BI-D1870 is a highly specific, blood-brain-permeable, and ATP-competitive inhibitor of the N-terminal AGC kinase domain of RSK, with IC50 values ​​of 31 nM, 24 nM, 18 nM, and 15 nM for RSK1, RSK2, RSK3, and RSK4.

RSK3:18 nM (cell free), RSK2:24 nM (cell free), RSK1:31 nM (cell free), RSK4:15 nM (cell free)

方法：将HEK-293细胞与 BI-D1870（10 μM，15分钟，总共4小时） 孵育为了检验BI-D1870是否能抑制细胞中的RSK活性，研究BI-D1870对HEK-293细胞中RSK催化其已知底物磷酸化的影响，使用PMA作为激动剂激活ERK1/ERK2和RSK亚型。
结果：用 BI-D1870 孵育细胞可极大地抑制 PMA 诱导的 GSK3α 和 GSK3β 磷酸化 ，相比之下，BI-D1870在任何时间点对PMA诱导的ERK1/ERK2激活（由Raf和MKK1催化）或CREB在Ser位点的磷酸化几乎没有影响。[1]
方法：BI-D1870（1,2,3,4,5.28或者48小时） 处理口腔癌细胞系 (SCC2095、SCC4、SCC9、Ca922 和 HSC-3) 和NHOK细胞，MTT检测这些细胞的生长情况。
结果： BI-D1870 对 OSCC 细胞表现出剂量反应性抗增殖作用。[3]
方法：用 BI-D1870 （0.5,2μM）处理转染免疫荧光 GFP-LC3 的细胞 48 小时，并在共聚焦显微镜下观察；使用 DAPI 染色定位细胞核，用 BI-D1870 处理 48 小时的观察细胞中 LC3B 的蛋白质印迹。
结果：BI-D1870 诱导自噬，LC3B-II 的蛋白质印迹表明这种诱导具有剂量依赖性。[3]

方法：在用 MOG 肽免疫小鼠两天后，BI-D1870（0.5 mg/kg) 腹腔注射到小鼠体内，每隔一天重复注射一次，持续 11 天。
结果：小鼠表现出延迟神经缺陷；BI-D1870 治疗对体重减轻有中等程度的保护作用。[4]

Purified His6–RSK1, His6–RSK2 or GST–RSK21–389:S381E (1–2 units/ml) were 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 were terminated and analyzed as described previously. The amount of enzyme that catalyzed the phosphorylation of 1 nmol of substrate peptide in 1 min was termed one unit. In order to assay RSK and MSK1 in HEK-293 or Rat-2 cell lysates, these kinases were immunoprecipitated from the cell lysates (0.1 mg of lysate protein for RSK and 0.3 mg for MSK1) and assayed as described previously, except that for RSK assays the immunoprecipitates were 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].

The rat embryo fibroblast cell line, Rat-2 cells were cultured on 10 cm-diameter dishes in Dulbecco's Modified Eagle's medium supplemented with 10% (v/v) FBS. HEK-293 cells were cultured on 10 cm-diameter dishes in Dulbecco's Modified Eagle's medium supplemented with 10% FBS and 1×antimycotic/antibiotic solution. Prior to stimulation, cells were cultured in the absence of serum for 16 h. Inhibitors were dissolved in DMSO at a 1000-fold higher concentration than they were used at. These inhibitors, or the equivalent volume of DMSO as a control, were added to the tissue culture medium 30 min prior to stimulation unless indicated otherwise. The final concentration of DMSO in the culture medium was 0.1% and had no effect on agonist-induced activation or phosphorylation of any of the substrates examined. The cells were stimulated with the indicated agonists and lysed in 1 ml of ice-cold Lysis Buffer and centrifuged at 16000 g at 4 °C for 5 min. The supernatants were frozen in liquid nitrogen and stored at ?80 °C until use. Protein concentrations were determined using the Bradford method with BSA as the standard [1].

Myelin oligodendrocyte glycoprotein (MOG) peptide 35–55. (MEVGWYRSPFSRVVHLYRNGK) (BEX) was used to induce EAE in C57/BL6J mice. Mice were injecteds.c. with 200 g of MOG peptide in100 L of PBS emulsified in 100 L complete Freund's adjuvant (CFA) that was further supplemented with five mg mL?1 Mycobacterium tuberculosis (H37Ra). In addition, 500 ng pertussis toxin was injected i.p. on days zero and two. The RSK inhibitor (BI-D1870; 0.5 mg kg?1) was injected i.p. into mice two days after immunization with MOG peptide, and injection was repeated every other day for 11 days. Mice that received only dimethyl sulfoxide (DMSO) solution were used as controls. Paralysis was evaluated according to the following scale: zero, no disease; one, tail limpness; two, hind limb weakness; three, hind limb paralysis; four, forelimb weakness; five, quadriplegia; six, death. For histological analysis, CNS samples were fixed with 4% paraformaldehyde and sliced at 4 m, and then hematoxylin & eosin (H & E) staining was performed [4].