生物活性

BI-D1870isa potent ATP-competitive pan-RSK inhibitor and induces apoptosis invariouscancer cells as well. BI-D1870 is a cell permeable, ATP-competitive inhibitor of the four isoforms of ribosomal S6 kinase (p90 RSK) (IC50 at 100µM ATP: RSK1 = 31nM, RSK2 = 24nM, RSK3 = 18nM, RSK4 = 15nM). It does not significantly inhibit 10 other AGC kinases, and over 40 other protein kinases at 100X concentrations. BI-D1870 acts on the N-terminal AGC kinase domain and completely prevents PMA-induced TSC2 phosphorylation.

Kinase activities of BI-D1870

体外研究

体内研究

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

激酶实验

Kinase assay for recombinant Rsk2, Mst1, Lok andSlk[2]

Recombinant Rsk2 (12ng/reaction), GST-Mst1(35ng/reaction), GST-Lok (375ng/reaction) and rSlk (200ng/reaction), diluted in50mM Tris–HCl (pH7.5), 0.1mM EGTA, were treated with various amounts of BI-D1870for 10 min at 20 ^oC and then added to a reaction mix containing 50mMTris–HCl pH7.5, 0.1mM EGTA, 100μM γ ³²P-ATP (25μM for Lok and Slk)10mM MgCl₂, 2mM DTT and the substrates; 30μM CROSSTIDE (GRPRTSSFAEG)for Rsk2, 0.33mg/ml myelin basic protein for Mst1, 100μM AXLTIDE(KKSRGDYMTMQIG) for Lok and 1.25mg/ml bovine histone H1 for Slk. The kinasereaction for Lok and Slk was supplemented with 12.5mM sodium glycerophosphateand 0.25mM sodium orthovanadate. The reactions were incubated for 15–20 min at30◦C then terminated and analysed.

细胞实验

Cellculture[3]

SCC4, SCC9, and SCC2095 human oral cancercells were cultured in DMEM/F12 medium. Ca922 and HSC-3 cells were cultured inMEM and DMEM medium, respectively. All culture medium for cancer cells wassupplemented with 10 % fetal bovine serum and penicillin (100U/mL)/streptomycin (100 μg/mL). NHOK were maintained in keratinocyte serum-freemedium. All cell lines were cultured at 37 °C in a humidified incubatorcontaining 5 % CO₂.

MTTassay

Measurement of cell growth was assessedusing the MTT [3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium  bromide] assay in six replicates. The cells (5× 10³/200μL) were seeded in 96-well, flat-bottom plates for 24 h andthen exposed to various concentrations of test agents for the indicated timeintervals. After removing the culture medium, 200μL of the medium containing MTTat a concentration of 0.5 mg/mL was added, and the cells were incubated at 37°C for 2 h. The medium was removed, and the reduced MTT dye in each well wasdissolved in 200μL DMSO. Absorbance was determined with a multimode microplatereader Synergy HT at570 nm.

Apoptosisassay

Cells (2 × 10⁵/2 mL) were platedand treated with the indicated concentration of BI-D1870 or DMSO for 48 h, andthe cells were washed twice with ice-cold phosphatebuffered saline (PBS) andcollected by trypsinization. After 1,200 rpm for 5 min at room temperature, thecells were stained with Annexin V and propidium iodide (PI) (1μg/mL) andanalyzed using BD FACSAria flow cytometer.

动物实验

EAE[4]

Myelin oligodendrocyte glycoprotein (MOG)peptide 35–55(MEVGWYRSPFSRVVHLYRNGK) (BEX) was used to induce EAE inC57/BL6Jmice. Mice were injected s.c. with 200μg of MOG peptide in 100μLofPBS emulsified in 100μL complete Freund’s adjuvant (CFA) thatwas further supplemented withfive mg/mL Mycobacterium tuber-culosis. In addition, 500ng pertussistoxin wasinjected i.p. on days zero and two. The RSK inhibitor (BI-D1870; 0.5 mg/kg) wasinjected i.p. into mice two days after immunization with MOG pep-tide, andinjection was repeated every other day for 11 days. Mice that received onlydimethyl sulfoxide (DMSO) solution were used as controls. Paralysis wasevaluated according to the following scale: zero, no disease; one, taillimpness; two, hind limb weakness; three, hind limb paralysis; four, fore limbweakness; five, quadriplegia; six, death. For histological analysis, CNSsamples were fixed with4% paraformaldehyde and sliced at 4μm,and then hematoxylin & eosin (H & E) staining was performed.

ELISA

Supernatants were collected after theindicated periods of cell culture and were analyzed for IL-17A and IFN-γ with an ELISA kit according to the manufacturer’s instructions.

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] Sapkota GP, Cummings L, Newell FS, et al. BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo. Biochem J. 2007;401(1):29-38.
[2] Edgar Alexander J, Trost M, Watts C, Zaru R. A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870. Bioscience Reports. 2014;34(1):29-41.
[more]

体内溶解度
29 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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