The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF. The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures. The model was then refined using molecular mechanics and molecular dynamics. The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model. These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments. The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals. The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans. All 14 azole antifungals are shown to have a similar docking mode in the active site. The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate. The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132. The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.

In a continuing effort to develop highly potent azole antifungal agents, the three-dimensional quantitative structure-activity relationship methods, CoMFA and CoMSIA, were applied using a set of novel azole antifungal compounds. The binding mode of the compounds at the active site of lanosterol 14alpha-demethylase was further explored using the flexible docking method. Various hydrophobic, van der Waals, pi-pi stacking, and hydrogen bonding interactions were observed between the azoles and the enzyme. Based on results from the molecular modeling, a receptor-based pharmacophore model was established to guide the rational optimization of the azole antifungal agents. Thus, a total of 57 novel azoles were designed and synthesized by a three-step optimization process. In vitro antifungal assay revealed that the antifungal activities of these novel azoles were greatly improved, which confirmed the reliability of the model from molecular modeling.

In this study, we aimed at investigating effects of lncRNA ADAMTS9-AS2 on lung cancer progression through regulating miR-223-3p and TGFBR3 expressions. Expressions of ADAMTS9-AS2 in lung cancer tissues and cell lines were determined by reverse transcriptase polymerase chain reaction (qRT-PCR). TargetScan and miRcode were used to predict the targeting relationships, respectively. The luciferase reporter system was used to verify that the relationship among ADAMTS9-AS2, TGFBR3 and miR-223-3p. Western blot assay tested the protein level changes in TGFBR3. Cell proliferation was determined by CCK-8 assay. Cell cycle and cell apoptosis were detected by flow cytometry assay, and migration and invasion were determined by transwell assay. Tumor xenograft model was developed to study the influence of ADAMTS9-AS2 on tumor growth in vivo. qRT-PCR results demonstrated that lncADAMTS9-AS2 was lowly expressed in lung cancer tissues. High expression of ADAMTS9-AS2 in lung cancer cells significantly reduced proliferation ability and inhibited migration, as well as elevating their apoptosis rate. In vivo assay found that ADAMTS9-AS2 suppressed the lung tumor growth. Bioinformatics predicted that miR-223-3p bound directly to the ADAMTS9-AS2 and TGFBR3, which was later confirmed by luciferase reporter system. ADAMTS9-AS2 transfection increased TGFBR3 mRNA and protein expressions in lung cancer cells, but miR-223-3p transfection significantly decreased them. Besides, our results showed that miR-223-3p induced cellular apoptosis while TGFBR3 group showed the complete opposite effect. It was proved that ADAMTS9-AS2 and TGFBR3 were the direct genes of miR-223-3p. MiR-223-3p promotes proliferation, migration and invasion of lung cancer cells by targeting TGFBR3. Therefore, ADAMTS9-AS2, miR-223-3p and TGFBR3 may provide potential targets for the treatment of lung cancer patients. © 2018 IUBMB Life, 70(6):536-546, 2018.

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.