BI-D1870 is a specific, cell permeable and ATP-competitive inhibitor of RSK1/2/3/4 (p90 ribosomal S6 kinase) with the potential to treat autoimmune encephalomyelitis (EAE). In cell-free assays, it inhibits RSK1/2/3/4 with IC50 values of 31 nM, 24 nM, 18 nM, and 15 nM, respectively. and exhibits ten to one hundred times more selectivity for RSK than for MST2, GSK-3, CK1, MARK3, and Aurora B. Serine/threonine kinases known as RSKs play a role in a number of cellular functions, such as growth, survival, and motility. Additionally, BI-D1870 demonstrated in vitro strong antiproliferative activity.
Physicochemical Properties
| Molecular Formula | C19H23F2N5O2 |
| Molecular Weight | 391.4150 |
| Exact Mass | 391.181 |
| Elemental Analysis | C, 58.30; H, 5.92; F, 9.71; N, 17.89; O, 8.18 |
| CAS # | 501437-28-1 |
| Related CAS # | 501437-28-1 |
| PubChem CID | 25023738 |
| Appearance | Pale orange to brown solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 579.3±60.0 °C at 760 mmHg |
| Flash Point | 304.2±32.9 °C |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C |
| Index of Refraction | 1.585 |
| LogP | 3.53 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 28 |
| Complexity | 542 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | 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] |
| 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) |
| Chemical Name | 2-((3,5-difluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one |
| Synonyms | 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 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
RSK4 (IC50 = 15 nM); RSK3 (IC50 = 18 nM); RSK2 (IC50 = 24 nM); RSK4 (IC50 = 15 nM); RSK1 (IC50 = 31 nM) The target of BI-D1870 is the p90 ribosomal S6 kinase (p90RSK) family, including RSK1, RSK2, and RSK3. The IC50 values are as follows: IC50 for RSK1 is approximately 30 nM, IC50 for RSK2 is approximately 100 nM, and IC50 for RSK3 is approximately 60 nM. BI-D1870 shows very low inhibitory activity against other kinases such as ERK1/2 and Akt (IC50 > 10 μM) [1]/[2] ; The main target of BI-D1870 is still p90RSK, and it exerts its effect by inhibiting the activity of p90RSK in T cells [3] |
| ln Vitro |
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] 1. Enzyme activity inhibition experiment: BI-D1870 specifically inhibits the kinase activity of recombinant p90RSK family members (RSK1, RSK2, RSK3) in vitro, with IC50 values consistent with the target inhibition data. It has no significant inhibitory effect on the activity of other tested kinases (e.g., ERK1/2, Akt), showing good kinase selectivity [1]/[2] 2. HeLa cell experiment: After treating HeLa cells with BI-D1870 at different concentrations (0.1 μM, 1 μM, 10 μM) for 24 hours (with DMSO as solvent control), Western blot analysis showed that the phosphorylation level of S6 (a specific substrate of RSK) was significantly reduced in a concentration-dependent manner. Specifically, the phosphorylation level of S6 (p-S6) was obviously decreased when the concentration of BI-D1870 reached 1 μM, while the total S6 protein level remained unchanged, indicating that BI-D1870 inhibits RSK activity in HeLa cells [1]/[2] 3. COS-7 cell transfection experiment: After transfecting COS-7 cells with RSK1-expressing plasmid (24 hours after transfection), treating the cells with BI-D1870 at concentrations of 0.03 μM, 0.1 μM, 0.3 μM, and 1 μM, Western blot results showed that the phosphorylation level of overexpressed RSK1 (p-RSK1) was inhibited in a concentration-dependent manner, and the IC50 value was consistent with the in vitro enzyme activity assay, confirming that BI-D1870 can specifically inhibit the activity of RSK1 in cells [1]/[2] 4. Human peripheral blood T cell experiment: Purified human peripheral blood T cells were activated and cultured in medium containing IL-2, then treated with BI-D1870 at concentrations of 1 μM, 5 μM, and 10 μM (with 0 μM as control) for 48 hours. ELISA detection showed that the levels of T cell activation-related cytokines (IL-2 and IFN-γ) in the cell culture supernatant were significantly reduced in a concentration-dependent manner; Western blot analysis revealed that the phosphorylation level of RSK (p-RSK) in T cells was obviously inhibited, while the total RSK level was not affected; Flow cytometry detection showed that the expression of T cell activation markers (CD25 and CD69) was down-regulated, indicating that BI-D1870 can inhibit T cell activation and cytokine production by suppressing RSK activity [3] |
| ln 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. 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]. |
| Enzyme Assay |
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. 1. Preparation of materials: Recombinant kinase domains of p90RSK family members (e.g., RSK1 catalytic domain) were prepared first; RSK-specific substrate peptide (final concentration 200 μM), ATP (final concentration 10 μM), and buffer solution (containing Tris-HCl, MgCl2, and DTT) were prepared for the reaction system [1]/[2] 2. Reaction system setup: Different concentration gradients of BI-D1870 (ranging from 0.1 nM to 10 μM) were added to the reaction system, and blank control (without BI-D1870) and positive control (known RSK inhibitor) were set up. Each concentration was repeated three times. The recombinant RSK kinase was then added to initiate the reaction, and the system was incubated at 30°C for 30 minutes [1]/[2] 3. Detection and analysis: After the incubation, the reaction was terminated by adding a termination reagent. The amount of phosphorylated substrate peptide was detected by a radioactivity-based method (using radiolabeled ATP and scintillation counting) to calculate the kinase activity. The IC50 value of BI-D1870 against RSK was calculated based on the change in kinase activity at different drug concentrations [1]/[2] |
| 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. 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. 1. HeLa cell culture and treatment assay: - Cell seeding: HeLa cells were seeded in 6-well culture plates and cultured in DMEM medium containing 10% FBS at 37°C with 5% CO2 until the confluence reached 70%-80% [1]/[2] - Drug treatment: BI-D1870 was diluted to concentrations of 0.1 μM, 1 μM, and 10 μM with culture medium, and the cells were treated with these dilutions; DMSO (the solvent of BI-D1870) was used as a negative control. The treatment duration was 24 hours [1]/[2] - Protein extraction and detection: After treatment, the cells were collected, and total cellular protein was extracted. Western blot was performed using specific primary antibodies against phosphorylated S6 (p-S6) and total S6, followed by incubation with fluorescent secondary antibodies. The band intensity was quantified using an imaging system, and the ratio of p-S6 to total S6 was calculated to evaluate the inhibitory effect of BI-D1870 on RSK activity [1]/[2] 2. COS-7 cell transfection and treatment assay: - Cell transfection: Plasmids encoding RSK1 were transfected into COS-7 cells using a transfection reagent. The cells were cultured for 24 hours after transfection to allow expression of RSK1 [1]/[2] - Drug treatment: BI-D1870 was added to the transfected COS-7 cells at concentrations of 0.03 μM, 0.1 μM, 0.3 μM, and 1 μM, and the cells were cultured for a specific period [1]/[2] - Protein detection: Total protein was extracted from the cells, and Western blot was used to detect the phosphorylation level of RSK1 (p-RSK1) and total RSK1 level. The inhibitory effect of BI-D1870 on the activity of overexpressed RSK1 was analyzed based on the changes in p-RSK1 level [1]/[2] 3. Human peripheral blood T cell assay: - T cell isolation and activation: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors, and purified T cells were obtained by magnetic bead sorting. The T cells were cultured in medium containing IL-2 for activation [3] - Drug treatment: Activated T cells were treated with BI-D1870 at concentrations of 1 μM, 5 μM, and 10 μM (0 μM as control) for 48 hours [3] - Detection: ELISA was used to measure the concentrations of IL-2 and IFN-γ in the cell culture supernatant; Western blot was performed to detect the levels of p-RSK and total RSK in T cells; Flow cytometry was used to analyze the expression of T cell activation markers (CD25 and CD69) [3] |
| Animal Protocol | 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. |
| References |
[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. |
| Additional Infomation |
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] 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] 1. BI-D1870 is a selective p90RSK inhibitor designed based on the ATP-binding site of the RSK kinase domain. It exerts its inhibitory effect by competing with ATP for binding to the ATP-binding site of RSK, thereby blocking the kinase activity of RSK [1]/[2] 2. BI-D1870 is widely used as a tool compound in basic research to explore the role of RSK in cellular processes such as cell proliferation, survival, and differentiation, and to clarify the signal pathways mediated by RSK [1]/[2] 3. In T cells, BI-D1870 inhibits T cell activation and cytokine production by suppressing p90RSK activity, which suggests that BI-D1870 may have potential therapeutic applications in autoimmune diseases or inflammatory diseases. However, it has not yet entered clinical research and is still in the basic research stage [3] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~78 mg/mL (~199.3 mM) Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
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. 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. 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. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5548 mL | 12.7740 mL | 25.5480 mL | |
| 5 mM | 0.5110 mL | 2.5548 mL | 5.1096 mL | |
| 10 mM | 0.2555 mL | 1.2774 mL | 2.5548 mL |