生物活性

RO4929097 is a inhibitor of the γ secretase (GS) complex which is composed of four proteins: presenilin, nicastrin, APH-1 and PEN-2. RO4929097 causes a decreased amount of Aβ peptides to be secreted into the culture medium in HEK293 cells (EC50= 14 nM). It is a strong inhibitor of Notch processing (EC50= 5 nM) in the Notch cell-based reporter assay.

The IC50 of ant-proliferation in NEM78 glioma cells is 2.4μM.[2]

体外研究

体内研究

30% PEG400+0.5% Tween80+5% Propylene glycol

激酶实验

In Vitro γ-Secretase Assay[1][3]

The cell-free assay forγ-secretase was done as described. ADNA fragment encoding amino acids

596–695 of the 695-aa isoform of APP (APP695) and the Flag sequence (DYKDDDDK) at the C terminus was 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 γ-secretase cleavage site). This DNA fragment was inserted into the prokaryotic expression vector pET2–21b. The recombinant protein, C100Flag, was overproduced in Escherichia coli and purified by Mono-Q column chromatography. C100Flag (1.7mM) was incubated with cell membranes (0.5mg/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 (50mM Pipes, pH 7.0/5 Mm MgCl2/5mM CaCl2/150mM KCl) at 37°C. The reactions were stopped by adding RIPA (150mM NaCly1.0% NP-40y0.5% sodium deoxycholatey0.1% SDS/50mM TriszHCl, pH 8.0) and boiling for 5 min. The samples were centrifuged and the supernatant solutions were assayed for the Aβpeptides by ECL. The Aβ40- and Aβ42-related products fromγ-secretase-mediated processing of C100Flag possess a Met at the N terminus and are thus defined as M-Ab40 and M-Ab42, respectively. Likewise, supernatant solution (0.125mg/ml) from CHAPSO-extracted HeLa cell membranes (solubilizedγ-secretase) was incubated with C100Flag (1.7mM) in buffer B containing 0.25% CHAPSO and subsequently assayed for M-Ab40 and M-Ab42 by using ECL.

细胞实验

Cell culture[4]

B-RAF mutant cutaneous melanoma cell lines; SK-Mel 32 and SK-Mel 267 (both wild type for PTEN), SK-Mel 39 (PTEN mutant) and SK-Mel 133 (PTEN null) were maintained in RPMI with 50 U/ml each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum, and incubated at 37˚C in 5% CO2. MDAMB 468 vector and MDAMB 468 PTEN-inducible cells were a maintained in Dulbecco’s Modified Eagle’s (DME) media with 50 U/ml each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum, and incubated at 37˚C in 5% CO2.

Clonogenic cell proliferation assay

Cells were plated, in triplicate, onto 100-mm dishes at 1,000 per dish and were treated with the indicated concentrations of RO4929097 for 24 h. Cells were then cultured in drug-free medium for 10 to 14 days. The resulting colonies were scored after staining with 0.01% crystal violet.

Colorimetric cell proliferation assay

Briefly, 2,000 cells were plated in 100μl volume per well of a 96-well plate, and treatments were carried out 24 h after plating. After an incubation period of six days with RO4929097 (5μM) and temozolomide (300μM), alone or in combination, the media were replaced with MEM without phenol red with 10% serum and 10% Cell Counting Kit-8 (CCK-8) solution and cells were further incubated at 37˚C for 1 to 4 h. In this assay, the amount of formazan dye generated by the activity of dehydrogenases in the cells is quantified and is directly proportional to the number of living cells. The optical density at 450 nm to determine the cell viability was measured using Spectra Max 340 PC.

动物实验

Reagents[2]

RO4929097 was dissolved in a vehicle containing 1% hydroxypropyl cellulose, 0.2% Tween 80 in purified water for in-vivo studies.

Evaluation of RO4929097 activity in vivo

Antitumor activity of RO4929097 alone or in combination was evaluated against advanced-stage tumors in Swiss athymic mice 6–8 weeks of age. Animals bearing subcutaneous IGREP37, IGREP83 ependymoma, and IGRG121 glioma tumors of 100–300mm3 were randomized to treatment groups on day0 (=day of treatment start) which was on 67 days, 38 or67 days, and 13 days, respectively, after xenograft transplantation. Treatment groups received RO4929097 at 10 or30 mg/kg/day in 0.2ml/oral gavage 4 days/week for 3 or4 weeks, local irradiation 1 Gy daily for 5 days using an X-ray tube MXR 225–22 or radiotherapy and RO4929097 in equivalent doses and schedules. Rapamycin was administered intravenously at5mg/kg for 4 consecutive days and 2 weeks. Control animals were treated with drug vehicle. Clinical status and mortality were monitored daily. Tumor volumes were calculated according to the following equation: V (mm3) = width2(mm2) ×length (mm)/2. The study ended after 150 days or when tumor volumes reached 1500–2000mm3. Statistical difference between the treatment and the control groups was estimated by the nonparametric Kruskal–Wallis test using GraphPad Prism software (version 3.0).

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Luistro L, He W, Smith M, et al. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties. Cancer Res. 2009;69(19):7672-7680.
[2] Dantas-Barbosa C, Bergthold G, Daudigeos-Dubus E, et al. Inhibition of the NOTCH pathway using gamma-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors. Anticancer Drugs. 2015;26(3):272-283.
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体内溶解度
约16.5 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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