The endophytic fungus XJ-AC03, which was isolated from the healthy roots of Aconitum leucostomum, produced aconitine when grown in potato dextrose agar (PDA) medium. The presence of aconitine was confirmed by the chromatographic and spectroscopic analyses. The yield of aconitine was recorded as 236.4 μg/g by high performance liquid chromatography (HPLC). The mass spectrometry was shown to be identical to authentic aconitine. Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal aconitine indicated that the fungal aconitine produced an NMR spectrum identical to that of authentic aconitine. Strain XJ-AC03 was identified as Cladosporium cladosporioides by its characteristic culture morphology and ITS rDNA sequence analysis.

Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family 38 and is involved in the biosynthesis and turnover of N-linked glycoproteins process. Locoweeds, which contain swainsonine (SW) that inhibits LAM, are the main poisoning plants in many regions of the world, and thereby resulting in animal poisoning or even death. Based on regions of protein sequence conservation between LAM from Bos taurus and Homo sapiens, we cloned cDNA encoding Capra hircus LAM (chLAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant chLAM was activated 1.6 and 1.2-fold with Zn 2+ and Ca 2+ , respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant chLAM is lower than wild type chLAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also of a potential strategy for a novel therapy for locoweed poisoning.

Nicotinic acetylcholine receptor (nAChR) is a target for insect-selective neonicotinoid insecticides (NNs), exemplified by imidacloprid (IMI). In the present study, 78 IMI derivatives reported as inhibitors of Drosophila melanogaster nAChR (Dm-nAChR) and Musca domestica nAChR (Md-nAChR) were used for three-dimensional quantitative structure-activity relationship (3D-QSAR) studies. Two optimal models with good predictive power were obtained: Q(2) = 0.64, R(2)(pred) = 0.72 for Dm-nAChR, and Q(2) = 0.63, R(2)(pred) = 0.62 for Md-nAChR. In addition, homology modeling, molecular dynamic (MD) simulation, and molecular docking also showed that amino acids located within loops A, C, D and E play key roles in the interaction of Dm-/Md-nAChR with NNs. This is highly consistent with the results of graphical analysis of 3D-QSAR contour plots. Mutation analysis also implicates the Y/S mutation within loop B as being associated closely with NN resistance in Drosophila and Musca. The results obtained lead to a better understanding not only of interactions between these antagonists and Dm-/Md-nAChR, but also of the essential features that should be considered when designing novel inhibitors with desired activities.

In order to obtain structural features of 3-arylpyrimidin-2,4-diones emerged as promising inhibitors of insect γ-aminobutyric acid (GABA) receptor, a set of ligand-/receptor-based 3D-QSAR models for 60 derivatives are generated using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The statistically optimal CoMSIA model is produced with highest q2 of 0.62, r2ncv of 0.97, and r2pred of 0.95. A minor/bulky electronegative hydrophilic polar substituent at the 1-/6-postion of the uracil ring, and bulky substituents at the 3′-, 4′- and 5′-positions of the benzene ring are beneficial for the enhanced potency of the inhibitors as revealed by the obtained 3D-contour maps. Furthermore, homology modeling, molecular dynamics (MD) simulation and molecular docking are also carried out to gain a better understanding of the probable binding modes of these inhibitors, and the results show that residues Ala-183(C), Thr-187(B), Thr-187(D) and Thr-187(E) in the second transmembrane domains of GABA receptor are responsible for the H-bonding interactions with the inhibitor. The good correlation between docking observations and 3D-QSAR analyses further proves the model reasonability in probing the structural features and the binding mode of 3-arylpyrimidin-2,4-dione derivatives within the housefly GABA receptor.

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