生物活性

FH535 is a sulfonamide-based and cell-permeable compound that exhibits a dual pathway inhibition against Wnt/β-catenin and PPAR. FH535 and its structurally similar analog, PPAR inhibitor GW9662, both exhibit inhibition of coactivator/PPAR binding, though only FH535 blocks β-catenin/PPAR interaction. Further, the antagonistic activity of FH535 does not involve the covalent modification of non-covalent interaction with the PPR ligand-binding site cysteine residue, which has been experimentally shown to be crucial for GW9662 action. β-Catenin/Tcf Inhibitor, FH535 is an inhibitor of GRIP1, PPAR-β, and PPAR-γ.

Cell viability of FH535

体外研究

体内研究

激酶实验

High-throughputLibrary Screen[1]

Three copies of the optimized or mutatedTcf-binding element from TOPFLASH or FOPFLASH driving a secreted alkalinephosphatase reporter gene were cloned into pCEP4 plasmid, replacing thecytomegalovirus promoter. The plasmids were transfected into HepG2 cells, andhygromycin-resistant clones were pooled. Library screening was done at 20μmol/Lconcentration in HepG2 serum-free media. Hits were tested in the HCT116 cellline for inhibition of TOPFLASH luciferase activity but not for inhibition of areporter activity controlled from h-actin promoter.

Transfectionand Reporter Gene Assays

TransIT-LT1 transfection reagent was used accordingto the manufacturer’s instructions. After transfection of reporter constructs(18–24 h), cells were trypsinized and replated in drug-containing assay platesfor another 24 h. Alkaline phosphatase and luciferase activities were measuredusing CSPD Emerald-II or steadylite-HTS, respectively. When an internal controlfor transfection efficiency was required, the pCMV/β-galactosidasevector was cotransfected with the luciferase reporter constructs andβ-galactosidaseactivity was assayed with Galacto- Light Plus. All assays were done intriplicate using a 96-well plate format and a TopCount-NXT luminescence counter.

细胞实验

Cellculture[3]

Acute promyelocytic leukemia (APL) cellline (HL60), chronic myeloid leukemia (CML) in blast crisis cell line (K562),acute monocytic leukemia cell line (THP1), and immortalized T cell line(Jurkat, clone E6-1) were procured. Histopaque-1077 density gradientcentrifugation was performed on total blood to collect peripheral bloodmononuclear cells (PBMC). All the cell lines and PBMC were cultured in completemedium, containing Iscove’s modified Dulbecco’s medium (IMDM), 1× penicillin/ streptomycin,and 10 % fetal bovine serum (FBS) (HL60 was cultured in complete medium containing20 % FBS), at 37 °C under 5 % CO2. Medium was changed every 3–4days. All cell lines were used at the density of 105cells/mlthroughout all experiments. PBMCs were used at the density of 4 × 105cells/ml.

Small‑molecule inhibitors

Wnt inhibitors IWP2, XAV939, FH535 andBCR-ABL tyrosine kinase inhibitor imatinib (IM) were reconstituted in DMSO to10 mM stock solutions.

Cellproliferation assays

HL60, K562, THP1, and Jurkat were culturedin 96-well plates at 2 × 104cells/well in the presence of 1μM of XAV939,IWP2, FH535 and IM when specified. Viable cell count was performed using trypanblue staining after 24 h of treatment. For 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazoliumbromide (MTT) assay, 5 × 104cells/well were seeded in 24-wellplates containing above-mentioned cell growth medium. MTT was added to theculture 3 h before the end of the treatment to a final concentration of 0.5mg/ml. Cells from each well were transferred to 1.5ml microcentrifuge tube andcentrifuged. Supernatant was discarded and pellet containing formazan crystalwas dissolved in DMSO. One hundred microliter of DMSO was transferred into96-well plate in triplicate. The absorbance was measured at 562 nm using amicroplate reader.

动物实验

Nude mouse tumor xenograft model and treatment[4]

Four-week-old female BALB/c athymic nude micewere purchased. PANC-1 cells stably expressing firefly luciferase were injectedinto the left flanks of the mice in a total volume of 100μL (0.5 × 107cells),and the mice were randomly assigned to a dimethyl sulfoxide (DMSO)[intraperitoneally injected with 100μL DMSO/DMEM (1:1)] or FH535 group[intraperitoneally injected with 25 mg/kg FH535 dissolved in 100μL DMSO/DMEM(1:1)]. Treatment was conducted every 2 days for 20 days; tumor volume wasmeasured with a caliper using the formula: volume = length × width2/2.At the end of the experiment, the mice were anaesthetized and given D-luciferinin PBS. Twenty minutes after the injection, bioluminescence was imaged with acharge-coupled device camera. Then, the tumor tissue was stripped andformalin-fixed, paraffin-embedded, cut into4-μm sections, andimmunohistochemically stained.

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] Handeli S, Simon JA. A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities. Mol Cancer Ther. 2008;7(3):521-529.
[2] Gedaly R, Galuppo R, Daily MF, et al. Targeting the Wnt/beta-catenin signaling pathway in liver cancer stem cells and hepatocellular carcinoma cell lines with FH535. PLoS One. 2014;9(6):e99272.
[more]

体内溶解度

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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