An aminopeptidase inhibitor
Bestatin is an aminopeptidase inhibitor originally isolated from S. olivoreticuli.1 It inhibits aminopeptidase B (IC50 = 0.05 ?g/ml), aminopeptidase N (IC50 = 16.9 ?M), leucine aminopeptidase (IC50 = 0.01 ?g/ml), and the aminopeptidase activity of leukotriene A4 (LTA4) hydrolase (Kapp = 172 nM).1,2,3 It is selective for these aminopeptidases over aminopeptidase A, trypsin, chymotrypsin, elastase, papain, pepsin, and thermolysin.1 Bestatin inhibits the production of LTB4 in erythrocytes when used at a concentration of 70 ?M.3 It increases the expression of Akt, inhibits proliferation, migration, and invasion, and induces autophagy and apoptosis in 5637 bladder cancer cells.4 Bestatin (5 and 15 mg/kg) decreases serum levels of LTB4 and reduces tumor growth in a patient-derived xenograft (PDX) mouse model of colorectal cancer.5
1.Umezawa, H., Ayoagi, T., Suda, H., et al.Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetesJ. Antibiot. (Tokyo)29(1)97-99(1976) 2.Melzig, M.F., and Bormann, H.Betulinic acid inhibits aminopeptidase N activityPlanta Med.64(7)655-657(1998) 3.?rning, L., Krivi, G., and Fitzpatrick, F.A.Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymesJ. Biol. Chem.266(3)1375-1378(1991) 4.Wang, X., Liu, Y., Liu, W., et al.Ubenimex, an APN inhibitor, could serve as an anti?tumor drug in RT112 and 5637 cells by operating in an Akt?associated mannerMol. Med. Rep.17(3)4531-4539(2018) 5.Zhao, S., Yao, K., Li, D., et al.Inhibition of LTA4H by bestatin in human and mouse colorectal cancerEBioMedicine44361-374(2019)
Kinase experiment: | Cells are harvested, washed, and lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40). Total cell protein is quantified using the Bradford assay and 1-mg/mL protein aliquots are made. Ten microliters of total cell protein is mixed with 290 μL of substrate solution (0.1 mg/mL dithiothreitol [DTT], 0.1 mg/mL albumin, and 1 mM alanine-β-naphthylamide). Fluorometric measurements (340 nm excitation, 400 nm emission) are made after 15 and 30 min. The slope of the line between the 15- and 30-min measurements is used to represent aminopeptidase activity. Total cell protein is preincubated with bestatin, amastatin, puromycin, EDTA, and/or ZnCl2 for 20 min before the fluorometric aminopeptidase assay. |
Cell experiment: | Growing cells (1×106 to 2×106 cells/mL) are diluted to 1.0×103 cells/mL and transferred (3 mL) into a well in a 12-well multiwell plate (2.5-cm diameter/well). Cells are treated with 0, 10, 50, 100, 300, or 600 μM Bestatin and allowed to grow at 21°C shaking at 180 rpm for 48 h. A hemocytometer is used to measure cell density after 0, 24, and 48 h. |
Animal experiment: | Bestatin is dissolved in PBS. The agent (doses of 10, 1, and 0.1 mg/kg) is injected i.p. to non-cyclophosphamide-treated mice, 5 or 10 times at 24-h intervals before SRBC immunization. The mice are immunized 24 h after the last dose of bestatin. Pharmacological immunosuppression is induced by a single intraperitoneal injection of cyclophosphamide administered at a dose of 350 mg/kg, 12 days before SRBC immunization. Bestatin at the doses of 1 and 0.1 mg/kg is injected to cyclophosphamide-immunosuppressed mice i.p. five times at 48-h intervals or 10 times at 24-h intervals before SRBC immunization. The first dose of bestatin is administered 24 h after cyclophosphamide, while the last dose of the drug is injected 24h before SRBC immunization. |
参考文献: [1]. Hossain A, et al. Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice. Exp Eye Res. 2016 Aug;149:100-6 | |
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