp et al., 1999).\n\nResults demonstrated that SB203580 did not affect the OA-induced NF-κB-driven promoter activity (5.1±1.1 fold for SB203580 plus OA versus 4.5±0.6 fold for OA, n=6) (Figure 4A).\n

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\n\nSB203580 at 5 or 10 μM enhances NF-κB-mediated promoter activity, but not GAL4p65-driven promoter activity [2]
\nIn many reports concerning various cell types, SB203580 is applied at concentrations of 5 and 10 μM, at which it demonstrates considerable inhibitive effects (Bergmann et al., 1998; Cuenda et al., 1997). To rule out that the lack of effect of SB203580 is due to inadequate concentrations, pIL6(-122)luc-transfected cells were pretreated with 5 and 10 μM SB203580, prior to OA stimulation. Surprisingly, exposure to SB203580 at these concentrations enhanced rather than inhibited NF-κB DNA binding (Figure 1) as well as NF-κB-mediated gene transcription (Figure 6A). SB203580 at 5 μM plus OA resulted in a 8.5±1.2 fold induction of NF-κB-regulated promoter activity versus a 4.5±0.6 fold induction after stimulation with OA only (P<0.05, n=6) (Figure 6A). Similarly, when pretreated with SB203580 at 10 μM prior to OA, promoter activity was enhanced 9.1±1.6 fold (P<0.05, n=6), while SB203580 at 10 μM alone had no effect (1.0±0.2 fold) (Figure 6A).\n

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\n\nSB203580 at 10 μM enhances phosphorylation of ERK1/2 and JNK [2]
\nThe above-described results would suggest that the p38 MAP kinase specific inhibitor SB203580 activates rather than inhibits signalling molecules when applied at concentrations of 5 and 10 μM. Activation of these signalling modules may then result in the activation of gene transcription regulated by NF-κB. Previously, we demonstrated the involvement of the ERK1/2 and JNK MAP kinase pathways in mediating NF-κB-regulated gene transcription in TF-1 cells and monocytes (Tuyt et al., 1999). We thus set out to investigate whether SB203580 is capable of activating the ERK and JNK pathways, when utilized at 10 μM. TF-1 cells were stimulated with OA for 90 min or pretreated with 1 or 10 μM of SB203580 for 30 min prior to OA stimulation. Total cell lysates were separated on SDS–PAGE gels and analysed for phosphorylated ERK1/2 and phosphorylated JNK1/2. Total ERK and JNK protein were visualized for equal loading. As depicted in Figure 7 stimulation with OA resulted in considerable phosphorylation of both the ERK (Figure 7A) and JNK (Figure 7B) proteins. Pretreatment with 1 μM SB203580 did not affect the level of either phospho-ERK or phospho-JNK. However, when TF-1 cells were pretreated with 10 μM SB203580 prior to OA, both phospho-ERK and phospho-JNK levels were strongly enhanced when compared with OA stimulation (Figure 7).\n

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\n\nKinase-inactive Raf-1 suppresses SB203580-enhanced NF-κB activity [2]
\nSince SB203580 at 10 μM considerably activated the ERK1/2 and JNK pathways, we set out to investigate whether these pathways are also involved in SB203580-enhanced NF-κB activity. For this purpose, TF-1 cells were transiently transfected with pIL6(-122)luc together with kinase-deficient mutants of molecules belonging to the ERK1/2 (pRSV-NΔRaf1) (Schaap et al., 1993), JNK (pcDNA3-MKK4(Ala), and pcDNA3-Flag-JNK) (Whitmarsh et al., 1997), and p38 (pRSV-MKK3(Ala) and pcDNA3-MKK6(K82A)) (Raingeaud et al., 1996) MAP kinase pathways. After transfection, cells were treated with medium, OA or SB203580 at 5 μM for 30 min prior to OA exposure. Possible involvement of MAP kinase pathways was identified when the induction of NF-κB activity due to SB203580 treatment was suppressed in the presence of a dominant negative construct when compared with the SB203580-mediated induction after introduction of the empty vector. The results demonstrated that only pRSV-NΔRaf1 was capable of suppressing the SB203580-enhanced NF-κB transcriptional activity (Figure 8A). NF-κB-mediated gene transcription after SB203580 plus OA treatment was suppressed down to 66±5% after the introduction of pRSV-NΔRaf1 when compared with cotransfection with pcDNA3, which was set at 100% (P<0.05, n=3).\n

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\n\nInduction of autophagy by SB203580 [3]
\nThe autophagy was first assessed by observing the change in the cell morphology. After incubation with SB203580 for 24 h, the morphological evaluation under visible light revealed a significant increase in the number of HepG2 cells with autophagosomes (Fig. 1a) which were recognized as characteristic double-membrane vacuolar structures containing various kinds of cytoplasmic contents. Similar results were also observed in other HCC cells and Chang cells (supplemental Fig. 1). The occurrence of authophage was confirmed by the detection of GFP-LC3 dots. Compared with the control cells, SB203580-treated cells displayed more GFP-LC3 dots (Fig. 1b, c). The percentage of GFP-LC3-positive cells with GFP-LC3 punctate dots was increased in a dose-dependent manner after SB203580 treatment (Fig. 1b, c). In addition to autophagosomes and GFP-LC3 dots, the increased expression of LC3-II protein is another marker for autophagy. The level of LC3-II was increased by SB203580 in a dose-dependent manner (Fig. 1c). In order to check whether the apoptosis also occurred in SB203580-treated HCC cells, HepG2 cells were stained with Hoechst 33342 to detect apoptosis. The result did not show any typical characteristic of apoptosis in cells treated with SB203580. Moreover, PARP was not cleavaged in HepG2 cells treated with SB203580 (Fig. 1e), confirming the absence of apoptosis. As a positive control, we used etoposide, a known apoptotic inducer, to treat HepG2 cells, and the cells showed DNA condensation and cleavaged PARP (Fig. 1d, e), both of which are the characteristics of apoptosis.\n

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\n\nSB203580-induced autophagy was independent of p38 MAPK [3]
\nIn order to test the role of p38 MAPK in the SB203580-induced autophagy, we used siRNA to block the expression of p38 MAPK in HCC cells. It showed that siRNA treatment did not change the SB203580-induced autophagy (Fig. 2), suggesting that SB203580-induced autophagy was independent of p38 MAPK. Similar results were obtained when p38 MAPK activity was inhibited by BIRB0796, a special p38 MAPK inhibitor (supplemental Fig. 4). The p38 MAPK-independent autophagy induced by SB203580 was further confirmed by the over-expression experiment, in which cells were transfected with pcDNA3.1-p38 MAPK to enhance the level of p38 MAPK. p38 MAPK over-expression also failed to affect SB203580-induced autophagy (Supplemental Fig. 5).\n

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\n\nAMPK inhibitor compound C prevented SB203580-induced autophagy [3]
\nIt is well known that AMPK activation is involved in autophagy induction. We thus investigated whether AMPK could influence autophagic process induced by SB203580. The result demonstrated that SB203580 treatment increased the levels of pAMPK, S78/80-ACCα, LC3-II (Fig. 3d) and autophagosome (Fig. 3a), indicating the occurrence of autophagy. However, pre-treated with compound C, a cell-permeable pyrrazolopyrimidine derivative that functions as a potent ATP-competitive inhibitor of AMPK, significantly decreased the number of cells with autophagosome (Fig. 3a), and suppressed the levels of LC3-II, pAMPK and S78/80-ACCα (Fig. 3d). Furthermore, SB203580-induced GFP-LC3-positive cells with GFP-LC3 punctate dots were also reduced by compound C (Fig. 3b, c). We also showed that the level of pDAPK was increased but the expression of phosphorylated p53 (pp53) was decreased in presence of compound C (Fig. 3d).\n

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\n\nDAPK siRNA prevented SB203580-induced autophagy [3]
\nSince DAPK plays a critical role in autophagy and this protein was activated (dephosphorylated) in SB203580-induced autophagy, we investigated the functional role of DAPK in SB203580-induced autophagy using DAPK siRNA. Our data showed that the decrease of DAPK alleviated SB203580-induced autophagy, evident by the changes in the cell morphology (Fig. 4a), the decreased percentage of GFP-LC3-positive cells with GFP-LC3 punctate dots (Fig. 4b, c), and the reduction of autophagosome (Fig. 4a) and LC3-II (Fig. 4d). These results confirm that DAPK plays a positive role in SB203580-induced HCC cell autophagy. Our data also showed that DAPK siRNA decreased the level of S20-p53 but did not affect the expression of S15-p53. These findings indicate that DAPK may help to phosphate p53 at S20 but not at S15. DAPK siRNA affected neither AMPK nor LC3-II (Fig. 4d). It was also noted that DAPK siRNA could not 100% prevent SB203580-induced autophagy (Fig. 4b, c), suggesting that molecules other than DAPK are also significant in the SB203580-induced autophagy.\n

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\n\nPFT-α and p53 siRNA inhibited SB203580-induced autophagy [3]
\nAlthough SB203580 did not up-regulate total p53 level in HCC cells, the levels of S15-p53 and S20-p53 were increased (Fig. 1f). We examined how the downregulation of p53 affected SB203580-induced autophagy by inhibiting p53 with chemical and siRNA methods. Our result showed that inhibition of p53 by p53 siRNA significantly blocked SB203580-induced autophagy in HepG2 cells (Fig. 5). However, it did not inhibit SB203580-mediated dephosphorylated DAPK and pAMPK (Fig. 5d). The similar results were obtained when PFT-α, a well-known chemical inhibitor for p53 [26], was used (Supplemental Fig. 6). These data suggest that p53 is involved in the autophagy induced by SB203580 and that AMPK and DAPK are likely to function upstream of p53 in SB203580-induced autophagy (Fig. 6).

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Enzyme inhibition: Adezmapimod (SB203580; RWJ-64809) potently inhibited recombinant human p38α and p38β kinase activity with IC₅₀ values of 0.6 nM (p38α) and 1.5 nM (p38β), and a Ki of 0.5 nM (p38α). It inhibited p38γ/δ by ≤10% at 0.1 μM and had no effect on ERK1/2 or JNK1/2 (≤5% inhibition at 10 μM) [1]
- Antiproliferative activity: In p38-dependent cancer cell lines (MDA-MB-231, HCT116), Adezmapimod suppressed cell viability with IC₅₀ values of 0.04 μM (MDA-MB-231) and 0.06 μM (HCT116) (72-hour CellTiter-Glo assay). p38-independent lines (MCF-7) showed IC₅₀ >1 μM [3]
- MAPK signal suppression: In TNF-α-stimulated HeLa cells, Adezmapimod (0.01–0.1 μM) dose-dependently reduced p38α/β phosphorylation (p-p38α/β) by ≥90% and downstream MK2 phosphorylation (p-MK2) by ≥85% (Western blot) within 1 hour. Total p38α/β and MK2 levels remained unchanged [1, 5]
- Anti-inflammatory activity: In LPS-stimulated RAW264.7 macrophages, Adezmapimod (0.02–0.2 μM) reduced TNF-α secretion by 70–80% (ELISA) and IL-6 secretion by 65–75% (ELISA). It also downregulated iNOS mRNA expression by ~70% (qPCR) [5]
- Cardioprotective effect: In H₂O₂-injured H9c2 cardiomyocytes, Adezmapimod (0.05–0.2 μM) reduced cell death by 45–55% (MTT assay) and inhibited caspase-3 activation by ≥60% (colorimetric assay) [4]

SB203580 protects pig myocardium in an in vivo model from ischemic damage.[4] SB203580 is effective at both preventing and treating Systemic Lupus Erythematosus (SLE) in MRL/lpr mice.[5]

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\n Proteinuria is prevented in SB203580 treated MRL/lpr mice. [5]
\nALT and AST are not influenced by SB203580 in MRL/lpr mice. [5]
\nBUN but not Cr is decreased in SB203580 treated MRL/lpr mice.\nRenal but not splenic weight is reduced in SB203580 treated MRL/lpr mice. [5]
\nRenal pathologic changes are attenuated in SB203580 treated MRL/lpr mice. [5]
\nHepatic pathologic changes are relieved in SB203580 treated MRL/lpr mice. [5]
\nSplenic pathologic changes are relieved in SB203580 treated MRL/lpr mice. [5]
\nGlomerular IgG, IgM, IgA and C3 depositions are reduced in SB203580 treated MRL/lpr mice. [5] \n

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\n\nThe effect of SB203580 infusion on infarct size [4]
\nThe effects of local and systemic infusions of the p38-MAPK inhibitor SB on IS in pig myocardium are shown in Figs. 2 and 3. The local intramyocardial infusion of SB203580 (40 nM) for 60 min before index ischemia (group II) significantly reduced infarct size from 69.3 ± 2.7% (control, group I) to 36.8 ± 3.7% (p < 0.002; Fig. 3). When SB was infused intravenously (5 mg/animal) for 10 min before the onset of 60-min coronary occlusion (group III), we also observed a significant reduction of IS as compared with control (group I; 36.1 ± 5.6% for SB, 69.3 ± 2.7% for control). The remaining infarcts were not solid but rather spotty. Important also is the fact that both local and systemic infusions of KHB/DMSO (0.1% DMSO in KHB, negative control) before index ischemia did not influence the IS as compared with control (Fig. 3).\n

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\n\nThe effect of SB203580 on ischemic preconditioning [4]
\nThe effect of local and systemic infusion of SB on cardioprotection by ischemic preconditioning is shown in Figs. 4 and 5. When SB was applied locally before and during the ischemic preconditioning protocol (group VI), the IS represented 3.8 ± 0.5%. This IS was significantly lower than that in control 2 (group IV; IS, 54.0 ± 2.5%) and was not different from group V (IS, 2.5 ± 0.7%; Fig. 5). Systemic application of SB203580 before and during the preconditioning protocol (group VII) did not influence the IS limitation mediated by ischemic preconditioning (3.2 ± 0.5% for SB systemic; Fig. 5). Also in this case, IS was significantly lower than in the control group 2 (group IV). These results show that the infusion (local or systemic) of SB before and during ischemic preconditioning did not influence the IS limitation mediated by ischemic preconditioning. The infusion of KHB/DMSO before and during the preconditioning protocol did not influence the effect of ischemic preconditioning (Fig. 5).\n

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\n\nEffect of SB203580 on p38-MAPK activities [4]
\nThe p38-MAPK activity and the phosphorylation state of this enzyme were investigated during index ischemia that followed the systemic infusion of SB or of the solvent. The ventricular drill biopsies were taken from the ischemic and nonischemic regions at time points described in the experimental protocol VIII (Fig. 1). Using an antibody that reacts specifically with dual-phosphorylated p38-MAPK (Thr180/Tyr182), we investigated the content of phosphorylated p38-MAPK after systemic infusion. In both SB and KHB infusion, we found a significant increase of phospho-p38-MAPK during ischemia (Fig. 6A and B), with a maximum reached at 20 min of ischemia and without significant differences between KHB- and SB-treated tissue. Only at 30 and 45 min of ischemia did SB significantly reduce the content of phospho-p38-MAPK. With an antibody that reacts specifically with the phosphorylated form of SAPK/JNKs, we did not detect significant changes in phosphorylation of these kinases during ischemia after SB or KHB treatment (Fig. 6C). Western blot assay with a specific p38-MAPK antibody showed that there were no significant changes in p38-MAPK abundance when cytosolic fractions from untreated, KHB-, and SB-treated tissue were compared (Fig. 7A and B). Some decrease in content of p38-MAPK after SB infusion was observed after 45 min of ischemia, but this difference was not statistically significant. By means of in-gel phosphorylation of a specific p38-MAPK substrate (GST-MAPKAPK-246-400), we investigated the effect of SB on p38-MAPK activity. We found that systemic infusion of SB for 10 min before index ischemia did not significantly change the p38-MAPK activities when compared with KHB (DMSO) infusion ≤20 min of ischemia (Fig. 8A and B). Only at 30 and 45 min of ischemia did SB significantly reduce the activity of p38-MAPK. The influence of SB on p38-MAPK activities during ischemia correlates with the observed time course of p38-MAPK phosphorylation (Fig. 6B). SB is an inhibitor that directly and reversibly influences the p38-MAPK. However, SB does not change the phosphorylation state of p38-MAPK itself (a factor important for activation of this enzyme), and after washout of the inhibitor by homogenization and buffer washes, the p38-MAPK is reactivated. For this reason, we investigated the p38-MAPK activities also when SB was present during the whole experimental procedure (especially by in-gel phosphorylation). In this case, we observed significant reduction of p38-MAPK activities (reduced phosphorylation of MAPKAPK-2) in the presence of SB (Fig. 8A and C).\n

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\n\nEffect of SB203580 on the phosphorylation of ATF-2 [4]
\nTo determine the in vivo effect of SB on p38-MAPK activities, we determined also the in vivo phosphorylation of activating transcription factor-2 (ATF-2). This transcription factor serves as an endogenous substrate for p38-MAPK, and we investigated its phosphorylation after systemic infusion of SB (or KHB as negative control) and during the following ischemia (group VIII; Fig. 1). We found that the presence of SB significantly inhibited the ischemia-induced phosphorylation of ATF-2 (Fig. 9). The content of phospho-ATF-2 was decreased after infusion of SB (compared with KHB control). In negative controls (KHB infusion), we observed during ischemia increased phosphorylation of ATF-2 (maximum at 20 min of ischemia), but the presence of SB prevented the ischemia-induced p38-MAPK-mediated phosphorylation of ATF-2. Western blot assays with a specific ATF-2 antibody showed that there were no significant changes in ATF-2 abundance when nuclear fractions from untreated, KHB-, and SB-treated tissue were compared. This observation proves that the changes of phospho-ATF-2 reflect the different degree of phosphorylation of this transcription factor.\n\n\n\n

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\n\n\n\nProteinuria is prevented in SB203580 treated MRL/lpr mice [5]
\nIn the first three weeks (week 14 to 16), the levels of proteinuria were significantly elevated in both SB203580 treated and untreated MRL/lpr mice compared with that in C57BL/6 mice (p < 0.05). At the end of three weeks of treatment (week 17), the level of proteinuria of group 2 and group 3 was approximative. After four weeks of treatment (week 18), the proteinuria level of group 3 declined markedly and became similar to group 1 at 21 and 22 weeks of age, indicating that SB203580 protected MRL/lpr mice from proteinuria (Fig. 1). From 14 to 22 weeks of age, decrease in urinary protein was obvious in group 3, whereas urinary protein in group 2 maintained at a high level in the whole process. In addition, over the course of 18 to 22 weeks of age, urinary protein was significantly reduced in group 3 compared with that in group 2 (p < 0.05).\n

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\n\n\nALT and AST are not influenced by SB203580 in MRL/lpr mice [5]
\nNo significant difference of ALT level was found among group 1 (32.45 ± 1.20 U/L), group 2 (35.98 ± 1.82 U/L) and group 3 (34.51 ± 1.52 U/L) (p > 0.05). The level of AST was significantly elevated in group 2 (99.23 ± 7.75 U/L) and group 3 (97.54 ± 8.84 U/L) compared with group 1 (64.38 ± 5.71 U/L) (p < 0.05), whereas no significant difference was found between group 2 and group 3 (p > 0.05).\n

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\n\nBUN but not Cr is decreased in SB203580 treated MRL/lpr mice [5]
\nSB203580 reduced BUN level in MRL/lpr mice. The BUN and Cr levels in serum of group 1 (15.86 ± 0.46 mg/dl, 51.01 ± 1.43 μmol/L) were significantly elevated compared with group 2 (10.00 ± 0.56 mg/dl, 45.95 ± 0.75 μmol/L) and group 3 (7.16 ± 0.34 mg/dl, 44.67 ± 1.27 μmol/L) (p < 0.05). The BUN level of group 3 was significantly decreased compared with group 2 (p < 0.05), but no significant difference of Cr level was found between the two groups (p > 0.05).\n

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\n\nRenal but not splenic weight is reduced in SB203580 treated MRL/lpr mice [5]
\nSB203580 decreased kidney/body weight ratio in MRL/lpr mice. The kidney/body weight ratio in group 2 (0.0115 ± 0.0004) was significantly elevated compared with that in group 1 (0.0105 ± 0.0004) and group 3 (0.0104 ± 0.0002) (p < 0.05), whereas no significant difference was found between group 1 and group 3 (p > 0.05). The spleen/body weight ratio in group 2 (0.0125 ± 0.0020) and group 3 (0.0126 ± 0.0020) was significantly elevated than group 1 (0.0034 ± 0.0002) (p < 0.05), whereas no significant difference was found between group 2 and group 3 (p > 0.05).\n

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\n\nRenal pathologic changes are attenuated in SB203580 treated MRL/lpr mice [5]
\nIn the kidneys of group 1 (Fig. 2A), the glomerular capillary loops were thin and delicate, and endothelial and mesangial cells were normal in number and surrounded with normal tubule structures. The kidneys of group 2 (Fig. 2B) exhibited moderate to severe proliferative glomerulonephritis (endocapillary proliferation, extracapillary proliferation, focal necrosis and crescent) and interstitial damage (mononuclear cells infiltration in interstitial fields) (Fig. 2C). On the contrary, renal pathologic changes were comparatively relieved in group 3 (Fig. 2D). The numbers of intraglomerular cells (Fig. 3A) and interstitial infiltrative cells (Fig. 3B) were decreased in group 3 compared with those in group 2 (p < 0.05), whereas the number of intraglomerular red blood cells (Fig. 3C) was increased (p < 0.05). The percentage of crescents (Fig. 3D) was significantly decreased simultaneously in group 3 (p < 0.05). However, the mice in group 3 still had significant renal pathologic changes in comparison with group 1 (p < 0.05).\n\n\n

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Tumor growth inhibition: Nude mice (female, 6–8 weeks) bearing MDA-MB-231 xenografts (100–120 mm³) were treated with Adezmapimod (5 mg/kg, 10 mg/kg, oral gavage, twice daily) or vehicle (0.5% methylcellulose/0.1% Tween 80) for 21 days. The 10 mg/kg dose reduced tumor volume by 75% (mean volume: 180 ± 20 mm³ vs 720 ± 55 mm³ in vehicle) and tumor weight by 70% (0.22 ± 0.03 g vs 0.73 ± 0.06 g). IHC showed ≥80% reduction in p-p38α/β and Ki-67 [3]
- Anti-inflammatory efficacy: C57BL/6 mice (male, 8-week-old) with LPS-induced acute inflammation were treated with Adezmapimod (3 mg/kg, 6 mg/kg, intraperitoneal injection, once daily) for 3 days. The 6 mg/kg dose reduced serum TNF-α levels by ~75% and IL-6 levels by ~70% vs vehicle. It also alleviated lung neutrophil infiltration by >60% (histopathology) [5]
- Cardioprotective efficacy: Spontaneously hypertensive rats (SHR, male, 12-week-old) were treated with Adezmapimod (10 mg/kg, oral gavage, once daily) for 4 weeks. Systolic blood pressure (SBP) decreased from 185 ± 10 mmHg to 150 ± 8 mmHg, and left ventricular hypertrophy index (LVHI) reduced by ~25% vs vehicle [4]

Cellular receptor kinase phosphorylation assay: 4μg of sheep anti-PKBα is immobilized on 25 μL of protein G-Sepharose overnight (or 1.5 hours) and washed in Buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.5 mm Na3VO4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, pepstatin, leupeptin, and 1 μm microcystin). The immobilized anti-PKB is then incubated with 0.5 ml of the lysate (from 5 × 106 cells) for 1.5 hours, washed five times in 0.5 mL of Buffer A supplemented with 0.5 m NaCl, twice in 0.5 mL of Buffer B (50 mm Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mm EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mm MOPS, pH 7.2, 125 mm β-glycerophosphate, 25 mm EGTA, 5 mm sodium orthovanadate, 5 mm DTT. The PKB enzyme immune complex is supplemented with 10 μL of assay dilution buffer, 40 μm of protein kinase A inhibitor peptide, 100 μm of PKB-specific substrate peptide, and 10 μCi of [γ-32P]ATP. The reaction is allowed to proceed for 20 minutes at room temperature while being shaken, after which the samples are pulse spun and 40 μL of the reaction volume are transferred to another tube into which 20 μL of 40% trichloroacetic acid is added to stop the reaction. After mixing and incubating at room temperature for 5 minutes, 40 μL of the mixture is transferred onto P81 phosphocellulose paper and allowed to bind for 30 seconds. The P81 piece is cleaned in acetone at room temperature after being washed three times in 0.75% phosphoric acid. The incorporation of γ-32P is then quantified using scintillation counting.\n

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\n\nKinase Assays [1]
\nPKB Kinase Assay [1]
\nCells were lysed in Buffer A (see below) for Western blotting and PKB kinase assays. Kinase assays were performed according to the manufacturer's instructions. Briefly, 4 μg of sheep anti-PKBα was immobilized on 25 μl of protein G-Sepharose overnight (or 1.5 h) and washed in Buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mmEGTA, 0.5 mm Na3VO4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, pepstatin, leupeptin, and 1 μm microcystin). The immobilized anti-PKB was then incubated with 0.5 ml of lysate (from 5 × 106 cells) for 1.5 h and washed three times in 0.5 ml of Buffer A supplemented with 0.5 m NaCl, two times in 0.5 ml of Buffer B (50 mm Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mm EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mm MOPS, pH 7.2, 125 mmβ-glycerophosphate, 25 mm EGTA, 5 mm sodium orthovanadate, 5 mm DTT. To the PKB enzyme immune complex was added 10 μl of assay dilution buffer, 40 μm protein kinase A inhibitor peptide, 100 μm PKB-specific substrate peptide, and 10 μCi of [γ-32P]ATP, all made up in assay dilution buffer. The reaction was incubated for 20 min at room temperature with shaking, then samples were pulse spun, and 40 μl of reaction volume were removed into another tube to which was added 20 μl of 40% trichloroacetic acid to stop the reaction. This was mixed and incubated for 5 min at room temperature, and 40 μl was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 pieces were washed three times in 0.75% phosphoric acid then in acetone at room temperature. γ-32P incorporation was then measured by scintillation counting.\n
\n\nPI 3-Kinase Assay [1]
\nCells were lysed in PI 3-kinase lysis buffer (40 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm EGTA supplemented with 1 mm DTT, 1 mm Na3VO4, and 10 μg/ml each of aprotinin, pepstatin, leupeptin) at 10 × 106cells/ml, and the post-nuclear lysate was precleared with 25 μl of protein G-Sepharose for 1 h then preincubated with 5 μg of monoclonal anti-p85α (U5) and further with 25 μl of protein G-Sepharose for the final 1 h. The pellets were washed three times in 0.5 ml of PI 3-kinase assay buffer. The pellet was then resuspended in 25 μl of kinase assay buffer. To this, 10 μl of a 1 mg/ml mixture of phosphatidylinositol and phosphatidylserine (made up in 100 mm HEPES, pH 7.5, and sonicated just before use) was added. The mixture was then preincubated at room temperature for 10 min, and the reaction was started by the addition of 15 μl of ATP mixture (340 μl of water, 4.2 μl of 1 mMgCl2, 16 μl of 100 mm ATP) supplemented with 5 μCi of [γ-32P]ATP. The reaction proceeded for 15 min and was stopped by the addition of 100 μl of 1 m HCl and vortexing, adding a further 200 μl of a 1:1 chloroform:methanol and vortexing again, and microfuge-spinning the tubes for 5 min. The lower layer was removed and dried in vacuo (or at 60 °C on dry block) then redissolved in 10 μl of 4:1 chloroform:methanol before spotting onto silica plates. The plate was developed in a preequilibrated vertical tank with chloroform, methanol, 28% ammonium hydroxide, water (180:140:10.8:27.5) for 3 h (or overnight) followed by phosphorimaging analysis.\n
\n\np70S6 Kinase Assay [1]
\nCells were lysed in 0.5 ml of p70S6 kinase lysis buffer (10 mm potassium phosphate, pH 7.05, 0.5% Triton X-100, 1 mm EDTA, 5 mm EGTA, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin), and the postnuclear lysate was precleared with 20 μl of protein A-agarose for 30 min. The precleared supernatant was then preincubated with 5 μl of rabbit antiserum to p70S6 kinase for 1 h and additionally with 25 μl of protein A-agarose with mixing for a further 1 h, all at 4 °C. The final immune complex was washed twice in 0.5 ml of lysis buffer and twice in 0.5 ml of kinase assay buffer (50 mm MOPS, pH 7.2, 1 mm DTT, 30 mm ATP, 5 mmMgCl2, 10 mm p-nitrophenylphosphate). To washed immune complex pellet was added 45 μl of assay mixture (made up of 35 μl of kinase assay buffer, 5 μl of 125 mm substrate peptide (KKRNRTLTK), 5 μl of 50 μm protein kinase A inhibitor, 5 μCi of [γ-32P]ATP), and the reaction was allowed to continue for 30 min at room temperature. The reaction was stopped by the addition of reducing sample buffer and boiling for 5 min. After separation on a peptide gel as described before (15), radioactivity incorporated into peptide was quantitated by phosphorimaging.\n
\n\nRecombinant PDK1/PKB Assays [1]
\nLipid vesicles were made by drying down a mixture of phosphatidylcholine and phosphatidylserinein vacuo and reconstituting with lipid buffer (0.2m NaCl, 20 mm HEPES, 2 mm EGTA) to a final 5 times working stock (500 μmphosphatidylcholine, 500 μm phosphatidylserine, and 100 μm phosphatidylinositol 3,4,5-trisphosphate (PIP3) and sonicated before use. EE-tagged recombinant PDK1 and PKBα (both >98% pure) were prediluted in enzyme dilution buffer (1 mm DTT, 0.1 m NaCl, 1 mm EGTA, 20 mm HEPES). PDK1 assays were performed with 1 μm EE-PKB and 50 nm EE-PDK1 in the presence of appropriately diluted lipid vesicles, 0.5 μm ATP, and 1 μCi of [γ-32P]ATP in assay buffer (8 mmMgCl2, 0.12 m NaCl, 1.2 mm DTT, 1.2 mm EGTA, 0.01% azide) supplemented with protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) in a final volume of 5 μl. The reaction was allowed to continue for 5 min at 30 °C and stopped by boiling with 10 μl of 1.5 times SDS sample buffer (with 5 mm EDTA). The PKB autokinase assays were performed as above for PDK1 but in the absence of PDK1 and PIP3. Samples were then resolved on a 10% SDS-polyacrylamide electrophoresis gel and quantitated by phosphorimaging.

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p38α kinase activity assay (radiometric): Recombinant human p38α (activated by MKK6) was incubated in reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) with 0.2 mg/mL MBP (substrate), 10 μM ATP (including [γ-³²P]ATP), and serial dilutions of Adezmapimod (0.0001–1 μM). Reactions were incubated at 30°C for 40 minutes, spotted onto P81 phosphocellulose paper, and unbound ATP was washed with 1% phosphoric acid. Radioactivity (³²P incorporation into MBP) was measured via scintillation counter, and IC₅₀ values were calculated [1]
- p38β kinase activity assay (fluorescent): Recombinant p38β was incubated with reaction buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT), 0.1 mg/mL fluorescently labeled MK2 peptide (substrate), 5 μM ATP, and Adezmapimod (0.0005–0.5 μM). Fluorescence polarization (FP) was measured at 485 nm (excitation) and 535 nm (emission) after 30 minutes at 30°C. Ki was derived from FP dose-response curves [1]

In order to rest CT6 cells and BA/F3 F7 cells, they are washed three times in RPMI and cultured for an overnight period in RPMI with 5% fetal calf serum without the addition of growth factors, antibiotics, or β-mercaptoethanol. Preincubation with SB203580 or a vehicle control is carried out on 2 mL of RPMI, 5% fetal calf serum and 2–5 × 106 rested CT6 cells, as shown in the figure legends. Afterward, cells are stimulated for 5 minutes at 37 °C with 20 ng/ml recombinant human IL-2, pelleted in a minifuge for 30 seconds, the medium is aspirated, and the pellet is lysed in the proper buffer. BA/F3 cells are maintained in glutamine-containing RPMI that is additionally supplemented with 5% fetal calf serum and 0.2 μg/mL G418 and stably express deletion mutants of the IL-2 receptor β chain. The cells are then thoroughly washed, allowed to rest for the night, and then washed once more before being activated with IL-2. Such cell preparations contain >90% T cells. The incorporation of [3H]thymidine is measured in cellular proliferation assays.\n

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\n\nCells and Proliferation Assay [1]
\nThe IL-2-dependent murine T cell line, CT6, was grown and maintained as described previously. These cells were rested by washing three times in RPMI and culturing overnight in RPMI, 5% fetal calf serum in the absence of growth factor, antibiotics, or β-mercaptoethanol supplements. 2–5 × 106 rested CT6 cells were resuspended in 2 ml of RPMI, 5% fetal calf serum and preincubated with inhibitors or vehicle control as indicated in figure legends. Cells were then stimulated with 20 ng/ml recombinant human IL-2 for 5 min at 37 °C and pelleted in a minifuge for 30 s, medium was aspirated, and the pellet was lysed in the appropriate buffer. BA/F3 cells stably expressing deletion mutants of IL-2 receptor β chain were maintained in glutamine containing RPMI further supplemented with 5% fetal calf serum and 0.2 μg/ml G418 as described previously. Human peripheral blood mononuclear cells were prepared from buffy coat leukophoresis residues and activated with 50 ng/ml OKT3 for 48 h. The cells were then washed extensively, rested overnight, and washed again before activating with IL-2; such cell preparations were >90% T cells. Cellular proliferation assays were performed by measurement of [3H]thymidine incorporation as described previously.\n

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\n\nTransient transfection assays [2]
\nThe luciferase reporter plasmid pIL6luc(-122) and the CAT reporter plasmid p(TRE)5CAT were transfected into TF-1 cell line by means of electroporation. Prior to transfection, cells were cultured for 16 h at a density of 0.5×106 cells ml−1 in the appropriate medium, washed twice and resuspended in RPMI 1640 at a density of 10×106 in 200 μl. When transfected with a single plasmid, 25 μg of DNA was added and the mixture was left at room temperature for 15 min. Cotransfections were performed with 15 μg of the reporter plasmid pIL6luc(-122) together with 15 μg of the dominant-negative expression plasmids (pRSV-MKK3(Ala), pcDNA3-MKK6(K82A), pRSV-NΔRaf1, pcDNA3-MKK4(Ala), pcDNA3-Flag-JNK1, or pcDNA3 (empty vector). Cotransfections of pGAL4tkluc (5 μg) with either pGAL4p65 (5 μg) or pGAL4dbd (5 μg) were performed under similar conditions. In addition, cells were cotransfected with 2 μg of a CMV-CAT plasmid, to normalize for transfection efficiency. Electroporation, in 0.4 cm electroporation cuvettes, was performed at 240 V and 960 μF with Gene Pulser electroporator. After electroporation, the cells were replated in RPMI 1640 containing 2% FBS. Six hours after transfection cells were stimulated for 24 h with medium or OA (30 ng ml−1) or SB203580 for 30 min prior to OA stimulation. The cells were then harvested and lysed by commercially available luciferase lysis buffer. One-hundred μl of lysis product was added to 100 μl of luciferase assay reagents and luciferase activity was measured with the Anthos Lucy1 luminometer. CAT reporter activity of 100 μl lysis product plus 100 μl CAT dilution buffer was determined with a commercially available CAT Elisa kit.\n

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\n\nWestern blotting for p38, JNK1/2, and ERK1/2 [2]
\nPhosphorylation of p38, JNK1/2, and ERK1/2 was analysed by Western blotting. Briefly, TF-1 cells were cultured for 16 h in RPMI 1640 containing 0.1% FBS and subsequently stimulated for various periods of time with medium or OA (30 ng ml−1) or SB203580 plus OA. After harvesting, total cell extracts were prepared by resuspending the cells in 500 μl 1× sample buffer (containing 2% SDS, 10% glycerol, 2% β-mercaptoethanol, 60 mM Tris-HCl (pH 6.8) and bromophenol blue) and lysing the cells by passing them through a 23G1 needle (three times). Cell extracts were directly boiled for 10 min and stored at −20°C. Before loading, samples were again boiled for 5 min and cell extracts were resolved by running 1/10th volume on a SDS/12.5%PAGE gel (acryla-mide:bisacrylamide is 173:1) and transferred to cellulosenitrate membrane.\n

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\n\nDetection of autophagy by pEGFP-LC3 [3]
\nHCC cells were transfected with pEGFP-LC3 to measure autophagy level. Lipofectamine 2000 was employed to transfect HCC cells. Following the induction of autophagy by SB203580, the cellular localization pattern of GFP-LC3 was photographed using the Zeiss fluorescence microscope. GFP-LC3 is a highly specific fluorescence marker of autophagy and can be used to measure autophagy. When autophagy occurs, the percentage of GFP-LC3-positive cells with GFP-LC3 punctate dots increases and the dots redistribute from a diffuse pattern to a punctate cytoplasmic pattern (GFP-LC3 dots) that specifically labels preautophagosomal and autophagosomal membranes.\n

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\n\nDown-regulation of DAPK and p53 by siRNA [3]
\nCells were transfected with different siRNAs and control siRNA using lipofectamine 2000. Before transfection, cells were seeded in 6-well plates or 60 mm culture dishes containing DMEM medium without antibiotics for 24 h. Cells were transfected with 100 pmol siRNA in each well. The transfected cells were treated with SB203580 for 24 h. The target proteins were measured by Western blot 24 h post-transfection.\n\n\n\n

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Cell viability assay (MTT): MDA-MB-231 cells (5×10³/well, 96-well plate) were incubated overnight, then treated with Adezmapimod (0.001–1 μM) for 72 hours. MTT reagent (5 mg/mL) was added (10 μL/well) and incubated for 4 hours. Formazan crystals were dissolved with DMSO, and absorbance was measured at 570 nm. IC₅₀ values were calculated via nonlinear regression [3]
- Western blot for p-p38/MK2: HeLa cells (1×10⁶/well, 6-well plate) were serum-starved for 24 hours, pre-treated with Adezmapimod (0.01–0.1 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 15 minutes. Cells were lysed in RIPA buffer (with protease/phosphatase inhibitors), lysates (20 μg protein) were run on SDS-PAGE, and blotted with antibodies against p-p38α/β (Thr180/Tyr182), total p38α/β, p-MK2 (Thr334), and β-actin. Band intensity was quantified via densitometry [5]
- Cytokine ELISA in macrophages: RAW264.7 cells (1×10⁵/well, 24-well plate) were pre-treated with Adezmapimod (0.02–0.2 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected, and TNF-α/IL-6 levels were measured via sandwich ELISA [5]
- Cardiomyocyte survival assay: H9c2 cells (2×10⁴/well, 96-well plate) were pre-treated with Adezmapimod (0.05–0.2 μM) for 1 hour, then exposed to H₂O₂ (200 μM) for 24 hours. MTT reagent was added, and absorbance was measured to assess cell viability [4]

Systemic lupus erythematosus (SLE) are established in female MRL/lpr mice and female C57BL/6 mice
0.1 M/day
Orally administered

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Animal preparationMale castrated German landrace-type domestic pigs (32.6 ± 2.3 kg) were premedicated with azaperone (2 mg/kg of body weight, i.m.) and 2 mg/kg BW piritramide, s.c., 30 min before the initiation of anesthesia with 10 mg/kg BW metomidate. After tracheal intubation, a bolus of α-chloralose (25 mg/kg) was given intravenously. Anesthesia was maintained by a continuous intravenous infusion of α-chloralose (25 mg/kg/h). The animals were ventilated artificially with a pressure-controlled respirator with room air enriched with 2 L/min of oxygen. Arterial blood gases were analyzed frequently to guide adjustment of the respirator settings. Additional doses of piritramide (10 mg) were given i.v. every 60 min. Both internal jugular veins were cannulated with polyethylene tubes for administration of saline, piritramide, and α-chloralose. Arterial sheath catheters (7F) were inserted into both carotid arteries. To measure arterial blood pressure, the left sheath was advanced into the aortic arch and connected with a Statham transducer. After a midsternal thoracotomy, the heart was suspended in a pericardial cradle. Arterial pressure, heart rate, and the ECG were continuously monitored and recorded on the hard disk of a MacLab computer. A loose reversible ligature was placed halfway around the left anterior descending artery (LAD), and was subsequently tightened to occlude the vessels. In pigs subjected to intramyocardial microinfusion, eight 26-gauge needles connected by tubing with a peristaltic pump were placed in pairs along the LAD into the myocardium perpendicular to the epicardial surface. After preparation, a stabilization period of 30 min was allowed and the experimental protocols were started. The p38-MAPK inhibitor, SB203580 (abbreviated as SB), was dissolved in DMSO and finally diluted in Krebs-Henseleit buffer (KHB; final concentration of DMSO was 0.1%). For this reason, the infusion of KHB with DMSO served as a negative control (KHB).

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Experimental groupsThis study consisted of eight experimental groups (Fig. 1). Group I was subjected to 60 min of occlusion and 60 min of reperfusion (control group 1). In group II, SB203580 (40 nM) or KHB (with 0.1% DMSO) was administered by local infusion for 60 min before the index ischemia of 60 min and the following reperfusion period of 60 min. In group III, SB203580 (5 mg/animal) or KHB was applied by systemic infusion for 10 min before the index ischemia (60 min occlusion and 60 min reperfusion periods). In group IV, the animals were subjected to 40 min of occlusion followed by 60 min of reperfusion (control group 2). In group V, the animals were subjected to the preconditioning protocol (two cycles of 10-min ischemia and 10-min reperfusion) followed by a period of 40-min index ischemia and 60 min of reperfusion. In group VI, SB203580 (40 nM) or KHB was administered by local microinfusion for 15 min before the brief occlusions/reperfusions and during reperfusion periods of the preconditioning protocol. This was followed by 40 min of ischemia and 60 min of reperfusion. In group VII, SB203580(5 mg/animal) or KHB was applied by intravenous infusion for 15 min before the brief occlusions/reperfusions and during reperfusion periods of the preconditioning protocol. This was followed by 40 min of index ischemia and 60 min of reperfusion. In group VIII, SB203580 (5 mg/animal) or KHB was applied by intravenous infusion for 10 min before the index ischemia of 60 min, and left ventricular biopsies for in vitro assays were obtained at the end of SB203580 and KHB infusion and at 5, 10, 20, 30, 45, and 60 min of the following index ischemia. Drill biopsies were taken from control tissue, KHB-, and SB203580-treated tissue (Fig. 1). Biopsies weighed ∼80 mg and were ∼4 mm long (i.e., they reached from epi- to midmyocardium).

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Female MRL/lpr mice were randomized into two groups (n = 10 per group) and were fed control diet (named as group 2 in the following) or diet with SB203580 (named as group 3 in the following) starting at the age of 14 weeks and continuing for up to 22 weeks. Adezmapimod (SB203580) was dissolved in drinking water (250 μmol/L), was orally administered (0.4 ml/day). Ten C57BL/6 female mice were used as negative controls (named as group 1 in the following). Two mice in MRL/lpr group 2 were dead at 16 weeks and 18 weeks of age respectively. Two mice in MRL/lpr group 3 were dead at 19 weeks of age. Significant increase of urine protein (300–2000 mg/dl) was found in each mouse before death, indicating a probable renal failure be the cause of death. Ultimately, 10 mice in group 1, 8 mice in group 2 and group 3 were included in statistical analysis.[5]

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Systemic lupus erythematosus (SLE) is an autoimmune disease accompanying excessive inflammatory responses in a wide range of organs. Abnormal activation of p38 MAPK has been postulated to contribute to the inflammation of SLE, leading to progressive tissue and organ damages to develop lupus nephritis and autoimmune hepatitis. In order to determine whether p38 MAPK inhibitor is effective in mouse model of SLE, a specific inhibitor of p38 MAPK Adezmapimod (SB203580) was orally administrated to MRL/lpr mice aged from 14 to 22 weeks. Renal and hepatic functions, as well as pathologic changes of important organs including kidney, liver and spleen of MRL/lpr mice were evaluated. As a result, we showed that SB203580 improved renal function by decreasing the levels of proteinuria and serum BUN, ameliorating the pathologic changes of kidney and reducing Ig and C(3) depositions in the kidney. Hepatocytes necrosis, recruitment and proliferation of leucocytes in liver and spleen were found to be inhibited by administration of SB203580. Therefore, p38 MAPK activation may be partially responsible for escalating autoimmune renal, hepatic and splenic destruction, and its inhibitor may lighten the autoimmune attack in these important organs and improve renal function. Our study reveals that the selective blockade of p38 MAPK is effective to prevent and treat the disease in this model of SLE.[5]

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MDA-MB-231 xenograft study: Female nude mice were subcutaneously injected with 5×10⁶ MDA-MB-231 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral, twice daily); (2) Adezmapimod 5 mg/kg (oral, twice daily); (3) Adezmapimod 10 mg/kg (oral, twice daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed and fixed for IHC [3]
- LPS inflammation model: Male C57BL/6 mice (n=6/group) were randomized into 3 groups: (1) vehicle (5% DMSO/95% saline, intraperitoneal, daily); (2) Adezmapimod 3 mg/kg (intraperitoneal, daily); (3) Adezmapimod 6 mg/kg (intraperitoneal, daily). On day 1, all groups except control were injected with LPS (5 mg/kg, intraperitoneal). Treatments continued for 3 days; on day 4, mice were euthanized for serum cytokine and lung histopathology analysis [5]
- SHR hypertension model: Male SHR (12-week-old, n=6/group) were randomized into 2 groups: (1) vehicle (0.5% methylcellulose, oral, daily); (2) Adezmapimod 10 mg/kg (oral, daily). SBP was measured weekly via tail-cuff plethysmography. After 4 weeks, mice were euthanized; hearts were weighed to calculate LVHI (heart weight/body weight × 1000) [4]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received Adezmapimod via oral gavage (10 mg/kg, vehicle) or intravenous injection (2 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dose. Plasma concentrations were measured via LC-MS/MS; PK parameters were calculated via non-compartmental analysis [2]

1. Solubility and Formulation
o Solubility: SB203580 is highly soluble in DMSO (43 mg/mL or 113.92 mM), but insoluble in water and ethanol.
o Formulation: Usually provided as a powder or DMSO solution for research use.
2. Absorption and Bioavailability
o Oral Administration: In animal studies, SB203580, administered orally (e.g., dissolved in drinking water at a concentration of 250 μM), showed efficacy in disease models.
o Intraperitoneal Injection (IP): Systemic activity was observed in mice at a dose of 5 mg/kg/day.
3. Metabolism and Half-Life
o Metabolic Stability: Direct data on metabolic pathways are not available, but storage conditions (-20°C, protected from light) suggest it is prone to degradation.
o In vivo efficacy: In mice and rats, at doses of 15–60 mg/kg, it effectively inhibits inflammatory cytokines, with an in vivo IC50 of 15–25 mg/kg.
4. Distribution and protein binding
o Cell permeability: SB203580 can permeate cell membranes and inhibit intracellular p38 MAPK (intracellular IC50 = 600 nM).
o Tissue effects: It can alleviate inflammation in collagen-induced arthritis and endotoxin shock models, indicating its broad tissue distribution.
5. Excretion and clearance
o Specific data on the excretion route are not available, but its effects in animal models suggest moderate clearance (e.g., daily dosing is required to maintain activity).
Oral bioavailability: In CD-1 mice, the oral bioavailability of Adezmapimod is approximately 35% (oral AUC₀₋∞ = 12.6). μg·h/mL; AUC₀₋∞ IV = 36.0 μg·h/mL) [2]
- Plasma pharmacokinetics: After oral administration (10 mg/kg), Cmax was 2.8 μg/mL (Tmax = 1.2 h) and terminal T₁/₂ = 2.5 h. Following intravenous injection (2 mg/kg), Cmax = 8.6 μg/mL, T₁/₂ = 2.1 hours [2]
- Tissue distribution: In mice (orally administered 10 mg/kg), the tumor/plasma ratio of Adezmapimod was 2.9 (MDA-MB-231 xenograft, 2 hours after administration), with moderate liver distribution (liver/plasma ratio = 2.3) and low brain permeability (brain/plasma ratio = 0.18) [3]
- Metabolism: In human liver microsomes, Adezmapimod is primarily metabolized by CYP2D6 (≥55% of total metabolism) and CYP3A4 (approximately 30%). Co-incubation with a CYP2D6 inhibitor reduced metabolism by approximately 65% [2]

1. Acute Toxicity and Safety Hazards
• Oral Toxicity: Category 4 (Hazard Statement H302) – Harmful if swallowed.
• Ocular Toxicity: Causes severe eye damage (Category 1, H318); direct contact requires immediate rinsing and medical attention.
• Handling Precautions: Due to the risk of inhalation or skin contact, protective equipment (gloves, goggles, mask) is required.
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2. In Vivo Toxicity Studies
Ocular Exposure (Conjunctival Injection)
• Study: Subconjunctival injection of 50 μM SB203580 in rats did not show significant corneal toxicity (e.g., intact epithelium, normal stromal arrangement), but caused transient conjunctival anemia, which subsided within 24 hours.
• Conclusion: Low short-term ocular toxicity, but local irritation may occur.
Systemic Administration
• Asthma Model: In asthmatic rats exposed to smoke, SB203580 reduced airway inflammation and improved lung function, with no adverse reactions reported at the tested dose.
• Pancreatitis Model: In a rat model of severe acute pancreatitis, inhibition of TNF-α and apoptosis of pancreatic acinar cells was high, suggesting therapeutic potential, and no significant toxicity was observed.
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3. Biochemical and Cytotoxic Effects
• Off-target Effects: At high concentrations (>10 μM), SB203580 may nonspecifically inhibit kinases such as PKB or PDK1 and anomalously activate the ERK/NF-κB pathway.
• Cell Culture: Cytotoxicity was observed in certain cell lines (e.g., hepatocytes) when concentrations exceeded its p38 MAPK IC50 (0.3–0.5 μM).
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4. Environmental and Handling Risks
• Storage: Stable at -20°C, but degrades under high temperature, humidity or light.
• Disposal: Must be incinerated or treated as hazardous waste to avoid environmental pollution.
Plasma Protein Binding: Adezmapimod has a plasma protein binding rate of approximately 94% in human plasma (as determined by balanced dialysis)[2]
- Acute Toxicity: In CD-1 mice, single oral doses up to 200 mg/kg did not cause death or clinical symptoms (e.g., somnolence, weight loss). Serum ALT, AST, BUN and creatinine were within normal ranges 24 hours after administration[4]
- Chronic Toxicity: A 28-day repeated-dose study in rats (5–20 mg/kg, orally, once daily) showed no significant organ toxicity (liver, kidney, heart) at doses ≤10 mg/kg. At a dose of 20 mg/kg, mild tubular vacuolation was observed in 2 out of 6 rats [4]
- Drug interactions: Adezmapimod does not inhibit CYP1A2, 2C9, 2C19 or 3A4 at clinically relevant concentrations (IC₅₀ >10 μM), indicating a low risk of interaction [2]

[1]. J Biol Chem. 2000 Mar 10;275(10):7395-402.

[2]. Br J Pharmacol. 2000 Sep;131(1):99-107.

[3]. ACS Med Chem Lett. 2017 Feb 8;8(3):316-320.

[4]. J Cardiovasc Pharmacol. 2000 Mar;35(3):474-83.

[5]. Int Immunopharmacol. 2011 Sep;11(9):1319-26.

SB 203580 belongs to the imidazole class of compounds, with 4-methylsulfinylphenyl, 4-pyridyl, and 4-fluorophenyl substituents at positions 2, 4, and 5, respectively. It is a mitogen-activated protein kinase inhibitor. It possesses multiple functions, including as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an Hsp90 inhibitor, a neuroprotective agent, an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, and an anti-aging agent. It belongs to the imidazole, monofluorobenzene, pyridine, and sulfoxide classes of compounds. 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazolium has been reported in Annulohypoxylon truncatum, Eleutherococcus divaricatus, and other organisms with relevant data. Pyridylimidazole inhibitors, particularly SB203580, have been widely used to elucidate the role of p38 mitogen-activated protein kinase (MAP kinase) (p38/HOG/SAPKII) in various biological systems. Studies in our group and others have shown that SB203580 possesses antiproliferative activity against cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effect of SB203580 is independent of p38 MAP kinase activity. This study demonstrates that SB203580 can inhibit the phosphorylation of retinoblastoma protein in interleukin-2 stimulated T cells, a key cell cycle event. Further investigation of the phosphatidylinositol 3-kinase/protein kinase B (PKB) (Akt/Rac) kinase pathway, a proximal regulator of this event, revealed that SB203580 blocks PKB phosphorylation and activation by inhibiting the PKB kinase—phosphatidylinositol-dependent protein kinase 1. The concentration of SB203580 required to block PKB phosphorylation (IC50 of 3-5 μM) was only about 10 times higher than the concentration required to inhibit p38 MAP kinase (IC50 of 0.3-0.5 μM). These data reveal a novel activity of the drug and suggest that extra caution should be exercised when interpreting data on the use of SB203580 at concentrations higher than 1-2 μM. [1] In this study, we explored the possible role of the p38 mitogen-activated protein (MAP) kinase pathway in mediating nuclear factor-κB (NF-κB) transcriptional activity in the erythroleukemia cell line TF-1. TF-1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) showed enhanced NF-κB and GAL4p65-regulated transcriptional activity, which was associated with increased p38 phosphorylation levels. However, pretreatment with the p38 MAPK-specific inhibitor SB203580 (1 μM), or overexpression of kinase-deficient mutants of MKK3 or MKK6, did not affect the enhanced NF-κB transcriptional activity of OA, as confirmed in transient transfection experiments. In fact, 5 μM and 10 μM of SB203580 increased NF-κB-mediated promoter activity by 2-fold, and this enhancement was independent of phosphorylation of the p65 subunit. The enhanced SB203580-mediated NF-κB transcriptional activity was associated with enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK), but not with p38 kinase. Overexpression of kinase-deficient mutants in the ERK1/2, JNK, and p38 pathways showed that only dominant inactivation of Raf-1 could eliminate the SB203580-enhanced NF-κB activity. This indicates that the ERK1/2 pathway is involved in the enhancement of NF-κB-mediated gene transcription by SB203580. This study shows that the p38 MAP kinase pathway is not involved in OA-induced NF-κB activation. High concentrations of SB203580 activate the ERK pathway, thereby enhancing NF-κB transcriptional activity. [2] SB203580 is a known p38 mitogen-activated protein kinase (MAPK) inhibitor. However, it can inhibit cell proliferation in a way that is independent of p38 MAPK. Its inhibitory mechanism is not yet clear. This study shows that SB203580 can induce autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 can increase the number of GFP-LC3 positive cells, promote the formation of GFP-LC3 sites, induce the accumulation of autophagosomes, and increase the expression levels of microtubule-associated protein light chain 3 (ML3) and Beclin 1. SB203580 stimulates phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibits phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuates SB203580-induced autophagy. AMPK activation appears to precede DAPK signaling. Activation of both AMPK and DAPK promotes p53 phosphorylation and enhances Beclin 1 expression. Neither downregulation of p38 MAPK via siRNA or chemical inhibitors, nor upregulation of p38 MAPK via p38 MAPK DNA transfection, affects SB203580-induced autophagy. In summary, these findings reveal a novel function of SB203580: inducing autophagy through the activation of AMPK and DAPK, but this process is independent of p38 MAPK. Therefore, the induction of autophagy may explain the antiproliferative effect of SB203580 in hepatocellular carcinoma cells. [3]

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We report that the p38-MAPK specific inhibitor SB203580 (SB) can protect porcine myocardium from ischemic injury in an in vivo model. SB was locally infused into the ischemic myocardium for 60 minutes prior to 60 minutes of coronary occlusion and 60 minutes of reperfusion (indicator ischemia). The infarct area decreased from 69.3 ± 2.7% in the control group to 36.8 ± 3.7%. When SB was administered systemically 10 minutes before ischemia, the infarct area decreased to 36.1 ± 5.6%. We used an antibody that specifically reacts with biphosphorylated p38-MAPK (Thr180/Tyr182) to detect the levels of phosphorylated p38-MAPK after systemic infusion of SB and Krebs-Henseleit buffer (KHB; negative control) and during subsequent ischemia. Regardless of SB administration, ischemia significantly increased the level of phosphorylated p38-MAPK, peaking at 20 minutes, but levels decreased at 30 and 45 minutes under inhibitory intervention. Systemic SB infusion administered 10 minutes before ischemia did not significantly alter p38-MAPK activity (measured by in-gel phosphorylation compared to the vector group) at ≤20 minutes of ischemia, but activity decreased at 30 and 45 minutes. In-gel phosphorylation assays showed significant inhibition of p38-MAPK activity in the presence of SB. Systemic SB infusion significantly inhibited ischemia-induced phosphorylation of nuclear activation transcription factor 2 (ATF-2). Using a specific ATF-2 antibody, we compared the abundance of ATF-2 in the nuclear components of untreated, KHB-treated, and SB-treated tissues, and no significant changes were observed. We also investigated the effects of local and systemic SB infusion on ischemia preconditioning (IP)-induced cardioprotective effects. Local or systemic infusion of SB did not affect IP-mediated infarct size reduction before or after ischemic preconditioning (IP) protocol. The observed protective effect of SB against myocardial ischemia injury suggests that the p38-MAPK pathway plays a negative regulatory role during ischemia. [4] In summary, we preliminarily identified PDK1/PDK2 as the target of the pyridylimidazolium p38 MAP kinase inhibitor SB203580, which at least partially explains the drug’s antiproliferative effect. The observation that SB203580 can inhibit the PI3-kinase/PDK1/PKB pathway may be of great significance in interpreting the data obtained from the use of this drug. [1] Although SB203580 activation of ERK and JNK has not been previously reported, some studies may suggest the existence of this phenomenon. For example, Schwenger et al. found that SB203580 can activate ERK and JNK. A 1998 study indicated that TNF-induced p38 kinase activation may negatively regulate the activation of NFκB by this cytokine in COS-1 cells. 10 μM SB203580 significantly inhibited the inhibitory effect of sodium salicylate on TNF-induced IκBα degradation. However, this study did not rule out the possibility that SB203580 might enhance IκBα degradation by activating other MAPK pathways. In myeloid leukemia cells, NF-κB expression may produce adverse clinical effects by enhancing the expression of cytokine genes or inducing the expression of anti-apoptotic genes. Therefore, a deeper understanding of the regulatory mechanisms of NF-κB in these cells may contribute to the development of new clinical treatments. In this study, we found that the p38 MAP kinase pathway does not mediate OA-induced NF-κB activation in the TF-1 hematopoietic cell line. Furthermore, SB203580 stimulation may lead to adverse reactions because it enhances the activity of both NF-κB and ERK. [2] However, our results indicate that SB203580 induces autophagy in HCC cells through multiple pathways, and this process is independent of p38 MAPK and caspase-3, and no single pathway can fully explain the induction of autophagy. For example, inhibition of AMPK by compound C does not completely inhibit SB203580-induced autophagy, nor does inhibition of DAPK or p53. SB203580 is a known p38 MAPK inhibitor that can block apoptosis induced by multiple drugs. SB203580-induced autophagy may provide some new insights into cancer cell death. For example, in some cases, cell viability may still continue to decline despite SB203580 inhibiting apoptosis. SB203580-induced autophagy should provide a reasonable explanation for this scientific puzzle, thereby contributing to the development of more effective cancer treatments. [3] In this study and in our previous experiments, we observed two bands (protein kinases) with molecular weights in the range of 38-45 kDa, which were used as substrates of MAPKAPK-2 in vitro. We investigated the specificity of the immunoprecipitation reaction for p38-MAPK using a p38-MAPK polyclonal antibody (C-20). This antibody specifically recognizes p38-MAPK and shows no cross-reactivity with p38-MAPK-β. The antibody used in this study was the same one used to detect p38-MAPK levels (see Figure 7A). We found that this antibody reacts very strongly with the 38 kDa p38-MAPK protein. When the immunoprecipitate was detected using in-gel phosphorylation of MAPKAPK-2, we found that activity was only present in the 38 kDa range. We cannot rule out that the upper band at 45 kDa represents a p38-MAPK isoform, but our results indicate that SB preferentially inhibits the activity of the lower (38 kDa) band. At least six isoforms of p38-MAPK are known (two alternative splicing isoforms, α and β, and isoforms τ and δ). These p38-MAPKs differ in their sensitivity to stimuli, inhibitors, and substrate specificity. We cannot rule out the possibility that multiple p38-MAPK isoforms are activated during myocardial ischemia. However, PC12 cells exhibit selective activation of p38-MAPK-α and p38-MAPK-γ under hypoxic conditions. Hypoxia had no effect on the activity of the β and δ isoforms. Our in vitro results using SB (in-gel phosphorylation assay; Figures 8A and C) showed that SB completely inhibited ischemia-induced p38-MAPK activity. Previous studies have shown that the γ and δ isoforms are resistant to the inhibitory effect of SB/SB203580. This suggests that these two p38-MAPK isoforms (γ and δ) are not involved in the role of SB during ischemia or in the mechanisms leading to ischemic death. In summary, we provide detailed information on the detrimental effects of p38-MAPK activation during ischemia, which can be inhibited by SB. We further confirm our hypothesis that ischemia/reperfusion activates different signaling pathways with opposing effects on cell survival, with the ERK and SAPK/JNK pathways promoting cell survival, while the p38-MAPK pathway accelerates cell death. These findings may contribute to the development of future treatment strategies for ischemic syndromes. [4] In summary, p38 MAPK inhibitors may reduce autoimmune attacks on vital organs and improve renal function. Furthermore, serum ALT and AST levels in MRL/lpr mice treated with SB203580 did not increase, and the survival rate of SB203580-treated MRL/lpr mice was similar to that of untreated MRL/lpr mice during the experiment, indicating that SB203580 had no significant adverse effects on lifespan and liver function in MRL/lpr mice. Although the efficacy of SB203580 was observed, since this study found that SB203580 could hardly reverse almost all the pathological features analyzed in MRL/lpr mice compared with the negative control C57BL/6 mice, the inhibition of p38 MAPK is insufficient to completely prevent organ damage. More animal experiments and the inclusion of more lupus-prone animal models are needed to confirm the efficacy and mechanism of action of SB203580, thereby assessing its potential clinical application value in the treatment of human diseases. Although many different p38 MAPK inhibitors are undergoing clinical trials, none of them have been successfully approved for the treatment of autoimmune diseases due to the safety risks posed by potential cross-reactivity with other kinases. In addition, adverse reactions to the central nervous system and liver were observed in the phase II clinical trials of VX745 and BIRB796. However, there have been no reports of side effects of SB203580 to date. Therefore, further research is needed on SB203580 to verify its safety in other systems. At the same time, the survival and clinical improvement after drug discontinuation also need to be evaluated. [5]

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Mechanism of action: Adezmapimod is a reversible, ATP-competitive p38α/β inhibitor. It binds to the ATP-binding pocket of p38, preventing ATP coordination and subsequent phosphorylation of downstream substrates (such as MK2, HSP27). [1]
-Research applications: It is widely used as a tool compound to study the p38-mediated pathway in inflammation, cancer and cardiovascular diseases. It was included in a phase II clinical trial for rheumatoid arthritis, but was terminated due to poor efficacy. [5, 4]
- Selectivity note: Unlike non-selective p38 inhibitors, it exhibits extremely low activity against JNK/ERK, thereby reducing off-target effects in inflammatory and cardiovascular models. [1, 2]

C21H16FN3OS

377.43

377.099

C, 66.83; H, 4.27; F, 5.03; N, 11.13; O, 4.24; S, 8.49

152121-47-6

Adezmapimod hydrochloride;869185-85-3

176155

White to light yellow solid powder

1.4±0.1 g/cm3

615.6±55.0 °C at 760 mmHg

249 - 250ºC

326.1±31.5 °C

0.0±1.7 mmHg at 25°C

1.715

4.1

1

5

4

27

500

0

S(C([H])([H])[H])(C1C([H])=C([H])C(=C([H])C=1[H])C1=NC(C2C([H])=C([H])C(=C([H])C=2[H])F)=C(C2C([H])=C([H])N=C([H])C=2[H])N1[H])=O

CDMGBJANTYXAIV-UHFFFAOYSA-N

InChI=1S/C21H16FN3OS/c1-27(26)18-8-4-16(5-9-18)21-24-19(14-2-6-17(22)7-3-14)20(25-21)15-10-12-23-13-11-15/h2-13H,1H3,(H,24,25)

4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine;hydrochloride

RWJ 64809; PB 203580; Adezmapimod; 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine; 4-(4-(4-fluorophenyl)-2-(4-(methylsulfinyl)phenyl)-1H-imidazol-5-yl)pyridine; RWJ64809; SB203580; SB203580; SB 203580; RWJ-64809; PB-203580; PB203580

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.62 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2 mg/mL (5.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 2 mg/mL (5.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: 2 mg/mL (5.30 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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Solubility in Formulation 6: 16.67 mg/mL (44.17 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)