Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized. Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal isolates. Especially, compounds a10, a13, and a14 exhibited superior or comparable antifungal activity to ravuconazole against all the tested fungi. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole moiety of ravuconazole with fluorophenylisoxazole resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole of ravuconazole with fluorophenylisoxazole resulted in antifungal triazoles with more effectiveness and a broader-spectrum.Download high-res image (134KB)Download full-size image

Based on our previous discovery and SAR study on the lead compounds 7d, 5 and berberine which can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, a series of 3-(benzo[d][1,3]dioxol-5-yl)-N-(substituted benzyl)propanamides were designed, synthesized, and evaluated for their in vitro synergistic activity in combination with fluconazole. The series 2a–f were designed by replacing the amide moiety of the lead compound 7d with retro-amide moiety, and compounds 2a and 2b showed more activity than the lead 7d. Furthermore, introducing biphenyl moiety into series 2d–f afforded series 3a–r, most of which exhibited significantly superior activity to the series 2d–f. Especially, compound 3e, at a concentration of 1.0 µg/ml, can enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans from 128.0 µg/ml to 0.125–0.25 µg/ml. A clear SAR of the compounds is discussed.A series of 3-(benzo[d][1,3]dioxol-5-yl)-N-(substituted benzyl)propanamides were designed, synthesized, and evaluated for their in vitro synergistic activity enhancing the susceptibility of fluconazole against fluconazole-resistant Candida albicans. Compound 3e, at a concentration of 1.0 µg/ml, can enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans from 128.0 µg/ml to 0.125–0.25 µg/ml. A clear SAR of the compounds is discussed.Download high-res image (198KB)Download full-size image

To identify effective and low toxicity synergistic antifungal compounds, 24 derivatives of chalcone were synthesized to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. The minimal inhibitory concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant Candida albicans. This was done via methods established by the clinical and laboratory standards institute (CLSI). Of the synthesized compounds, 2′-hydroxy-4′-methoxychalcone (8) exhibited the most potent in vitro (FICI = 0.007) effects. The structure activity relationship of the compounds are then discussed.

A series of caffeic acid amides were designed, synthesized, and their synergistic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The title caffeic acid amides 3–30 except 26 exhibited potent activity, and the subsequent SAR study was conducted. Compound 3, 5, 21, and 34c, at a concentration of 1.0 μg/ml, decreased the MIC80 of fluconazole from 128.0 μg/ml to 1.0–0.5 μg/ml against the fluconazole-resistant C. albicans. This result suggests that the caffeic acid amides, as synergists, can sensitize drug-resistant fungi to fluconazole. The SAR study indicated that the dihydroxyl groups and the amido groups linking to phenyl or heterocyclic rings are the important pharmacophores of the caffeic acid amides.A series of caffeic acid amides 3–13 were designed and synthesized through scaffold hopping from berbeine and 2, and their synergistic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The title caffeic acid amides 3–30 except 26 exhibited potent activity. The SAR study indicates that the dihydroxyl groups and the amido group in the caffeic acid amides are the important pharmacophores.

The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl2- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca2+ chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl2. Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans.

Lymphocytes are deeply involved in the initiation and perpetuation of inflammatory response in inflammatory bowel disease (IBD) and lymphocyte-derived proteins are associated with the pathogenesis of the disease. The aim of this study was to identify the altered protein profiles of lymphocytes from rats with colitis.Colitis models were induced by colonic administration of trinitrobenzene sulfonic acid (TNBS) in 50% ethanol in male SD rats. Seven days after administration of TNBS/ethanol, lymphocytes were harvested from mesenteric lymph nodes (MLNs) and proteins were extracted. Two-dimensional polyacrylamide gel electrophoresis and PDQuest 2D-image-analysis software were used to display and analyze the protein spots. The differentially-expressed proteins were identified by tryptic in-gel digestion and mass spectrometry. Real-time RT-PCR was used for selected transcripts to validate the findings of the proteomics analysis.A total of 1,100 protein spots including 26 proteins with at least a two-fold difference in abundance between colitis and control groups were identified. Among all the detected spots, 17 were up-regulated and 9 were down-regulated. It was found that the altered proteins included the regulators of the cell cycle and cell proliferation, signal transduction factors, inflammatory factors, apoptosis-related proteins and metabolic enzymes.In lymphocytes of rats with TNBS-induced colitis, 26 altered proteins were identified. They involve inflammation, apoptosis, metabolism, and regulation of the cell cycle, cell proliferation, and signal transduction.

Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17Δ/Δ mutant failed to catalyze the biochemical reaction from sulfite to H2S and hardly grew in media lacking Met and Cys. NaSH, the donor of H2S, dose-dependently improved the growth of ecm17Δ/Δ in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17Δ/Δ mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP–protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation.Highlights► Validate that ECM17 regulates the biosynthesis of Met and Cys in C. albicans. ► Met and Cys biosynthesis contributes to biofilm formation. ► Met and Cys insufficiency attenuates adhesion and filamentation. ► Met and Cys biosynthesis pathway is associated with cAMP–PKA pathway.