Nirogacestat (PF 03084014) is a specific γ-secretase inhibitor (IC50: 6.2 nM, in a cell-free assay).

γ-secretase (cell-free assay):6.2 nM

Nirogacestat抑制携带 Notch1 异二聚体化和 PEST 结构域突变的 HPB-ALL 细胞中 Notch 受体的裂解（IC50：13.3 nM）。同样，Nirogacestat分别降调 HPB-ALL 细胞内 Notch 目标基因 Hes-1（IC50<1 nM）和 cMyc 表达（IC50：10 nM）。通过诱导细胞周期停滞和凋亡，Nirogacestat抑制了一部分人类 T-ALL 细胞株（包括 HPB-ALL，DND-41，TALL-1 和 Sup-T1）的细胞生长（IC50s：30-100 nM）。此外，Nirogacestat减少了 HUVECs 的增殖（IC50：0.5 μM）并且减少了管腔形成（IC50：50 nM）。尽管 PF-03084014（1 μM）对 MX1 细胞无抗增殖作用，但它能够抑制迁移率达95%。

Nirogacestat(200 mg/kg, p.o.) 在HPB-ALL异种移植瘤中导致NICD抑制达到最大约80%。在150 mg/kg剂量下，Nirogacestat显示出强大的抗肿瘤活性，最大肿瘤生长抑制率达92%，伴随着NICD/Notch1、肿瘤有丝分裂指数（Ki67）以及凋亡（激活的caspase-3）染色显著减少。Nirogacestat(120 mg/kg) 在携带HCC1599肿瘤的乳腺癌鼠模型中诱导凋亡、抗增殖，减少肿瘤细胞自我更新能力、损害肿瘤血管并降低转移活性。在各种乳腺异种移植模型中，Nirogacestat显示出显著的抗肿瘤活性（TGI>50%）。

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 theβ-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)dim-ethylammonio]-1-propanesulfonate), or Triton X-100 (0, 0.125, 0.25, 0.5, or 1%) in buffer B (50 mM Pipes, pH 7.0y 5 mM 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 deoxycholate 0.1% SDS/50 mM Tris HCl, pH 8.0) and boiling for 5 min. The samples ae 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.

Cell lines: Human T-ALL cell lines HPB-ALL. Concentrations: ~1 μM. 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 PF-03084014 are done in DMSO,appropriate controls or designated concentrations of PF-03084014 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 Models: Human T-cell acute lymphoblastic leukemia xenografts HPB-ALL. Formulation: 0.5% methylcellulose. Dosages: 150 mg/kg,b.i.d. Administration: p.o.