| Over the past two decades, the incidence of invasive fungal infections and associated mortality has increased dramatically. However, the most commonly used clinical antifungal agents are on the existence of limited clinical efficacy, toxic side effects, a narrow spectrum and easy to produce antifungal drug resistance and other issues. Fungal drug resistance has been the main reason for the treatment failure of invasive fungal infections. In our previous research, we have found that some nature pruducts such as berberine, forsythiaside A, baicalein and mangiferin exhibited synergistic activity with fluconazole against drug-resistant fungi, which indicates these compounds can significantly improve the activity of fluconazole against resistant fungi. The compounds have similar pharmacophore groups besides their comparable efficacy, but the mechanism and targets remain unknown, thus makes it difficulty to search for novel antifungal agent further. Moreover, the distribution and efflux of azole antifungal drugs in the synergistic process need to be confirmed.Photo-affinity labeling (PAL) is an efficient and reliable tool used to identify, isolate, and characterize novel biological molecules and potential drug targets, particularly for the unknown target. In this regard, we apply PAL-CC-ABPP (Photoaffinity labeling click chemistry activity-based protein profiling) technology to making these compounds as photoaffinity probe. In this way, we expect to acquire the target information by bio-orthogonal technology, bioinformatics technology and genomics technology in later period.Based on our previous studies of target validation for berberine probe, we design, synthesize photoaffinity labeling probes of baicalein and azole antifungal drugs, and their function evaluation is also carried out.(1) A series of trifluoromethylphenyl diazirine intermediates, as photoaffinity labeling groups, were designed and synthesized including TPD-6, TPD-6a, TPD-6b, TPD-10a, TPD-10b, TPD-10c, TPD-A, TSL-7, which were characterized by MS and NMR.(2) Baicalein probes bearing trifluoromethylphenyl diazirine as photoaffinity labeling group, benzyl bromide as a reactive group, and propynyl group as potential reporter groups were designed and synthesized as BE-1, BE-2, BE-4, BE-5, BE-6, which were characterized by MS and NMR. The bioassary test showed that the MIC80 value of BE-2 was 8 μg/ml against fluconazole-resistant Candida albicans 103 in the presence of fluconazole (0.25μg/ml), and their FICI value was 0.039. BE-5 showed synergistic activity with fluconazole (0.25μg/ml) against fluconazole-resistant Candida albicans 103 at a concentration of 8 μg/ml, and their FICI value was 0.065. These result proved that probes BE-2 and BE-5 showned good synergistic antifungal activity.(3) Based on the novel in situ tag generation technology, utilizing a tandem photoactivatable unit that consists of a carbene precursor and a coumarin precursor reported in current years, we designed and synthesized BE-3 confirmed by NMR and MS. The bioassary test showed that the synergistic activity of BE-3 was equal to BE-2,which has a synergistical MIC80 value of 4μg/ml with fluconazole (1μg/ml) against fluconazole-resistant Candida albicans 103, and their FICI value was 0.039. Thus indicates that both BE-3 and BE-2 are active probes.(4) Taking trifluorormethylphenyl diazirine as photoaffinity labeling group and azole antifungal agents as parental compounds, we designed and synthesized small molecule probes FCZ-GB, FCZ-1, FCZ-2, FCZ-3, FCZ-4 and the corresponding activity test showed · that FCZ-GB, FCZ-3, FCZ-4 have certain activity against Candida albicans Y0109 with MICgo values of 0.25μg/ml,4μg/ml, and 0.5μg/ml, respectively.In order to evaluate the funtion of the title molecular probes, we conducted the research of target binding with probe BE-2. The click reaction was confirmed by MS. The binding proteins can be visualized on SDS-PAGE, thus indicated the probes can be used for target capturing for further. Preliminary mass spectral analysis of specific bands showed that the cysteine or lysine maybe the probe BE-2 binding amino acid residues of the target proteins. The above metioned tests provide basis for father study of BE-3 and BE-5. In addition, the FCZ-GB will be used for the mass spectrometry analysis of the target protein. For FCZ-3 and FCZ-4, we can combine photoaffinity labeling with bio-orthogonal imaging technology to conduct further target information exploringIn this thesis, a series of photoaffinity probes with synergistic activity with fluconazole against resistant fungi and azole antifungal photoaffinity probes were designed and synthesized. Their antifungal activity, protein binding function were determined and confirmed to be ideal probes for further study on the mechanism and target... |
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