Chymostatin

Chymostatin is a potent and selective inhibitor of the proteasome chymotrypsin-like activity, with Ki values of 9.36nM for chymotrypsin and 13.1nM for cathepsin D^[1]. Chymostatin also exhibits some inhibitory activity against trypsin and elastase^[2]. Chymostatin can easily cross cell membranes and is an important tool compound for studying the ubiquitin-proteasome pathway and protein degradation in various cellular processes^[3]. Additionally, Chymostatin has potential therapeutic applications in neurodegenerative diseases and cancer ^[4]

In vitro, pretreatment of HK2 cells with Chymostatin (50μM) for 1 hour, followed by treatment with palmitic acid (0.6mM) for 24 hours, significantly inhibited the protein expression of endoplasmic reticulum (ER) stress markers and reduced the levels of pro-apoptotic factors, thereby significantly alleviating cell apoptosis. Moreover, co-pretreatment of HK2 cells with Chymostatin (50μM) and Aliskiren (10nM) for 1 hour, followed by treatment with cholesterol (10μg/ml) for 6 days, also significantly inhibited the protein expression of ER stress markers and decreased the levels of apoptosis-related proteins, protecting cells from cholesterol-induced damage^[5]. Pretreatment of a co-culture system of HEK-293T cells and RBL-2H3 cells with Chymostatin (100μM) for 1 hour, followed by infection with SARS-CoV-2 pseudovirions, resulted in a significant inhibition of viral entry into cells by 52.1% compared to untreated cells^[6].

In vivo, Chymostatin (2mg/kg) was administered intraperitoneally 30 minutes after paraquat-induced acute lung injury in mice. Chymostatin significantly alleviated the pathological changes in lung tissues, decreased the lung wet-to-dry ratio, tissue cathepsin G activity, and serum concentrations of TNF-α, IL-1β, IL-6, and HMGB1, while increasing the serum concentration of endothelial cell-specific molecule-1 (endocan). Additionally, Chymostatin up-regulated endocan expression and down-regulated nuclear NF-κBp65 expression in the lung, thereby inhibiting the inflammatory response^[7]. Chymostatin (2mg/kg/h) was administered via intravenous infusion to rats with hypertension induced by unilateral nephrectomy and high-salt diet (UNX HS) and the Goldblatt renal artery stenosis (2K1C) model. Chymostatin significantly reduced plasma and tissue levels of angiotensin II (Ang II). In Goldblatt hypertensive rats, Chymostatin increased renal and hindlimb vascular resistance. In UNX HS hypertensive rats, Chymostatin blocked the solvent-induced decrease in outer medullary blood flow^[8].

References:
[1] Umezawa, H., Aoyagi, T., Morishima, H., et al. Chymostatin, a new chymotrypsin inhibitor produced by actinomycetes. J Antibiot (Tokyo). 1970 Aug;23(8):425-7.
[2] Akahoshi, F., Ashimori, A., Sakashita, H., et al. Synthesis, structure-activity relationships, and pharmacokinetic profiles of nonpeptidic difluoromethylene ketones as novel inhibitors of human chymase. J Med Chem. 2001 Apr 12;44(8):1297-304.
[3] Sacchetta P, Battista P, Santarone S, et al. Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family. Biochim Biophys Acta. 1990 Mar 1;1037(3):337-43.
[4] Satoh M, Yokosawa H, Ishii S. Membrane-bound trypsin-like enzyme functioning in degradation of dynorphin in neuroblastoma cells. Purification and characterization. J Biochem. 1988 Mar;103(3):493-8.
[5] Qiu M, Li S, Jin L, et al. Combination of Chymostatin and Aliskiren attenuates ER stress induced by lipid overload in kidney tubular cells. Lipids Health Dis. 2018 Jul 31;17(1):183.
[6] Liu S, Suzuki Y, Takemasa E, et al. Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex. Eur J Pharmacol. 2022 Sep 5;930:175169.
[7] Yang C, Song HW, Liu W, et al. Protective Effects of Chymostatin on Paraquat-Induced Acute Lung Injury in Mice. Inflammation. 2018 Feb;41(1):122-133.
[8] Roszkowska-Chojecka MM, Walkowska A, Gawryś O, et al. Effects of chymostatin, a chymase inhibitor, on blood pressure, plasma and tissue angiotensin II, renal haemodynamics and renal excretion in two models of hypertension in the rat. Exp Physiol. 2015 Sep;100(9):1093-105.

Chymostatin是一种选择性强的蛋白酶体糜蛋白酶样活性抑制剂，Chymostatin对胰凝乳蛋白酶和凝乳酶的Ki分别为9.36nM和13.1nM^[1]。Chymostatin对胰蛋白酶和弹性蛋白酶有抑制活性^[2]。Chymostatin能轻松穿过细胞膜，是研究泛素-蛋白酶体途径和各种细胞过程中蛋白质降解的重要工具化合物^[3]。此外，Chymostatin还具有潜在治疗神经退行性疾病和癌症的作用^[4]。

在体外，Chymostatin（50μM）预处理HK2细胞1h，随后以棕榈酸（0.6mM）处理24h，Chymostatin显著抑制内质网应激标志物的蛋白表达，同时降低促凋亡因子的水平，从而显著减轻细胞凋亡。此外，Chymostatin（50μM）与Aliskiren（10nM）联合预处理HK2细胞1h，随后以胆固醇（10μg/ml）处理6天，也能显著抑制内质网应激标志物的蛋白表达，并降低细胞凋亡相关蛋白的水平，保护细胞免受胆固醇诱导的损伤^[5]。Chymostatin（100μM）预处理HEK-293T细胞和RBL-2H3细胞共培养体系1h，随后以SARS-CoV-2假病毒（SARS-CoV-2S pseudovirions）感染，与未处理细胞相比，Chymostatin显著抑制了52.1%的病毒进入细胞^[6]。

在体内，Chymostatin（2mg/kg）在小鼠接受百草枯诱导的急性肺损伤后30分钟腹腔注射给药，Chymostatin显著减轻了肺组织的病理变化，降低了肺湿干比、组织蛋白酶G活性以及血清中TNF-α、IL-1β、IL-6和HMGB1的浓度，同时增加了血清中内皮细胞特异性分子-1（endocan）的浓度。此外，Chymostatin通过上调肺组织中endocan的表达和下调核NF-κBp65的表达，抑制了炎症反应^[7]。Chymostatin（2mg/kg/h）通过静脉输注给药，用于处理单侧肾切除后高盐饮食（UNX HS）诱导的高血压大鼠和Goldblatt肾动脉狭窄（2K1C）模型大鼠。Chymostatin显著降低了血浆和组织中血管紧张素II（Ang II）的水平。在Goldblatt高血压大鼠中，Chymostatin增加了肾脏和后肢血管阻力。在UNX HS高血压大鼠中，Chymostatin阻断了溶剂诱导的外髓血流减少^[8]。