Checkpoint kinase 1 (CHK1) inhibitor
Wee1 inhibitor

PKC inhibitor

When CHK1 is sufficiently blocked by PD 407824, p21 is significantly downregulated, activating CDK8/9 and SMAD2/3 depletion [1]. With an IC50 of 3.75 µM and 3.4 µM for Chk1 and WEE1, respectively, PD 407824 is more selective for these two proteins than PKC and Cdk4[3].

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PD407824 sensitizes C2C12 myoblasts to sub-threshold concentrations of BMP4. In a pld2-LucGFP reporter assay, the EC50 of PD407824 decreases as BMP4 concentration increases (EC50 = 12.3 μM in the absence of BMP4; EC50 = 0.75 μM in the presence of 0.1 ng/ml BMP4; EC50 = 0.12 μM in the presence of 1 ng/ml BMP4). [2]
PD407824 (1 or 10 μM) + BMP4 (10 ng/ml) treatment increased Id2 transcript levels 9.6 ± 2.2 fold and 11.5 ± 0.7 fold, respectively, compared to DMSO+10 ng/ml BMP4 (3.7 ± 1.0 fold) in C2C12 myoblasts. [2]
PD407824 + BMP4 quantitatively increased SMAD1/5/9 phosphorylation in C2C12 myoblasts, as shown by western blotting. PD407824 alone showed a trend to slightly decrease it, but this was not statistically significant. [2]
PD407824 (1 μM) + BMP4 (3 ng/ml) treatment in C2C12 myoblasts induced alkaline phosphatase (ALK) activity equal to that of cells treated with 10 ng/ml BMP4 alone. It also induced the expression of osteoblast markers (collagen I, osteopontin, osteocalcin) and mineralization, comparable to 10 ng/ml BMP4 treatment. [2]
PD407824 (1 μM) + BMP4 (3 ng/ml) treatment in human embryonic stem cell (hESC) H9 cells increased Id2 transcript levels 7.1 ± 0.3 fold higher than DMSO+3 ng/ml BMP4. It also increased transcript levels of the mesoderm marker Brachyury T. [2]
PD407824 (2.5 μM) + BMP4 (9 ng/ml) treatment in hESC H1 cells significantly increased the percentage of KRT7+ cytotrophoblast stem cells and the expression of cytotrophoblast markers TP63 and GCM1, comparable to treatment with 30 ng/ml BMP4. [2]
PD407824 functions through inhibiting CHK1, as another CHK1 inhibitor, CHIR124, mimics its effect by synergizing with BMP4 to upregulate Id2 in a dose-dependent manner and increase SMAD1/5/9 phosphorylation. [2]
Mechanistic studies revealed that PD407824 treatment significantly decreases p21 protein levels, which activates CDK8/9. CDK8/9 then phosphorylates the SMAD2/3 linker region (e.g., T179 for SMAD3), leading to decreased total SMAD2/3 protein levels. This frees up SMAD4 to bind with SMAD1/5/9, enhancing BMP signaling. Flavopiridol (a CDK inhibitor) or CDK9 knockout blocks this effect. [2]
PD407824 (2.5 μM) treatment in hESCs did not significantly affect the number of Ki67+ cells or induce γ-H2A.X+ cells, suggesting no significant effect on cell proliferation or DNA damage during differentiation. [2]

BMP Reporter Assay: C2C12 myoblasts stably expressing a pld2-LucGFP BMP reporter were seeded in 384-well plates. After 24 hours, the medium was changed to serum-free DMEM for 24 hours to reduce background. Cells were then treated with PD407824 at various concentrations (0-15 μM) with or without BMP4 (0.1 or 1 ng/ml). After 24 hours of treatment, cells were lysed and luciferase activity was measured using a luciferase substrate to determine the compound's effect on BMP pathway activation. [2]
Gene Expression Analysis (qPCR): C2C12 myoblasts or hESCs were treated with PD407824 (e.g., 1, 10 μM for C2C12; 0.1, 0.3, 1 μM for hESCs) alone or in combination with various concentrations of BMP4 for 24 hours. Total RNA was isolated and reverse transcribed. Quantitative real-time PCR (qPCR) was performed to measure the transcript levels of BMP target genes such as Id1, Id2, as well as mesoderm (Brachyury T), cardiomyocyte (TNNT2, MHY6, MLC2v), and cytotrophoblast (KRT7, TP63, GCM1) markers. Expression levels were normalized to a control gene and compared to DMSO-treated samples. [2]
Western Blotting: C2C12 myoblasts or hESCs were treated with PD407824 (e.g., 10 μM) with or without BMP4 (e.g., 3 ng/ml) for 24 hours. Cells were lysed in a complete lysis buffer. Proteins were resolved by electrophoresis, transferred to PVDF membranes, and probed with primary antibodies against phospho-SMAD1/5/9, total SMAD1, SMAD2/3, phospho-SMAD3 (T179), p21, and beta-ACTIN. Membranes were then incubated with secondary antibodies and developed using an ECL substrate to visualize protein levels and phosphorylation states. [2]
Osteoblast Differentiation: C2C12 myoblasts were treated with DMSO, PD407824 (e.g., 1 μM), BMP4 (3 ng/ml), PD407824 + BMP4, or a positive control of BMP4 (10 ng/ml) for 6-22 days. Alkaline phosphatase (ALK) activity was assessed by staining or a fluorescence-based substrate assay after 6 days. Expression of osteoblast markers (collagen I, osteopontin, osteocalcin) was analyzed by qPCR. Mineralization was evaluated by Alizarin Red S staining after 22 days of treatment. [2]
Cardiomyocyte Differentiation: hESC H9 cells were formed into embryoid bodies (EBs) and treated with DMSO or PD407824 (0.1, 0.3, 1 μM) combined with BMP4 (3 ng/ml) for 4 days. EBs were then cultured in cardiac differentiation medium. Beating colonies were counted at day 10. On day 12, cells were fixed for immunocytochemistry with anti-TNNT2 antibody or dissociated for intracellular flow cytometry to quantify the percentage of TNNT2+ cardiomyocytes. RNA was also isolated for qPCR analysis of cardiomyocyte markers. [2]
Trophoblast Differentiation: hESC H1 cells were treated with DMSO or PD407824 (2.5 μM) combined with various concentrations of BMP4 (0-30 ng/ml) in MEF-conditioned medium for 7 days. Cells were then fixed and stained with anti-KRT7 antibody for image analysis or dissociated for intracellular flow cytometry to quantify the percentage of KRT7+ cytotrophoblast cells. RNA was isolated for qPCR analysis of cytotrophoblast markers. [2]
Co-Immunoprecipitation (Co-IP): C2C12 cells were treated with BMP4 (3 ng/ml) in the presence or absence of PD407824 (10 μM) for 24 hours. Proteins from cell lysates were precipitated with an anti-SMAD4 antibody. The immunoprecipitated complexes were then analyzed by western blotting using an anti-SMAD1 antibody to detect the interaction between SMAD1 and SMAD4. [2]
CRISPR Knockout Experiments: C2C12 cells were infected with lentivirus expressing Cas9 and sgRNAs targeting CHK1, p21, CDK9, or GFP (control). After puromycin selection and subcloning, knockout clones (e.g., CHK1+/-, CHK1-/-; p21+/-, p21-/-; CDK9+/-, CDK9-/-) were validated by western blotting. These cells were then treated with BMP4 (e.g., 1 or 3 ng/ml) and analyzed for Id2 expression by qPCR or for osteoblast differentiation capacity to study the genetic requirement of these genes in the PD407824 mechanism. [2]

In hESC differentiation studies, treatment with PD407824 (2.5 μM) did not significantly affect the number of Ki67+ proliferating cells. [2]
Treatment with PD407824 (2.5 μM) did not induce γ-H2A.X+ cells, a marker for DNA damage, suggesting it does not affect DNA repair during hESC differentiation. [2]

[1]. 4-Phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione inhibitors of the checkpoint kinase Wee1. Structure-activity relationships for chromophore modification and phenyl ring substitution. J Med Chem. 2006 Aug 10;49(16):4896-911.

[2]. Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation. Cell Rep. 2016 May 31;15(9):2063-75.

[3]. Synthesis and structure-activity relationships of N-6 substituted analogues of 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of Wee1 and Chk1 checkpointkinases. Eur J Med Chem. 2008 Jun;43(6):1276-96. Epub 200.

PD407824 was identified as a "BMP sensitizer" from a high-throughput chemical screen using C2C12 myoblasts with a BMP-responsive luciferase reporter, where it was found to increase cell sensitivity to sub-threshold levels of BMP4. [2]
The mechanism of action involves inhibition of CHK1, leading to down-regulation of p21. This activates CDK8/9, which phosphorylate the linker region of SMAD2/3, targeting them for proteasomal degradation. The subsequent depletion of SMAD2/3 reduces competition for SMAD4, allowing more SMAD1/5/9-SMAD4 complexes to form and enhance BMP target gene expression. [2]
PD407824 is shown to be a useful pharmacological reagent for directing the differentiation of human embryonic stem cells (hESCs) towards mesoderm (and subsequently cardiomyocytes) and cytotrophoblast stem cell fates, by synergizing with low, sub-threshold concentrations of BMP4. [2]
The study suggests that CHK1 inhibitors, like PD407824, could be a stable and cost-effective alternative or adjunct to recombinant BMP proteins in stem cell differentiation protocols and potentially in clinical applications for bone repair, as they have already been used in Phase I/II clinical trials for cancer. [2]

C20H12N2O3

328.32

328.085

622864-54-4

4369491

Light yellow to yellow solid powder

1.507g/cm3

1.81

3.333

3

3

1

25

573

0

IAUZTOZLTFSMIE-UHFFFAOYSA-N

InChI=1S/C20H12N2O3/c23-11-6-7-14-13(8-11)16-15(21-14)9-12(10-4-2-1-3-5-10)17-18(16)20(25)22-19(17)24/h1-9,21,23H,(H,22,24,25)

9-hydroxy-4-phenyl-6H-pyrrolo[3,4-c]carbazole-1,3-dione

PD0407824 PD 0407824 PD-0407824

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 : ~250 mg/mL (~761.45 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 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: ≥ 2.08 mg/mL (6.34 mM) (saturation unknown) 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.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)