Nirogacestat (PF-03084014)

Catalog No.S8018 Batch:S801801

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Technical Data

Formula

C27H41F2N5O
Molecular Weight 489.64 CAS No. 1290543-63-3
Solubility (25°C)* In vitro DMSO 97 mg/mL (198.1 mM)
Ethanol 97 mg/mL (198.1 mM)
Water Insoluble
In Vivo (Add solvents to the product individually and in order.)
Homogeneous suspension
CMC-NA
≥5mg/ml Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description Nirogacestat (PF-03084014, PF-3084014) is a selective gamma-secretase inhibitor with an IC50 of 6.2 nM in a cell-free assay, and it induces apoptosis. Phase 2.
Targets
gamma-secretase
(cell-free assay)
6.2 nM
In vitro Nirogacestat (PF-03084014) inhibits Notch receptor cleavage in cellular assays using HPB-ALL cells that harbour mutations in both the heterodimerization and PEST domains in Notch1 with an IC50 of 13.3 nM. It downregulates Notch target genes Hes-1 and cMyc expression in HPB-ALL cells with IC50s of <1 nM and 10 nM, respectively, and inhibits cell growth of a subset of human T-ALL cell lines (HPB-ALL, DND-41, TALL-1, and Sup-T1) through induction of cell cycle arrest and apoptosis with IC50s of 30–100 nM. This compound reduces proliferation of HUVECs with an IC50 of 0.5 μM, and decreases lumen formation with an IC50 value of 50 nM. It (1 μM) has no antiproliferative effect in MX1 cells; however, it inhibits migration by 95%.
In Vivo Nirogacestat (PF-03084014) orally administered in a single dose of 200 mg/kg causes maximal NICD inhibition for ∼80% in xenograft HPB-ALL tumours. It shows robust anti-tumour activity in this mode with a maximal tumour growth inhibition of ∼92% at a dose of 150 mg/kg, accompanied by a significant reduction of NICD/Notch1, tumour mitotic index (Ki67), and apoptosis (activated caspase-3) staining. At 120 mg/kg, this compound induces apoptosis, antiproliferation, reduces tumour cell self-renewal ability, impairs tumour vasculature, and decreases metastasis activity in breast cancer HCC1599 tumour-bearing mice. Its treatment displays significant anti-tumour activity in various types of the breast xenograft models with a TGI value of at least 50%.

Protocol (from reference)

Kinase Assay:[3]
  • γ-secretase assay

    A DNA fragment encoding amino acids 596 - 695 of the 695-aa isoform of APP (APP695) and the Flag sequence (DYKDDDDK) at the C terminus is generated by PCR amplification with suitably designed oligonucleotides and the APP695 cDNA. The Met that serves as the translation start site is residue 596 of APP695 (the P1 residue with respect to the β-secretase cleavage site). This DNA fragment is inserted into the prokaryotic expression vector pET2-21b. The recombinant protein, C100Flag, is overproduced in Escherichia coli [strain BL21(DE3)] and purified by Mono-Q column chromatography. C100Flag (1.7 μM) is incubated with cell membranes (0.5 mg/mL) in the presence of CHAPSO, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or Triton X-100 (0, 0.125, 0.25, 0.5, or 1%) in buffer B (50 mM Pipes, pH 7.0y 5mM MgCl2/5 mM CaCl2/150 mM KCl) at 37°C. The reactions are stopped by adding RIPA (150 mM NaCl/1.0% NP-40/0.5% sodium deoxycholatey 0.1% SDS/50 mM Tris HCl, pH 8.0) and boiling for 5 min. The samples are centrifuged and the supernatant solutions are assayed for the Aβ peptides by ECL. The Aβ40- and Aβ42-related products from γ-secretase-mediated processing of C100Flag possess a Met at the N terminus and are thus defined as M-Aβ40 and M-Aβ42, respectively. Likewise, supernatant solution (0.125 mg/mL) from CHAPSO-extracted HeLa cell membranes (solubilized γ-secretase) is incubated with C100Flag (1.7 μM) in buffer B containing 0.25% CHAPSO and subsequently assayed for M-Aβ40 and M-Aβ42 by using ECL. This compound, Nirogacestat (PF-03084014), is used in the experimental context.
Cell Assay:[1]
  • Cell Lines

    Human T-ALL cell lines HPB-ALL

  • Concentrations

    ~1 μM

  • Incubation Time

    7 days

  • Method

    Cells are seeded in 96-well plates at 10,000 cells/well in growth media supplemented with 10% fetal bovine serum. Serial dilutions of Nirogacestat (PF-03084014) are done in DMSO, appropriate controls or designated concentrations of this compound are added to each well, and cells are incubated at 37℃ for 7 days (final DMSO content 0.1%). Resazurin at a final concentration of 0.1 mg/mL is added to the cells and plates are incubated for 2 to 4 hours. Fluorescent signals are read as emission at 590 nm after excitation at 560 nm.
Animal Study:[1]
  • Animal Models

    Human T-cell acute lymphoblastic leukemia xenografts HPB-ALL

  • Dosages

    150 mg/kg

  • Administration

    p.o. twice daily

References

  • https://pubmed.ncbi.nlm.nih.gov/20530712/
  • https://pubmed.ncbi.nlm.nih.gov/22806875/
  • https://pubmed.ncbi.nlm.nih.gov/10801983/

Customer Product Validation

The expression of p-EGFR and NICD was assessed by western blot after treatment for 3 d.

Data from [ , , Cell Prolif, 2018, 51(3):e12424 ]

(F) Tumour tissues were further analysed to detect the efficacy of the inhibitors by IHC staining using p-EGFR and Notch1. (4Fa) Notch1 staining was mostly confined on the cell membrane. (4Fc) After Erlotinib treatment, the Notch1 protein was cleaved and translocated to the nucleus. (4Fb) p-EGFR was overexpressed in the control group. (4Fd) Erlotinib treatment inhibited p-EGFR expression. (4Fe-f) PF-03084014 treatment reduced both Notch1 and p-EGFR expression. (4Fg-h) Synthetic therapy reduced the activation of Notch1 and EGFR pathway. Scale bar = 50 μm. *P < .05, **P < .01, ***P < .001

Data from [ , , Cell Prolif, 2017, doi: 10.1111/cpr.12424 ]

Sellecks Nirogacestat (PF-03084014) Has Been Cited by 21 Publications

Calorie restriction activates a gastric Notch-FOXO1 pathway to expand ghrelin cells [ J Cell Biol, 2024, 223(10)e202305093] PubMed: 38958606
Activation of the γ-secretase/NICD-PXR/Notch pathway induces Taxol resistance in triple-negative breast cancer [ Biochem Pharmacol, 2024, 230(Pt 2):116577] PubMed: 39427919
Multiomic analyses reveal new targets of polycomb repressor complex 2 in Schwann lineage cells and malignant peripheral nerve sheath tumors [ Neurooncol Adv, 2024, 6(1):vdae188] PubMed: 39620202
Inhibition of Notch Signaling Enhances Antitumor Activity of Histone Deacetylase Inhibitor LAQ824 [ Int J Mol Sci, 2023, 24(17)13660] PubMed: 37686467
Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia [ Cancers (Basel), 2023, 15(6)1883] PubMed: 36980769
In vitro effects of gamma-secretase inhibition in HPV-positive and HPV-negative head and neck squamous cell carcinoma [ Invest New Drugs, 2023, 41(2):193-201] PubMed: 36809443
Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals [ J Clin Invest, 2022, 132(24)e162720] PubMed: 36282594
DT7 peptide-modified lecithin nanoparticles co-loaded with γ-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemia and reduce gastrointestinal toxicity [ Cancer Lett, 2022, 533:215608] PubMed: 35240234
BRAFV600E;K601Q metastatic melanoma patient-derived organoids and docking analysis to predict the response to targeted therapy [ Pharmacol Res, 2022, 182:106323] PubMed: 35752358
Chemical induction of gut β-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes [ Mol Metab, 2022, 66:101624] PubMed: 36341906

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SHIPPING AND STORAGE
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