The Effect of Different Species Aminopeptidase N Structure on the Activity Screening of Aminopeptidase N Inhibitor

Abstract

Aminopeptidase N (APN, EC 3.4.11.2) is a type 2 transmembrane zinc-dependent metallopeptidase of the superfamily of gluzincins, 1,2) which forms a noncovalently bound homodimer on the cellular membrane. It has a conserved pentapeptide consensus sequence HEXXH representing a zinc binding motif in its extracellular metalloprotease domain. It expresses in many cells and tissues, including myeloids, fibroblasts, brain cells, hepatocytes, osteoclasts, endometrial cells, epithelial cells of the liver, kidney, intestine, and in synaptic membranes of central nervous system. 3,4) Because of its broad expression, the function of APN strongly depends on its location. The expression of APN is up-regulated in many types of cancer cells, and APN plays a crucial role in tumor progression, such as cell-cell contact, proliferation, tumor invasion and metastasis, and angiogenesis.5-7) Therefore, APN as a therapeutic target for treatment of cancer was presented.In recent years, many natural/synthetic APN inhibitors have been reported and proved to be clinically efficacious for the treatment of tumor. Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine, obtained from culture filtrates of Streptomyces olivoreticuli, 8) is a low-molecularmass dipeptide. And Bestatin is also a potent competitive inhibitor (K i ϭ1.4-4.1ϫ10 Ϫ6 M) of APN with antitumor activity and immunomodulating activity. However, Bestatin has significant bone marrow and mucosal toxicity.9,10) One of Lisoserine derivatives 16l was synthesized by our group showed similar enzyme activity inhibition to that of Bestatin. We found that the enzyme activity inhibition of 16l was similar to Bestatin determined by microsomal aminopeptidase N from porcine kidney microsomes which was commonly used in activity screening of APN inhibitor, [11][12][13] and the inhibition constant, K i , was also similar. However, the enzyme activity inhibition and cell viability inhibition of 16l determined using human tumor cells were greatly lower than Bestatin. And there was significant difference in cell migration and invasion inhibition. Therefore, we proposed that the crystal structure of APN is different between homo sapien and porcine. Computer aided drug design verified that because of the difference in structure, the binding pattern of 16l in the active site of homo sapien and porcine APN are different. The activity screening of APN inhibitor using aminopeptidase N from porcine should be only preliminary determination. The real activity screening should be determined using homo sapien aminopeptidase N. MATERIALS AND METHODS ChemicalsThe chemical structures of 16l and Bestatin are shown in Fig. 1. Bestatin was obtained from Apeloa Kangyu Pharmaceutical Co., Ltd., Zhejiang, China.Cell Culture Human promyelocytic leukemia cells HL-60, human ovarian clear cell adenocarcinoma cell line ES-2 and mouse melanoma cell line B16 were maintained in RPMI-1640 supplemented with 10% fetal calf serum (FCS). These cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 .Membra...

A novel aminopeptidase N inhibitor developed by virtual screening approach

“…As shown in Figure 4, the inhibition constants (K i ) of compound 2 and Bestatin against APN from porcine kidney microsomes (L6007, Sigma) were 2.79 ± 0.32 lM and 3.37 ± 0.24 lM, respectively. 14,15 It confirmed that the inhibitory activity of compound 2 was indeed better than the control Bestatin.…”

“…15 So here we also investigated the inhibitory activity of compound 2 to human APN expressed on ES-2 (human ovarian clear cell carcinoma cells) and HL-60 (human promyelocytic leukemia cells) which expressed high level of APN. As displayed in Figure 5, both Bestatin and compound 2 indicated dose-dependent inhibitory activities against ES-2 and HL-60 at the concentration range of 0.512-1600 lM.…”

Aminopeptidase N (EC 3.4.11.2) inhibitors (2006 – 2010): a patent review

“…It is necessary to examine the APN inhibitory effects of the compounds. On one side, human APN is a membrane-binding protein, while porcine APN is a plasma soluble one, which means that they would have different conformation in the active sites [64]. Factually, in our laboratory, some compounds performed quite different inhibitory effects towards human APN and porcine APN, compared to bestatin as the positive control, respectively, while some performed quite similar results.…”

New Method for Detecting the Suppressing Effect of Enzyme Activity by Aminopeptidase N Inhibitor

“…In mouse xenograft models, the tumor size was significantly reduced by combining the CD13 inhibitor with reactive oxygen species (ROS)-inducing chemo/radiation therapy. 10) CD13 is a potential candidate for development of targeted anticancer drugs, and synthesis of CD13 inhibitors will be of great significance. A convenient and reliable method for detecting the suppressing effect of enzyme activity by the CD13 inhibitor would be useful for the study of CD13 inhibitors.…”