E 64d

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.