RSK4 (IC50 = 15 nM); RSK3 (IC50 = 18 nM); RSK2 (IC50 = 24 nM); RSK4 (IC50 = 15 nM); RSK1 (IC50 = 31 nM)

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. In human embryonic kidney 293 cells and Rat-2 cells, BI-D1870 is cell permeant and inhibits RSK-mediated phorbol ester- and EGF-induced phosphorylation of glycogen synthase kinase-3β and LKB1. Six other AGC kinases' agonist-triggered phosphorylation of their substrates is unaffected by BI-D1870. Furthermore, BI-D1870 does not block the phosphorylation of CREB brought on by either phorbol ester or EGF.[1]

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.

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Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by the infiltration of TH1 and TH17 cells into the CNS. Ribosomal S6 kinase 2 (RSK2; RPS6KA3) regulates TH17 differentiation by attenuating RORγt transcriptional activities and IL-17A production. The pan-RSK inhibitor BI-D1870 also inhibits TH17 differentiation, but the effect of BI-D1870 in vivo remains unclear. Here, we generated mice with experimental autoimmune encephalomyelitis (EAE) and treated them with BI-D1870. BI-D1870 administration protected mice from EAE by reducing the infiltration of TH1 and TH17 cells into the CNS and decreasing mRNA levels of Ccr6 in TH17 cells. These results suggest that RSK inhibition is a promising strategy for the treatment of MS[3].

Purified His6–RSK1, His6–RSK2 or GST–RSK2 1–389: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. As previously mentioned, reactions are stopped and examined. One unit of an enzyme is the quantity required to catalyze the phosphorylation of 1 nmol of a substrate peptide in one minute. In order to assay RSK and MSK1 in HEK-293 or Rat-2 cell lysates, these kinases are immunoprecipitated from the cell lysates (0.1 mg of lysate protein for RSK and 0.3 mg for MSK1) and for RSK assays the immunoprecipitates are washed twice with Buffer A containing 1 mM ATP and twice with Buffer A prior to the assay, as a precaution to ensure dissociation of BI-D1870 from the RSK isoforms.

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. In 96-well flat-bottom plates, the cells (5 103/200 L) are seeded for 24 hours before being exposed to varying concentrations of test agents for the durations of time specified. Following the removal of the culture medium, 200 L of medium containing MTT at a concentration of 0.5 mg/mL is added, and the cells are incubated at 37°C for 2 hours. Each well's reduced MTT dye is dissolved in 200 L of DMSO after the medium has been removed. Synergy HT multimode microplate reader at 570 nm is used to measure absorbance.

Myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 MEVGWYRSPFSRVVHLYRNGK) (BEX) is used to induce EAE in C57/BL6J mice. Mice are given s.c. injections of 200 g of MOG peptide emulsified in 100 μL of PBS, 100 l of complete Freund's adjuvant (CFA), and five mg/mL of Mycobacterium tuberculosis. Additionally, on days 0 and 2, 500 ng of pertussis toxin is intravenously injected. Two days after receiving the MOG peptide vaccination, mice are given an intraperitoneal injection of the RSK inhibitor (BI-D1870; 0.5 mg/kg), which is repeated every other day for 11 days. As controls, mice are given only dimethyl sulfoxide (DMSO) solution. On a scale from zero (no disease) to six (death), paralysis is rated as follows: one (tail limpness), two (hind limb weakness), three (hind limb paralysis), four (fore limb weakness), five (quadriplegia), and six (no disease). Hematoxylin and eosin (H & E) staining is carried out on CNS samples after they have been cut at a thickness of 4 m and fixed with 4% paraformaldehyde for histological analysis.

[1]. Biochem J. 2007 Jan 1; 401(Pt 1): 29–38.

[2]. Biochem J . 2007 Jan 1;401(1):29-38.

[3]. Immunobiology . 2016 Feb;221(2):188-92.

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly 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 (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, 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 (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.[1]

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Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly 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 (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, 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 (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.[2]

C19H23F2N5O2

391.4150

391.181

C, 58.30; H, 5.92; F, 9.71; N, 17.89; O, 8.18

501437-28-1

501437-28-1

25023738

Pale orange to brown solid powder

1.3±0.1 g/cm3

579.3±60.0 °C at 760 mmHg

304.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.585

3.53

2

8

5

28

542

0

FC1C(=C(C([H])=C(C=1[H])N([H])C1=NC([H])=C2C(=N1)N(C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C([H])(C([H])([H])[H])C(N2C([H])([H])[H])=O)F)O[H]

DTEKTGDVSARYDS-UHFFFAOYSA-N

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)

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

BI D1870; BID1870; 501437-28-1; 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one; 2-(3,5-difluoro-4-hydroxyanilino)-5,7-dimethyl-8-(3-methylbutyl)-7H-pteridin-6-one; BI-D 1870; 2-[(3,5-DIFLUORO-4-HYDROXYPHENYL)AMINO]-7,8-DIHYDRO-5,7-DIMETHYL-8-(3-METHYLBUTYL)-6(5H)-PTERIDINONE; 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one.; MFCD11223662; BI-D1870

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~78 mg/mL (~199.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)