PAC-1

• Catalog No.: S2738
• CAS Number: 315183-21-2
• M. F.: C23H28N4O2
• M. W.: 392.49402
• Storage: -20°C

SYNONYMS

• IUPAC name: 2-(4-benzylpiperazin-1-yl)-N'-[(1E)-[2-hydroxy-3-(prop-2-en-1-yl)phenyl]methylidene]acetohydrazide
• IUPAC Traditional name: 2-(4-benzylpiperazin-1-yl)-N'-[(1E)-[2-hydroxy-3-(prop-2-en-1-yl)phenyl]methylidene]acetohydrazide
• Synonyms: Procaspase activating compound 1

DATABASE IDS

• CAS Number: 315183-21-2

PROPERTIES

• Target: Caspase
• Salt Data: Free base
• Storage Condition: -20°C

DETAILS

Description (English)

Research Area

• Description: Cancer

Biological Activity

• Description: PAC-1 is a potent procaspase-3 activator with EC50 of 0.22 μM.
• Targets: Procaspase-3
• IC50: 0.22 μM ^[1]
• In Vitro: PAC-1 activates procaspase-7 in a less efficient manner with EC50 of 4.5 μM. Elevated caspase 3 level in cancer cell lines allows PAC-1 to selectively induce apoptosis in a manner proportional to procaspase-3 concentration with IC50 of 0.35 μM for NCI-H226 cells to ~3.5 μM for UACC-62 cells. PAC-1 induces apoptosis in the primary cancerous cells with IC50 values of 3 nM to 1.41 μM, more potently than in the adjacent noncancerous cells with IC50 of 5.02 μM to 9.98 μM, which is also directly related to the distinct procaspase-3 concentration. ^[1] PAC-1 activates procaspase-3 by chelating zinc ions, thus relieving the zinc-mediated inhibition and allowing procaspase-3 to auto-activate itself to caspase-3. ^[2] PAC-1 is capable to induce cell death in Bax/Bak double-knockout cells and Bcl-2 and Bcl-xL-overexpressing cells with the same efficacy as its wild-type counterpart in a delayed manner. PAC-1 induces cytochrome c release in a caspase-3 independent manner, which subsequently triggers downstream caspase-3 activation and cell death. PAC-1 can not induce cell death and caspase-3 activation in Apaf-1 knockout cells, suggesting that apoptosome formation is essential for caspase-3 activation by PAC-1-mediated cell death. ^[3]
• In Vivo: Administration of PAC-1 at 5 mg with low and steady releasing significantly inhibits the growth of ACHN renal cancer xenograft in mice. Oral administration of PAC-1 (50 or 100 mg/kg) significantly retards tumor growth of NCI-H226 lung cancer xenograft in a dose-dependent manner, and markedly prevents the cancer cells from infiltrating the lung tissue. The in vivo anti-tumor effect of PAC-1 is ascribed to procaspase-3 activation and subsequently apoptosis induction consistent with the activity in vitro. ^[1]
• Features: PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3

Protocol

• Protocol: Kinase Assay ^[1]
• In vitro procaspase-3 activation: Procaspase-3 is expressed and purified in Escherichia coli. Various concentrations of PAC-1 are added to 90 μL of a 50 ng/mL of procaspase-3 in caspase assay buffer in a 96-well plate, The plate is incubated for 12 hours at 37 °C. A 10 μL volume of a 2 mM solution of caspase-3 peptidic substrate acetyl Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNa) in caspase assay buffer is then added to each well. The plate is read every 2 minutes at 405 nm for 2 hours in a Spectra Max Plus 384 well plate reader. The slope of the linear portion for each well is determined, and the relative increase in activation from untreated control wells is calculated.
• Protocol: Cell Assay ^[1]
• Cell Lines: U-937, HL-60, CRL-1872, ACHN, NCI-H226, Hs888Lu, Hs578Bst, MCF-10A, SK-MEL-5, BT-20, MDA-MB-231, UACC-62, SK-N-SH, B16-F10 , Hs 578t, and PC-12
• Concentrations: Dissolved in DMSO, final concentrations ~100 μM
• Incubation Time: 72 hours
• Methods: Cells are exposed to various concentrations of PAC-1 for 72 hours. Cell death is quantified by the addition of MTS/PMS CellTiter 96 Cell Proliferation Assay reagent. The plates are incubated at 37 °C for approximately 1 hour (until the colored product formed), and the absorbance is measured at 490 nm.
• Protocol: Animal Study ^[1]
• Animal Models: Ovariectomized female athymic BALB/c (nude, nu/nu) mice injected subcutaneously with ACHN cells, male athymic BALB/c nude mice injected subcutaneously with NCI-H226 cells, and male athymic BALB/c–/– mice injected intravenously with NCI-H226 cells
• Formulation: Mixed with cholesterol and pelleted into a 3-mm-diameter 20-mg (total weight) pellet, or dissolved in a mixture of 24:1 vegetable oil/DMSO
• Doses: ~100 mg/kg
• Administration: Pellet implantation subcutaneously or oral gavage

References

• References: [1] Putt KS, et al. Nat Chem Biol, 2006, 2(10), 543-550.
• References: [2] Peterson QP, et al. J Mol Biol, 2009, 388(1), 144-158.
• References: [3] Seervi M, et al. Cell Death Dis, 2011, 2, e207.

REFERENCES

• Putt KS, et al. Nat Chem Biol, 2006, 2(10), 543-550.
• Peterson QP, et al. J Mol Biol, 2009, 388(1), 144-158.
• Seervi M, et al. Cell Death Dis, 2011, 2, e207.