In order to postulate its binding mode in APN

In order to postulate its binding mode in APN, CKI 7 dihydrochloride 13v was docked into the active site of APN (PDB code: 2DQM) using Sybyl_X 1.3. As illustrated in Fig. 5A, the catalytic zinc ion of APN was chelated by the hydroxamate moiety of 13v, and the S1, S1′ and S2′ pockets of APN were occupied by the leucine residue, the two phenyl groups of 13v, respectively. Moreover, the binding mode of 13a (Fig. 5B) was also postulated and compared to that of 13v (Fig. 5A). It could be found that both 13a and 13v chelated the zinc ion via the hydroxamate group and occupied the S1 pocket via the leucine residue. Note that compound 13a could only occupy the S1′ pocket with its single phenyl group, while compound 13v could occupy the S1′ and S2′ pockets simultaneously. This might rationalize the better APN inhibitory activity of 13v.
Conclusion In summary, one novel series of triazole derivatives was designed, synthesized and evaluated as APNIs. In the enzymatic assay against APN, all leucine ureido-based compounds presented much better inhibitory activities than the positive control bestatin. The best compound 13v demonstrated more potent in vitro anti-angiogenesis activity than bestatin in both HUVECs tubular structure formation assay and rat aortic ring model. What's more, 13v exhibited promising anti-metastasis effects in a mouse H22 pulmonary metastasis model, which deserve further research and development as anti-metastatic lead compound.
Experiment section
Introduction Fermented soybean products, such as soy sauce, soybean paste, Douchi, Sufu and Tianyou, play a key role in traditional fermented foods industry in China. These products are usually produced with high salinity (5–20%, w/v) using Aspergillus oryaze 3.042 (A. oryzae 3.042) as a main fermentation strain. The annual production of soy sauce exceeded 50% of the total condiment output in China, reaching 9.38 million tons in 2014 (http://www.askci.com/news/chanye/2015/02/04/165020koaw.shtml). Nevertheless, the quality and the raw material utilisation rate of fermented soybean products, especially soy sauce, are low, which result in poor competitiveness in both domestic and foreign markets. The above two parameters of soy sauce, soybean paste, Douchi, Sufu and Tianyou depend greatly upon the salt-tolerance of proteases secreted by A. oryzae 3.042 (Lee et al., 2010, Wang et al., 2013), but salt-tolerance of the dominant proteases, specifically the neutral proteases secreted by A. oryzae 3.042, is poor (Wang et al., 2013). The physicochemical properties and salt-tolerance molecular mechanisms of several known salt-tolerant proteases from A. oryzae remain unclear (Wang et al., 2013). A. oryzae, especially A. oryzae 3.042 and A. oryzae RIB40, are widely accepted as ‘generally recognised as safe’ (GRAS) due to their long history of safe applications in the manufacture of traditional fermented foods in East Asia and pharmaceutical-grade enzymes (Maeda et al., 2005, Vishwanatha et al., 2009). The entire A. oryzae RIB40 genome had been published, and 130 protease-like genes were identified, but most were not characterised (Machida et al., 2005). Amongst these genes, about 20 encoded proteases had aminopeptidase activity (Kusumoto et al., 2008). Researchers had isolated aspartyl aminopeptidases (AAP) from A. oryzae (Kusumoto et al., 2008), yeast (Yokoyama, Kawasaki, & Hirano, 2006), Escherichia coli (Watanabe, Tanaka, Akagawa, Mogi, & Yamazaki, 2007) and Lactobacillus delbrueckii (Stressler et al., 2016). The aforementioned studies demonstrated that AAPs isolated from different microorganisms were salt-tolerant and included varying numbers of isomeric subunits. They also showed the differential effects of metal ions and inhibitors on the activities of AAPs from various microorganisms. Notably, AAP can specifically release glutamic acid and aspartic acid from peptides that contain glutamic acid and aspartic acid residues in their N-termini. These two amino acids, which impart an umami taste with low thresholds, play a key role in fermented soybean products (Stressler et al., 2016). Currently, little is known about the salt-tolerance mechanisms of AAP, and such a capacity is important to maintain, activate and/or modulate the activity of AAP during manufacture of fermented soybean products in high salinity.