Studies On The Chemical Diversity And Bioactivity Of Secondary Metabolites From Fungi

The development in identifying lead molecules for drug discovery is well dependent on the microbial diversity and its chemical diversity. A study on manipulating the culture conditions of fungi, as well as the endophytes from different plants, was carried out to explore chemical diversity and search for antitumor metabolites. It can be summarized as two parts: By changing cultural conditions and addition of enzyme inhibitors, the silent biosynthetic pathway of the cytochalasins producing strain KLA03 might be activated and produced more active cytochalasins analogs. Meanwhile, the study of various metabolites of different endophytes was performed. All of the compounds isolated were evaluated for their growth inhibition against several cancer cell lines.Firstly, our previous work had reported the isolation of cytochalasins Z10–Z15 from the marine-derived fungus Spicaria elegans KLA03. These new skeleton cytochalasins showed obviously cytotoxicity and PTK inhibition activity and might be developed as leading compounds for antitumor drug discovery. By manipulating culture conditions and addition of enzyme inhibitor, the secondary metabolites of KLA03 was drastically shifted and resulted in another 13 new cytochalasans (1-9 and 28-31). In a new soybean powder medium, the strain KLA03 mainly produced aspochalasins (1-17) using leucine rather than cytochalasins, which gave the evidence of the potential biosynthetic pathways of two kinds of cytochalasans by one strain. Moreover, Spicarin A (26) and B (27), two unusual isobenzofuran dimmer, and spicarin C (28) and D (29), two novel isobenzfuran-aspochalasin heterodimers, were found during the reculture of the strain KLA03 in a high glucose medium. Their plausible biosynthetic pathway were proposed, which showed the possibilities of a Diels-Alder reaction and a free radical addition reaction to theα,β- unsaturated ketone moiety of aspochalsins. Treatment of strain KLA03 with cytochrome P-450 inhibitor resulted in two new deoxy-cytochalasins, 7-deoxy-cytochalasin Z7 (30) and 7-deoxy-cytochalasin Z9 (31), which were recognized as plausible precursors of cytochalasins Z7 and Z9, respectively. The absolute configurations of open-chain cytochalsins Z10-Z15 and aspochalsins were determinated, which confirmed the biosynthetic pathway of cytochalasans via a hypothetic Diels-Alder-type reaction. Secondly, fourty-two mangrove endophytic fungi and 97 rhizospheric fungi were isolated from 3 different mangrove samples, and 17 active strains were obtained after the antitumor screening against P388 cell line. After choosing appropriate fermentation conditions, a cytochalsins producing strain W-8 and 5 other active strains, GQ-7,W-8,JP-1,HK-03-6,HK-16-4 were chosen as aimed strains. Moreover, two endophytes (CF005 and AC-6) of Cistanches were also fermented for searching for neuroprotective effect compounds. As a result, six new cytochalasins (32-37) and 2 new tripeptide (38 and 39) were isolated from strain W-6; seventy eight compounds were isolated from other 7 strains. Eighteen of them were new compounds, including 6 tertramic acids (45-50), a citrinin dimer (51), 3 phenol and quinone derivative (59-61), 3 sesquiterpenes (74-76), 2 isobenzofran isomers (82 and 83), and 3 other compounds (77, 95 and 100).Finally, the antitumor activity against several cancer cell lines of the 29 cytochalsans was assayed by MTT and SRB methods. Among them 4 new cytochalasins (32, 33, 35, 39) with significant cytotoxicities against A-549 cell line were found, showing IC50 values of 19.6, 5.6, 17.4, 7.9μM, respectively. Considering all of the cytochalasins obtained by our lab, the structure activity relationships were deduced as below: The epoxide group at C-6,7 and the enol ester moiety at C-21 might be two essential parts of the pharmacophore of highly active cytochalsins; to the moderate active cytochalasins, theα,β-unsaturated ketone moiety at C-19,20 was the essential part, and the hydroxyl group at the benzene ring shorted down its activity. The cytotoxicity results of 17 aspochalsins (1-17) strongly supported the conclusion that the theα,β-unsaturated ketone musty be the essential part of the pharmacophore of aspochalasins. The cytotoxicity results had a significant implication that the presence of a saturated fatty chain at C-8 might be an essential part of the pharmacophore of penicillenols (45-50), and the 1-hydroxyethyl group at the 5S-position might enhanced their activity. Compound 59 showed a significant cytotoxicity against P388 cell line with an IC50 value of 1.38μM. In addition, tyrosol (109) can promote an increase of intracellular cAMP special on GPCR12 transfected cells, such as CHO and HEK293, which means it may be a possible ligand for GPCR12. Chrysogenamide A (100) exhibited a neurocyte protection effect at 0.1 nM against oxidative stress-induced cell death in SH-SY5Y cells.