p38 MAPK (IC50 = 89 nM); TGF-β; Activin A; Enterovirus71
p38 mitogen-activated protein kinase (p38 MAPK); [1]
- p38 MAPK; no specific IC50, Ki, or EC50 values provided in the literature [2]
- p38 mitogen-activated protein kinase (p38 MAPK); [3]
- p38 mitogen-activated protein kinase (p38 MAPK); [4]
- p38 mitogen-activated protein kinase (p38 MAPK); [5]

PD169316 (10 μM) inhibits TGFβ and Activin A signaling but not BMP4 signaling in CaOV3 cells. In CaOV3 cells, PD169316 (0.2–20 μM) inhibits TGFβ-induced Smad2 nuclear translocation, Smad7 mRNA induction, and reporter gene activity[1]. In Nestin knockdown cells, PD169316 (10 μM) exhibits a noticeably higher rate of proliferation, and it can reverse the effect of Nestin knockdown on cell viability in the absence of EGF[2]. In PC12 cells, PD169316 significantly reduces p38 MAP kinase activity while having no effect on ERK activity. In differentiated PC12 cells, PD169316 (10 μM) inhibits the apoptosis that is brought on by the removal of trophic factors[3]. Without preventing upstream kinases from phosphorylating p38, PD169316 (10 μM, 30 min) selectively inhibits the kinase activity of the phosphorylated p38. Increased phospho p-38 levels in the presence of PD169316 are most likely caused by MAPK phosphatases blocking the negative feedback loop of p38 MAPK dephosphorylation[4].

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1. In human ovarian cancer cells, treatment with PD169316 at a concentration of 5 μM or higher dose-dependently inhibited transforming growth factor beta (TGFβ)-induced Smad signaling. This inhibition was manifested by reduced phosphorylation of Smad2 and Smad3, decreased nuclear translocation of Smad proteins, and down-regulated expression of the TGFβ target gene Smad7. Notably, PD169316 also abrogated Activin A-induced signaling but had no effect on bone morphogenetic protein (BMP) 4-induced signaling. It was confirmed that the inhibition of TGFβ signaling by PD169316 was not due to the abrogation of p38 MAPK activity, as blocking p38 MAPK activity with a dominant negative form of p38 MAPK did not affect TGFβ/Smad signaling [1]
2. In mouse neural progenitor cells (mNPCs) where Nestin was knocked down (resulting in cell cycle arrest and reduced proliferation), treatment with the PD169316 reversed the cell cycle arrest. Specifically, PD169316 increased the viability of Nestin-knockdown mNPCs, making their growth curve similar to that of control cells. Additionally, PD169316 significantly increased the number of neurospheres in Nestin-knockdown mNPCs in sphere-forming assays. Even in the absence of epidermal growth factor (EGF), PD169316 could rescue the reduced cell viability caused by Nestin knockdown [2]
3. In Rat-1 fibroblasts deprived of serum (resulting in elevated p38 activity and increased apoptosis) and differentiated PC12 cells deprived of nerve growth factor (NGF) (also leading to increased p38 activity and apoptosis), treatment with PD169316 blocked p38 enzyme activity by approximately 80% and simultaneously inhibited apoptosis by about 80%. In contrast, the MAP kinase kinase inhibitor PD98059 had no effect on apoptosis in Rat-1 fibroblasts and only partially blocked apoptosis in PC12 cells [3]
4. In Ishikawa cells stably expressing progesterone receptor A (PRA), pretreatment with PD169316 (10 μM) impaired the stabilization of PRA induced by MAPK kinase kinase 1 (MEKK1). This effect was observed through immunoblot analysis, which showed that PD169316 reduced the fold increase in PRA levels caused by MEKK1 transfection compared to control cells [4]
5. In cell models infected with Enterovirus 71 (EV71), PD169316 exhibited significant antiviral activity by inhibiting EV71 replication. Additionally, PD169316 reduced EV71-induced apoptosis in infected cells [5]

PD169316 PD169316 (1 mg/kg, intramuscular injection every day for 14 days straight) exhibits antiviral activity in a suckling mouse model[5].

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1. In suckling mice infected with EV71, administration of PD169316 dampened EV71 replication, reduced tissue damage (observed through pathological analysis of affected tissues), and inhibited the release of inflammatory cytokines. These effects collectively alleviated the severe diseases caused by EV71 infection in suckling mice [5]

The cells are incubated in the absence or presence of insulin (50 ng/mL) for 15 min at 37°C sixteen hours after the removal of serum from Rat-1 fibroblasts or NGF from differentiated PC12 cells. The cells are then solubilized in 400 μL of ice-cold immunoprecipitation buffer, which contains 10 mM Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, and 0.2 mM phenylmethylsulfonyl fluoride. The cell lysates are centrifuged to remove insoluble material, and 200 g of the supernatant protein (400 μL, total volume) is incubated with 1 μg of anti-p38 antibodies for 1 h at 4°C before being incubated with 30 μL of Protein G Plus/Protein A-agarose for an additional hour. The immunocomplexes are pelleted, washed three times in immunoprecipitation buffer, and then once in kinase ish buffer (50 mM β-glycerolphosphate, 1 mM EGTA, 20 mM MgCl2, and 100 μM sodium orthovanadate). The protein kinase assay is initiated by the addition of 20 μL of 2× reaction buffer (50 mMβ-glycerolphosphate, 1 mM EGTA, 20 mM MgCl2, 100 μM sodium orthovanadate, 0.1 mg/mL ATF-2 (N-terminal half), 50 μg/mL IP20, a peptide inhibitor of c-AMP dependent protein kinase, 200 μM ATP, and 0.9 mCi/mL [32P]ATP) to 20 μL of immune complex. The reaction is allowed to run for 10 minutes at 30 degrees Celsius before being stopped by adding 2× LaemmLi sample buffer. This reaction is then examined using 12% acrylamide gels and SDS-polyacrylamide gel electrophoresis. The gels are dried and then put through phosphoimaging after electrophoresis.

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1. For the detection of p38 MAPK activity in Rat-1 fibroblasts and PC12 cells: Cells were first treated with PD169316 at the appropriate concentration. Then, cell lysates were prepared, and immune complex assays were performed. In these assays, p38 MAPK was immunoprecipitated from the lysates using specific antibodies against p38 MAPK. The immunoprecipitated p38 MAPK was then incubated with a specific substrate (e.g., a peptide substrate) and ATP in a reaction buffer. After a certain incubation period, the amount of phosphorylated substrate was measured using techniques such as scintillation counting or Western blotting with phospho-specific antibodies to determine p38 MAPK activity. This assay showed that PD169316 inhibited p38 enzyme activity [3]

KBU cells are subjected to 12 ppm G. pps either alone or in combination with 1 hour of pre-exposure to the P38 inhibitor PD169316, and their proliferation (MTT), apoptosis (Ann V), and changes in the degree of protein modifications in the SAPK/JNK and P38 signaling pathways are assessed.

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1. Human ovarian cancer cell assay for TGFβ-induced Smad signaling: Human ovarian cancer cells were seeded in appropriate culture plates and cultured to reach the desired confluency. The cells were then divided into different groups: control group (no treatment), TGFβ-treated group, and TGFβ + PD169316 (at concentrations of 5 μM or higher) treatment groups. After incubation for a specific period, the cells were harvested. For the detection of Smad2 and Smad3 phosphorylation, Western blot analysis was performed using cell lysates and phospho-specific antibodies against Smad2 and Smad3. For nuclear translocation analysis of Smad proteins, immunofluorescence staining was conducted: cells were fixed, permeabilized, incubated with Smad-specific antibodies, followed by fluorescently labeled secondary antibodies, and then observed under a fluorescence microscope. For the analysis of Smad7 gene expression, reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR (qPCR) was used to measure the mRNA level of Smad7 [1]
2. Mouse neural progenitor cell (mNPC) assay: mNPCs were isolated and cultured in medium containing EGF and basic fibroblast growth factor (bFGF) to maintain their stemness. Nestin knockdown was achieved by infecting mNPCs with Nestin shRNA adenovirus (with scramble virus-infected and uninfected cells as controls). After 72 hours of infection, PD169316 was added to the culture medium of the Nestin-knockdown group. For cell viability analysis, MTT or CCK-8 assays were performed at specific time points to measure the absorbance values, which reflected cell viability. For sphere-forming assays, mNPCs were seeded in low-attachment plates at a low density, and after a certain culture period, the number and size of neurospheres were counted and measured. For cell cycle analysis, flow cytometry was used: cells were harvested, fixed, stained with propidium iodide (PI), and then analyzed to determine the percentage of cells in G1, S, and G2 phases [2]
3. Rat-1 fibroblast and PC12 cell apoptosis assay: Rat-1 fibroblasts were cultured in medium with serum, and then serum was withdrawn to induce apoptosis. Differentiated PC12 cells were cultured in medium containing NGF, and then NGF was withdrawn to induce apoptosis. PD169316 was added to the culture medium of the serum-deprived Rat-1 fibroblasts and NGF-deprived PC12 cells. After 16 hours of incubation, apoptosis was detected using methods such as flow cytometry (Annexin V-FITC/PI double staining) or TUNEL assay to count the number of apoptotic cells [3]
4. Ishikawa cell assay for PRA stability: Ishikawa cells stably expressing PRA were transfected with empty control vector or MEKK1 expression vector. After 24 hours of transfection, PD169316 (10 μM) was added to the culture medium. After a specific incubation period, cells were harvested, and Western blot analysis was performed using cell lysates and PRA-specific antibodies to detect the expression level of PRA. The band intensities were quantified to analyze the effect of PD169316 on MEKK1-induced PRA stabilization [4]
5. EV71-infected cell assay: Cells (e.g., Vero cells) were seeded in culture plates and cultured until confluent. EV71 was added to the cells at a specific multiplicity of infection (MOI) and incubated for a certain time to allow viral infection. Then, PD169316 was added to the culture medium at the appropriate concentration. After incubation for a specific period, the viral titer in the cell supernatant was determined using plaque assay: serial dilutions of the supernatant were added to confluent Vero cells, overlaid with agarose, and after incubation, the number of plaques was counted to calculate the viral titer. For the detection of EV71-induced apoptosis, flow cytometry (Annexin V-FITC/PI double staining) was used [5]

EV71-challenged suckling mouse model (7-day-old Kunming mice)[5].
1 mg/kg.
Intramuscular injection every day for 14 consecutive days.

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1. Suckling mouse model of EV71 infection: Suckling mice (e.g., 1-day-old or 3-day-old BALB/c mice) were used. EV71 was inoculated into the suckling mice via intraperitoneal injection or intracerebral injection at a specific viral dose to establish the EV71 infection model. PD169316 was prepared by dissolving it in an appropriate solvent (e.g., dimethyl sulfoxide (DMSO) followed by dilution with physiological saline to a suitable concentration). The drug was administered to the infected suckling mice via intraperitoneal injection at a specific dose and frequency (e.g., once a day for several consecutive days). During the experiment, the general condition of the mice (such as activity, feeding, and mortality) was observed and recorded. After the experiment, the mice were euthanized, and tissues (e.g., brain, heart, and limbs) were collected. Pathological sections of the tissues were prepared and stained with hematoxylin and eosin (HE) to observe tissue damage. The viral load in the tissues was detected using methods such as RT-PCR or virus isolation. The levels of inflammatory cytokines (e.g., TNF-α, IL-6) in the serum or tissue homogenates were measured using enzyme-linked immunosorbent assay (ELISA) [5]

[1]. The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor beta-induced Smad signaling in human ovarian cancer cells. Biochem Biophys Res Commun. 2003 Oct 17;310(2):391-7.

[2]. Suppression of Nestin reveals a critical role for p38-EGFR pathway in neural progenitor cell proliferation. Oncotarget. 2016 Dec 27;7(52):87052-87063.

[3]. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem. 1997 Aug 15;272(33):20490-4.

[4]. p38 and p42/44 MAPKs differentially regulate progesterone receptor A and B isoform stabilization. Mol Endocrinol. 2011 Oct;25(10):1710-24.

[5]. PD169316, a specific p38 inhibitor, shows antiviral activity against Enterovirus71. Virology. 2017 Aug;508:150-158.

4-[4-(4-fluorophenyl)-2-(4-nitrophenyl)-1H-imidazol-5-yl]pyridine is an imidazole compound. PD-169316 is a p38 MAP kinase inhibitor. 1. PD169316 belongs to the pyridylimidazolium class of compounds [3]. 2. When PD169316 is used in experiments at concentrations of 5 μM or higher, it can block the activity of the TGFβ signaling pathway. Therefore, when using this inhibitor, cellular activity must be carefully attributed entirely to the p38 MAPK signaling pathway [1]. 3. In Nestin-mediated regulation of mNPC proliferation, PD169316 works by targeting the p38-MAPK pathway, which is involved in the mechanism by which Nestin promotes mNPC proliferation and self-renewal [2]. 4. In studies of insulin-mediated cell survival, PD169316 and insulin have similar effects in blocking p38 activity and apoptosis, supporting the hypothesis that insulin promotes cell survival at least partially by inhibiting the p38 pathway [3]. 5. The regulation of PRA/PRB stability by PD169316 (targeting p38 MAPK) is independent of PR serine 294 phosphorylation, which was previously considered a major sensor for PR. [4] 6. The antiviral activity of PD169316 against EV71 is related to its regulation of the p38-MAPK signaling pathway, which may be involved in the inflammatory response caused by EV71 infection [5].

C20H13FN4O2

360.34

360.102

C, 66.66; H, 3.64; F, 5.27; N, 15.55; O, 8.88

152121-53-4

4712

white solid powder

1.4±0.1 g/cm3

583.1±50.0 °C at 760 mmHg

306.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.651

5.32

1

5

3

27

495

0

FC(C=C1)=CC=C1C2=C(C3=CC=NC=C3)NC(C4=CC=C([N+]([O-])=O)C=C4)=N2

BGIYKDUASORTBB-UHFFFAOYSA-N

InChI=1S/C20H13FN4O2/c21-16-5-1-13(2-6-16)18-19(14-9-11-22-12-10-14)24-20(23-18)15-3-7-17(8-4-15)25(26)27/h1-12H,(H,23,24)

4-[4-(4-fluorophenyl)-2-(4-nitrophenyl)-1H-imidazol-5-yl]pyridine

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)

DMSO: ~14 mg/mL ( ~38.6 mM)
Water: Insoluble
Ethanol: Insoluble

Solubility in Formulation 1: 1.25 mg/mL (3.47 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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 2: 1.25 mg/mL (3.47 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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 3: ≥ 1.25 mg/mL (3.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5%DMSO+40%PEG300+5%Tween 80+ 50%ddH2O: 0.7mg/ml (1.94mM)

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Solubility in Formulation 5: 5 mg/mL (13.88 mM) in 50% PEG300 50% Saline (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.)