Molecular Weight (MW)

391.42

Formula

C19H23F2N5O2

CAS No.

501437-28-1

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 78 mg/mL (199.3 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

Synonyms

BI D1870; BID1870; BI-D1870

Chemical Name: 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one.

InChi Key: DTEKTGDVSARYDS-UHFFFAOYSA-N

InChi Code: InChI=1S/C19H23F2N5O2/c1-10(2)5-6-26-11(3)18(28)25(4)15-9-22-19(24-17(15)26)23-12-7-13(20)16(27)14(21)8-12/h7-11,27H,5-6H2,1-4H3,(H,22,23,24)

SMILES Code: O=C1N(C)C2=CN=C(NC3=CC(F)=C(O)C(F)=C3)N=C2N(CCC(C)C)C1C

In Vitro

In vitro activity: BI-D1870 inhibits RSK1, RSK2, RSK3 and RSK4 with an IC50 of 10–30 nM, but does not significantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF -induced phosphorylation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB.

Kinase Assay: Purified His6-RSK1, His6-RSK2 or GST-RSK21-389:S381E (1-2 units/mL) are assayed for 10 min at 30°C in a 50 μL assay mixture in Buffer A containing 30 μM substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), 10 mM magnesium acetate and 100 μM of [γ-32P]ATP. Reactions are terminated and analysed. The amount of enzyme that catalysed the phosphorylation of 1 nmol of substrate peptide in 1 min is termed one unit.

Cell Assay: Measurement of cell growth is assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay in six replicates. The cells (5×103/200 μL) are seeded in 96-well, flat-bottom plates for 24 h and then exposed to various concentrations of test agents for the indicated time intervals. After removing the culture medium, 200 μL of the medium containing MTT at a concentration of 0.5 mg/mL is added, and the cells are incubated at 37°C for 2 h. The medium is removed, and the reduced MTT dye in each well is dissolved in 200 μL DMSO. Absorbance is determined with a multimode microplate reader Synergy HT at 570 nm.

In Vivo

BI-D1870 (0.5 mg/kg)-injected experimental autoimmune encephalomyelitis (EAE) mice exhibits a delayed neural deficit without obvious weight loss. Histopathological analyses shows inflammatory cell infiltration and demyelination in the spinal cord in control mice, but not in BI-D1870-treated mice. BI-D1870 protects against the infiltration of TH1 or TH17 cells into the CNS.

Animal model

Mice

Formulation & Dosage

Myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 MEVGWYRSPFSRVVHLYRNGK) (BEX) is used to induce EAE in C57/BL6J mice. Mice are injecteds.c. with 200 g of MOG peptide in 100 μL of PBS emulsified in 100 μL complete Freund’s adjuvant (CFA) that is further supplemented with five mg/mL Mycobacterium tuberculosis. In addition, 500 ng pertussis toxin is injected i.p. on days zero and two. The RSK inhibitor (BI-D1870; 0.5 mg/kg) is injected i.p. into mice two days after immunization with MOG peptide, and injection is repeated every other day for 11 days. Mice that receive only dimethyl sulfoxide (DMSO) solution are used as controls. Paralysis is evaluated according to the following scale: zero, no disease; one, tail limpness; two, hind limb weakness; three, hind limb paralysis; four, fore limb weakness; five, quadriplegia; six, death. For histological analysis, CNS samples are fixed with 4% paraformaldehyde and sliced at 4 μm, and then hematoxylin & eosin (H & E) staining is performed.

References

Biochem J. 2007 Jan 1; 401(Pt 1): 29–38; Immunobiology. 2016 Feb;221(2):188-92.