E 64d is a cell-permeable, irreversible, broad-spectrum cysteine protease inhibitor with platelet aggregation inhibitory activity[1]. E 64d is a non-selective covalent tissue protease inhibitor containing an epoxide ring as a warhead that can be nucleophilically attacked by Cys-SH and form a covalent bond at the catalytic site[2]. E 64d inhibits the accumulation of protease-resistant prion protein (PrP-res) in ScNB cells with an IC50 of 0.5±0.11μM[3].
In vitro, pretreatment of SH-SY5Y cells with E 64d (3µM) for 30min improved the amyloid beta-induced decrease in sAPPα secretion and reversed the ceramide-induced downregulation of protein kinase C[4]. Treatment of HT22 cells with E 64d (25μM) for 24h alleviated glutamate-induced apoptosis, reversed the decrease in mitochondrial membrane potential, and reduced oxidative stress[5].
In vivo, E 64d (9mg/kg/day) was intraperitoneally injected into arthritic mice for 10 days, which significantly reduced the severity of clinical arthritis and histopathology, and decreased the mRNA levels of cytokines IL-6 and IL-1β[6]. E 64d (1mg/kg) was intravenously injected into rats with spinal cord injury (SCI) for 7 days, which inhibited the inflammatory response caused by SCI, reduced the expression of calretinin 1 in spinal cord tissue, reduced neuronal apoptosis, and inhibited the activity of cyclooxygenase 2[7].
References:
[1] Chen Y, Li Q, Li Q, et al. p62/SQSTM1, a central but unexploited target: advances in its physiological/pathogenic functions and small molecular modulators[J]. Journal of medicinal chemistry, 2020, 63(18): 10135-10157.
[2] Cannalire R, Stefanelli I, Cerchia C, et al. SARS-CoV-2 entry inhibitors: small molecules and peptides targeting virus or host cells[J]. International Journal of Molecular Sciences, 2020, 21(16): 5707.
[3] Doh-Ura K, Iwaki T, Caughey B. Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation[J]. Journal of virology, 2000, 74(10): 4894-4897.
[4] Tanabe F, Nakajima T, Ito M. The thiol proteinase inhibitor E-64-d ameliorates amyloid-β-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells[J]. Biochemical and Biophysical Research Communications, 2013, 441(1): 256-261.
[5] Xie R J, Li T X, Qiao X Y, et al. The Protective Role of E‐64d in Hippocampal Excitotoxic Neuronal Injury Induced by Glutamate in HT22 Hippocampal Neuronal Cells[J]. Neural Plasticity, 2021, 2021(1): 7174287.
[6] Yoshifuji H, Umehara H, Maruyama H, et al. Amelioration of experimental arthritis by a calpain-inhibitory compound: regulation of cytokine production by E-64-d in vivo and in vitro[J]. International immunology, 2005, 17(10): 1327-1336.
[7] Zhang Z, Huang Z, Dai H, et al. Therapeutic Efficacy of E‐64‐d, a Selective Calpain Inhibitor, in Experimental Acute Spinal Cord Injury[J]. BioMed Research International, 2015, 2015(1): 134242.
E 64d是一种可渗透细胞的不可逆的广谱半胱氨酸蛋白酶抑制剂，具有抑制血小板聚集的活性[1]。E 64d是一种非选择性共价组织蛋白酶抑制剂，含有环氧化物环作为弹头，可以受到Cys-SH的亲核攻击并在催化位点形成共价键[2]。E 64d抑制蛋白酶抗性朊病毒蛋白（PrP-res）在ScNB细胞中的积累，IC50为0.5±0.11μM[3]。
在体外，E 64d（3µM）预处理SH-SY5Y细胞30min，改善了淀粉样蛋白β诱导的sAPPα分泌减少，逆转了神经酰胺诱导的蛋白激酶C下调[4]。E 64d（25μM）处理HT22细胞24h，减轻了谷氨酸诱导的细胞凋亡，逆转了线粒体膜电位的降低，减轻了氧化应激[5]。
在体内，E 64d（9mg/kg/day）通过腹腔注射治疗关节炎小鼠10天，显著减轻了临床关节炎和组织病理学严重程度，降低了细胞因子的IL-6和IL-1β的mRNA水平[6]。E 64d（1mg/kg）通过静脉注射治疗脊髓损伤（SCI）模型大鼠7天，抑制了SCI引起的炎症反应，降低了脊髓组织中钙蛋白1的表达，减少了神经元凋亡，抑制了环氧合酶2的活性[7]。