生物活性

TH588 is first-in-class nudix hydrolase family inhibitor that potently and selectively engage and inhibit the MTH1(IC50 = 5 nM) in cells. TH588 has been shown to selectively kill a variety of cancer cell lines and with in vivo activity shown for TH588 in SW480 colorectal and MCF7 breast tumour xenografts. TH588 is highly selective towards MTH1, with no relevant inhibition of other members of the nudix protein family or a panel of 87 enzymes, GPCRs, kinases, ion channels and transporter.

体外研究

体内研究

激酶实验

Enzyme assay for MTH1[1]

An MTH1 enzyme assay was performed withminor modifications. Briefly, test compounds were dissolved in an assay buffer[100mM Tris-acetate, 40mM NaCl, 10mM Mg(OAc)₂, 0.005% Tween-20, and2mM dithiothreitol (DTT), pH 7.5] containing His-tagged human recombinant MTH1protein (2nM). After addition of the mixed reagent from the PPi Light InorganicPyrophosphate Assay kit, either 8-oxo-dGTP (13.2μM) or 2-OH-dATP (8.3μM) wasadded as a substrate to initiate the enzymatic reaction. Relative MTH1 activitywas measured by monitoring the pyrophosphate (PPi) generated through MTH1-catalyzednucleotide triphosphate hydrolysis. The luciferase-mediated luminescence signalwas recorded for 30 min using a luminometer. Then, SigmaPlot was used forkinetic analysis to determine the inhibition patterns of NPD7155 and NPD9948and calculate their Ki values.

In vitro tubulin polymerization assay wasperformed using a tubulin polymerization assay kit according to themanufacturer’s instructions. Briefly, lyophilized porcine tubulin wassolubilized to a final concentration of 2 mg/mL in reaction buffer containing80mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES; pH 6.9), 2mM MgCl₂,0.5mM ethylene glycol tetraacetic acid (EGTA), 1mM guanosine-5’-triphosphate(GTP), 10μM fluorescent reporter, and 20% glycerol, and kept at 4 ^oC.Compounds (100 × DMSO stock solutions) were added to prewarmed half-area 96-wellblack plates. Cold tubulin solution was added to the wells. The plate contentswere mixed by shaking, and fluorescence (Ex 350 nm, Em 435 nm) was read everyminute for 1 h using a fluorescence microplate reader.

细胞实验

Cell culture[2]

The human pancreatic neuroendocrine BON1tumor cell line, the pancreatic islet tumor cell line QGP1, human bronchopulmonaryneuroendocrine NCI-H727 (H727) tumor cells and human midgut carcinoid GOT1cells were prepared. All cell lines used in this study were grown in DMEM/F12(1:1) (BON1, QGP1) or RPMI-1640 (NCI-H727, GOT1), supplemented each with 10%FBS, 100 unit / ml penicillin, 100μg / ml streptomycin and 1μg / mlamphotericin B at 37 ^oC and 5% CO₂. The GOT1 culturemedium was additionally supplemented with 5μg/mL apo-transferrin and 0.135IU/mL insulin.

The MTH1 inhibitor TH588, everolimus and5-fluorouracil (5-FU) were prepared. All three substances were dissolved indimethyl-sulfoxide, at 10mM stock concentration and stored at -20 ^oC.

Cellviability assessment

Cells were seeded into 96-well plates atdensities of 1,500 (BON1), 2,000 (QGP1 and NCIH727) and 30,000 (GOT1) cells perwell and grown for 24 h. Subsequently, cells were treated with differentconcentrations of TH588, either alone or in combination with 10nM everolimus or5μM 5-FU. Metabolic activity was assessed by the ªCell Titer Blue1º cellviability assay after 96 h and 144 h of incubation. Therefore, cells wereincubated for 4 h with Cell Titer Blue1solution and fluorescence was measuredat 560/590 nm using a GLOMAX plate reader.

Cellcycle analysis by flow cytometric analysis (FACS)

Cell cycle phase distribution was analyzedusing propidium iodide staining and flow cytometry. Cells were cultured in6-well plates for 24 h. Subsequently, medium was replaced by fresh medium andcells were incubated with different concentrations of TH588. After 72 h, cells werewashed with PBS and treated with 300μl trypsin for 5 min. at 37^oC.Cells were collected, washed and resuspended in 300μl propidium iodide.

动物实验

Mouse xenograft assay[3]

Cancer cells (3x10⁷ cells/mouse)were inoculated subcutaneously into the left axilla of 5‑week‑old female nude mice (n=30). Mice weremaintained under general animal husbandry conditions, except for those carryingMCF‑7 xenografts, which were administered daily with estradiol benzoate(3μg/mouse) for thecourse of the assay. Following the growth of tumors to a visible size (~2 mm inmean diameter; typically between 10 and 14 days following inoculation), micewere randomly grouped (5 mice/treatment) for daily subcutaneous TH588 (30mg/kg) or vehicle (2% dimethylsulfoxide, 10% ethanol, 10% cremophor and 10%Tween-80 in PBS) treatment. Tumor size was determined using a calliper threetimes/week and mouse body weight was recorded. The treatment period was between2 and 3 weeks depending on the tumor growth. The mice were sacrificed bycervical dislocation.

Invivo toxicity assay

Nude mice (6‑week‑old) with no tumor burden were administered with TH588 or vehicle asdescribed above. Mice were weighed daily and 300μl of blood was collected by retro‑orbital bleeding at the end of the experiment. Toxicity was assessedby determining the number of red and white blood cells, and platelets using anLH 750 Hematology Analyzer. The levels of alanine aminotransferase, aspartateaminotransferase and creatinine in serum were determined using a UniCel DxC 600Synchron Biochemical Analyzer.

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] Kawamura T, Kawatani M, Muroi M, et al. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival. Sci Rep. 2016;6:26521.
[2] Aristizabal Prada ET, Orth M, Nolting S, Spottl G, Maurer J, Auernhammer C. The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells. PLoS One. 2017;12(5):e0178375.
[3] Zhang X, Song W, Zhou Y, et al. Expression and function of MutT homolog 1 in distinct subtypes of breast cancer. Oncol Lett. 2017;13(4):2161-2168.

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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