Computational study of novel natural inhibitors targeting aminopeptidase N(CD13).

OBJECTIVES: To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development. RESULTS: Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances. CONCLUSION: This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement. MATERIALS AND METHOD: Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment.

CD13-specific ligand facilitates Xanthatin nanomedicine targeting dendritic cells for therapy of refractory allergic rhinitis.

Relapse in Allergic Rhinitis (AR) is triggered by various unclear mechanisms. Xanthium strumarium L. as a traditional folk medicine can inhibit inflammatory responses through multiple mechanisms. Xanthatin (XT) is a bioactive compound derived from Xanthium strumarium L, and we developed a polymeric micelle (PM) that is dendritic cells (DCs)-specific targeting delivery system loading XT (NGR-XT-PM) based on a cyclic peptide moiety (NGR) to render DCs maturation-resistant for therapy of refractory AR. A murine model of AR was employed to investigate the in vivo therapeutic efficiency and relapse rate compared with the commercial product Budesonide. The results showed intranasal administration of NGR-XT-PM presented significant anti-allergy effect with no recurrence, in contrast, all mice treatment with Budesonide relapsed. NGR-XT-PM could effectively reverse the Th1/Th2 imbalance by depleting the serum inflammatory levels (IgE, histamine and IL-4) and DCs surface costimulatory molecules (CD80, CD86 and I-A/I-E), and promote immune tolerance by upregulating the level of Treg cells and reducing the levels of Th2, Th9 and Th17 cells. Furthermore, we appealed to virtual screening of inflammatory targets and found XT blocking the COX-2/PGE2 signaling pathway, which is a key effector in immune responses. These indicated CD13-specific NGR could facilitate XT selectively targeting DCs for efficiently ameliorating refractory rhinitis, and NGR-XT-PM should be a potential anti-AR drug.

Molecular modeling of Bt Cry1Ac (DI-DII)-ASAL (Allium sativum lectin)-fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta.

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI-DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI-DII domains of Cry1Ac and lectin has been identified using protein-protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI-DII-lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI-DII-DIII) protein. Molecular mechanics/Poisson-Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein-protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic-aromatic, aromatic-sulphur, cation-pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac-APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac-APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding.

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.

The effect of different species aminopeptidase N structure on the activity screening of aminopeptidase N inhibitor.

Aminopeptidase N (APN) is a transmembrane metallopeptidase, which participates in the tumor progress such as proliferation, attachment, angiogenesis and tumor invasion. All of this makes APN as a good chemical therapeutic anti-tumor target. In the present study, we got a novel compound 16l which markedly inhibited the enzyme activity of porcine APN, and the inhibition constant, K(i), of 16l are similar to the positive medicine Bestatin determined using porcine APN. However, when tested using human tumor cells, 16l couldn't effectively inhibit the enzyme activity, cell viability, cell migration and invasion. 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 was different. The compound 16l could effectively inhibit the enzyme activity of porcine APN, but not homo sapien APN located on the surface of tumor cells. Therefore, 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.

Isolation and characterization of cDNA encoding chicken egg yolk aminopeptidase Ey.

Aminopeptidase Ey (EC 3.4.11.20) from chicken (Gallus gallus domesticus) egg yolk is a homodimeric exopeptidase with a broad specificity for N-terminal amino acid residues at P1 position of the substrate. Aminopeptidase Ey is a 300-k metalloexopeptidase, containing 1.0 g atom of zinc per mole of a subunit with a relative molecular mass of 150 k. A full-length cDNA was cloned from chicken (female) liver cDNA library. Analysis of the 3196-base pairs (bp) nucleotide sequence of the cDNA revealed a single open reading frame coding for 967 amino acid residues. The coding region of aminopeptidase Ey gene, apdE, occupies 2901 bp of the cDNA. The predicted amino acid sequence of the enzyme is 66, 65, 64 and 63% identical with those of aminopeptidases N (EC 3.4.11.2) from human, pig, rabbit and rat, respectively. Aminopeptidase Ey contains the metallo-binding sequence motif, His-Glu-Xaa-His, found in zinc metallopeptidases. Zinc binding sites, His-386, His-390 and Glu-409, and catalytic site, Glu-387, were conserved in the homologous aminopeptidases N.

Cloning and sequence analysis of the aminopeptidase N isozyme (APN2) from Bombyx mori midgut.

An aminopeptidase N (APN) isozyme having the molecular weight of 90 kDa, was released by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified homogeneously, from the brush border membrane of Bombyx mori. From the result of cDNA cloning, the primary structure of 90 kDa APN proved to consist of 948 amino acid residues, containing a typical metalloprotease-specific zinc-binding motif in the deduced sequence. Moreover, the primary sequence contained two hydrophobic segments on N- and C-termini. The N-terminal one showed characteristics of leader peptide for secretion and the C-terminal one contained a possible glycosylphosphatidylinositol (GPI) anchoring site, suggesting that the APN encoded by the cDNA is not only a zinc-binding enzyme, but also a GPI-anchored protein. The primary sequence is significantly homologous with those of insect and mammalian APNs, and contains four conserved segments around the zinc-binding motif, two potential N-glycosylation sites and four conserved Cys residues. The deduced primary sequence had 30.7% identity with that of B. mori 110 kDa APN, and did not contain the N-terminal and internal amino acid sequences of B. mori 100 kDa APN, revealing B. mori 90 kDa APN to be the third isozyme on the midgut brush border membrane. On the other hand, the primary sequence of 90 kDa APN showed high homology with Manduca sexta APN2 (65.1% identity) and Plutella xylostella APN2 (63.8% identity). It appears that the B. mori 90 kDa APN should be classified in the insect apn2 cluster and differentiated from insect apn1 and mammalian apn clusters by phylogenetic analysis. These results suggest that 90 kDa APN isozyme encoded by the cDNA is a product of B. mori apn2 gene.