Background
E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor1, was tested for ability to inhibit calpain activity in intact platelets.
E-64d inhibits severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 surface glycoprotein incorporation into pseudotyped vesicular stomatitis virus (VSV) particles in Vero cells, an effect that is reduced by expression of the serine protease TMPRSS2.
E-64c or E-64d also inhibited (lanes 3-8), demonstrating their effect on calpain. When the platelets were incubated with these inhibitors for I0 min and were then washed to remove extracellular inhibitor before lysis, neither E-64c nor leupeptin inhibited proteolysis, but E-64d did inhibit. E-64d was able to penetrate the platelet and was thus not removed by washing.E-64c failed to inhibit proteolysis in intact platelets, but E-64d, the permeant inhibitor, did? inhibit intracellular proteolysis.E-64c and E-64d were each able to inhibit the protease activity in lysed platelets. This protease activity has been attributed to calpain by its absolute dependence on Ca 2+and by inhibition by known inhibitors of calpain. E-64d is able to enter the? intact platelet: i) after washing to remove extracellular inhibitor, there was? no protease activity detected after platelet lysis, and ii) activation of? platelets preincubated with E-64d, but not E-64c, resulted in inhibition of? proteolysis by calpain activated in intact platelets by A23187 plus calcium.
E-64d是E-64c的膜透性衍生物，是一种巯基蛋白酶抑制剂[1]，经过测试后发现具有抑制血小板内钙蛋白酶活性的能力。
E-64d抑制严重急性呼吸综合征冠状病毒（SARS-CoV）和SARS-CoV-2表面糖蛋白在伪装成的副病毒性口炎症病毒（VSV）颗粒中的融合，在Vero细胞中减少了这种影响，这种影响可以通过表达丝氨酸蛋白酶TMPRSS2减轻。
E-64c或E-64d也能抑制钙蛋白酶的活性（3-8道），显示了它们对钙蛋白酶的影响。当血小板在这些抑制剂中孵育10分钟后，并在溶解之前被洗涤以去除细胞外抑制剂时，E-64c和白蛋白酶不会抑制蛋白质分解，但是E-64d会抑制蛋白质分解。E-64d能够渗透进入血小板，因此不会被洗涤去除。E-64c无法抑制完整的血小板内蛋白质分解，但是渗透性抑制剂E-64d却可以抑制细胞内蛋白质分解。E-64c和E-64d都可以抑制溶解的血小板中的蛋白酶活性。已知该蛋白酶活性完全依赖于Ca2+并且受到钙蛋白酶已知抑制剂的抑制，因此被归因于钙蛋白酶。E-64d能够进入完整的血小板：i）在洗涤以去除细胞外抑制剂后，溶解血小板中未检测到蛋白酶活性；ii）在A23187加钙预孵育E-64d的血小板中激活后，但不是E-64c预孵育的血小板，导致钙蛋白酶在完整的血小板中被抑制。
Reference:
1. M. Tamai, K. Matsumoto, S. Omura, I. Koyama, Y. Ozawa, K. Hanada J. Pharmacobio-Dyn., 9 (1986), pp. 672–677
2. E. B. McGowan, E. Becker, and T. C. Detwiler, INHIBITION OF CALPAIN IN INTACT PLATELETS BY THE THIOL PROTEASE INHIBITOR E-64d. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS , Vol. 158, No. 2, 1989
3. Carmen JC, Sinai AP. The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities. Front Microbiol. 2011 Jan 24;2:1.
Protocol
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Cell experiment: [1]
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Cell lines
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Platelets
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Preparation method
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The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months.
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Reaction Conditions
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50 μg/ml, 10 min
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Applications
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Platelets were activated with A23187 plus calcium, a condition known to lead to calpain-catalyzed proteolysis of ABP and talin. In the absence of inhibitor, A23187 led to complete degradation of ABP and talin. E 64d, the permeant inhibitor, did inhibit intracellular proteolysis. Some inhibition was observed at the lowest concentration tested, 20 μg/ml, and essentially complete inhibition was obtained with 50 μg/ml.
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Animal experiment: [2]
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Animal models
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Male Sprague–Dawley rats
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Dosage form
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Intraperitoneal injection, 4 μg
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Applications
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After E-64d treatment, mice were treated with penicillin to induce recurrent seizures. The result showed that E-64d remarkably reduced the aberrant mossy ?ber sprouting in the supragranular region of dentate gyrus and CA3 sub?eld of hippocampus. In rats without E-64d treatment, there was prominent aggregation of mossy ?ber terminals in the dentate gyrus and in the stratum pyramidale of CA3 sub?eld. In rats treated with E-64d, the aggregation of mossy ?ber terminals was remarkably decreased in both the supragranular region of dentate gyrus and CA3 sub?eld.
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Other notes
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Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.
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参考文献:
[1] McGowan E B, Becker E, Detwiler T C. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochemical and biophysical research communications, 1989, 158(2): 432-435.
[2] Ni H, Ren S, Zhang L, et al. Expression profiles of hippocampal regenerative sprouting-related genes and their regulation by E-64d in a developmental rat model of penicillin-induced recurrent epilepticus. Toxicology letters, 2013, 217(2): 162-169.
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