Bisindolylmaleimide X (hydrochloride)

Bisindolylmaleimide X (hydrochloride) is a novel, cell-permeable protein kinase C (PKC) inhibitor. Bisindolylmaleimide X also inhibits cyclin-dependent kinase 2 (CDK2) activity^[1-2]. Bisindolylmaleimide X regulates cell signaling by reversibly and competitively inhibiting PKC activity with ATP, while simultaneously affecting cell cycle progression by antagonizing CDK2. Bisindolylmaleimide X can be used in research on immune regulation and inflammation-related diseases^[3-4].

In vitro, Jurkat E6 cells were pretreated with Bisindolylmaleimide X (1 or 10μM) for 3-5 minutes, followed by stimulation with anti-CD3 antibody (1μg/ml). Bisindolylmaleimide X failed to inhibit the rise in intracellular calcium ion concentration ([Ca²⁺]i), phospholipase C activity, or divalent cation influx induced by T-cell receptor (TCR) stimulation. In the presence of extracellular calcium ions, Bisindolylmaleimide X attenuated the anti-CD3 antibody-induced rise in [Ca²⁺]i and enhanced capacitative calcium ion entry after depletion of intracellular calcium stores^[5]. Human laryngeal squamous cell carcinoma Hep2 cells were pretreated with Bisindolylmaleimide X (1μM) for 30 minutes, followed by treatment with ascorbic acid (2mM). Bisindolylmaleimide X significantly inhibited ascorbic acid-induced PKC activation, reduced the subsequent increase in cytosolic calcium ion levels, and thereby inhibited ascorbic acid-induced necrosis in Hep2 cells^[6].

In vivo, C57BL/6 male mice were pretreated with an intraperitoneal injection of Bisindolylmaleimide X (10mg/kg) 1 hour before injection of acetaminophen (APAP; 500mg/kg). Bisindolylmaleimide X significantly reduced the APAP-induced elevation in serum ALT (alanine aminotransferase) levels and decreased the area of centrilobular necrosis in the liver. Pretreatment with Bisindolylmaleimide X (10mg/kg) did not affect the APAP-induced depletion of glutathione (GSH) in the liver and mitochondria. Bisindolylmaleimide X also enhanced the phosphorylation of JNK and its translocation to mitochondria^[7]. Experimental autoimmune encephalomyelitis (EAE) model mice received Bisindolylmaleimide X (18.75μg/kg) via tail vein injection on day 3 and day 8 post-immunization. Bisindolylmaleimide X significantly alleviated the clinical score of experimental autoimmune encephalomyelitis^[8].

References:
[1] Song W, Yuan Y, Tan X, et al. Icariside II induces rapid phosphorylation of endothelial nitric oxide synthase via multiple signaling pathways. PeerJ. 2022 Oct 25;10:e14192.
[2] Brehmer D, Godl K, Zech B, et al. Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. Mol Cell Proteomics. 2004 May;3(5):490-500.
[3] Conroy LA, Merritt JE, Giafi CF, et al. Definition of early Ca2+ signals induced by anti-CD3 antibody in a human Jurkat T-cell line using a selective protein kinase C inhibitor, Ro 31-8425. Biochem Soc Trans. 1993 Nov;21(4):384S.
[4] Muid RE, Dale MM, Davis PD, et al. A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli. FEBS Lett. 1991 Nov 18;293(1-2):169-72.
[5] Baek MW, Cho HS, Kim SH, et al. Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling. J Cell Physiol. 2017 Feb;232(2):417-425.
[6] Levy O, Rothhammer V, Mascanfroni I, et al. A cell-based drug delivery platform for treating central nervous system inflammation. J Mol Med (Berl). 2021 May;99(5):663-671.
[7] Saberi B, Ybanez MD, Johnson HS, et al. Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways. Hepatology. 2014 Apr;59(4):1543-1554.
[8] Chen MW, Zhu H, Xiong CH, et al. PKC and Ras are Involved in M1 Muscarinic Receptor-Mediated Modulation of AMPA Receptor GluA1 Subunit. Cell Mol Neurobiol. 2020 May;40(4):547-554.

Bisindolylmaleimide X (hydrochloride) 是一种新型的、具有细胞渗透性的蛋白激酶C（PKC）抑制剂。Bisindolylmaleimide X也可抑制细胞周期蛋白依赖性激酶2(CDK2)活性^[1-2]。Bisindolylmaleimide X通过可逆性、ATP竞争性方式抑制PKC活性来调节细胞信号传导，同时通过拮抗CDK2以影响细胞周期进程。Bisindolylmaleimide X可用于免疫调节和炎症相关疾病的研究^[3-4]。

在体外，Bisindolylmaleimide X（1 or 10μM）预处理Jurkat E6细胞3-5分钟，随后用抗CD3抗体（1μg/ml）刺激，Bisindolylmaleimide X未能抑制T细胞受体（TCR）刺激引起的细胞内钙离子浓度（[Ca²⁺]i）升高、磷脂酶C活性或二价阳离子内流。然而，在有胞外钙离子存在时，Bisindolylmaleimide X可减弱抗CD3抗体诱导的[Ca²⁺]i升高，并在耗竭细胞内钙库后，增强电容性钙离子内流^[5]。Bisindolylmaleimide X（1μM）预处理人喉鳞状细胞癌Hep2细胞30分钟，随后用抗坏血酸（2mM）处理，Bisindolylmaleimide X显著抑制了抗坏血酸诱导的PKC激活，并降低了随后胞质钙离子水平的升高，从而抑制了抗坏血酸诱导的Hep2细胞坏死^[6]。

在体内，Bisindolylmaleimide X（10mg/kg）腹腔注射预处理C57BL/6雄性小鼠1小时，随后注射对乙酰氨基酚（APAP；500mg/kg），Bisindolylmaleimide X显著降低了由APAP引起的血清ALT（丙氨酸转氨酶）水平升高，并减少了肝脏中心小叶坏死面积。Bisindolylmaleimide X（10mg/kg）预处理并未影响APAP导致的肝脏和线粒体中谷胱甘肽（GSH）的耗竭。Bisindolylmaleimide X还增强了JNK的磷酸化及其向线粒体的转位^[7]。Bisindolylmaleimide X（18.75μg/kg）经尾静脉注射入实验性自身免疫性脑脊髓炎模型小鼠（EAE），并于免疫后第3天和第8天各给药一次。Bisindolylmaleimide X显著减轻了实验性自身免疫性脑脊髓炎的临床评分^[8]。