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Biological and chemical profiling of an Australian strain of the fungus Aspergillus unilateralis (MST-F8675), isolated from a soil sample collected near Mount Isa, Queensland, revealed a complex array of metabolites displaying broad chemotherapeutic properties. Noteworthy among these metabolites were a unique series of highly modified dipeptides aspergillazines A–E, incorporating a selection of unprecedented and yet biosynthetically related heterocyclic systems. Co-occurring with the aspergillazines was the recently described marine-derived fungal metabolite trichodermamide A (cf. penicillazine), whereas re-fermentation of A. unilateralis in NaCl (1%) enriched media resulted in co-production of the only other known example of this structure class, the marine-derived fungal metabolite trichodermamide B. Further investigation of A. unilateralis returned the known terrestrial fungal metabolite viridicatumtoxin as the cytotoxic and antibacterial principle, together with E-2-decenedioic acid, ferulic acid, (7E,7′E)-5,5′-diferulic acid and (7E,7′E)-8,5′-diferulic acid. The aromatic diacids have previously been reported from the chemical and enzymatic (esterase) treatment of plant cell wall material, with their isolation from A. unilateralis being their first apparent reported occurrence as natural products. Structures for all metabolites were determined by detailed spectroscopic analysis and, where appropriate, comparison to literature data and/or authentic samples.

Background: Englerin A (EA) is a natural product which has shown selective toxicity toward kidney tumor cells in the NCI60 panel. However, EA's targets and mechanism of action remain unknown. Methods: Cytotoxicity of EA toward a panel of kidney cancer cell lines was assessed in vitro by MTT and cell counting, and in vivo using a human clear cell kidney cancer xenograft mouse model. The protein kinase C family was predicted as a likely molecular target of EA using QSAR methodology. This was confirmed by PKC kinase activity assays. EA's mechanism of action was investigated using western-blot analysis, meso-scale discovery technology, and immunohistochemistry. Results: EA was cytotoxic in vitro and in vivo via induction of apoptosis. Comparison of EA's structure with other well-known compounds predicted the PKC family to be a potential drug target. Direct treatment of cell lysates with EA increased pan-PKC kinase activity, suggesting that EA directly activated at least one of the PKC isoforms. Further investigation showed that EA-dependant PKC activation inhibited the insulin pathway through inhibitory phosphorylation of Insulin Receptor Soluble 1 (IRS1) protein, and decreased phosphorylation status of Akt and GSK3beta. These data were reproduced in vivo, as phosphorylation of GSK3beta was reduced in RCC tumor treated with EA compared to tumor treated with vehicle. Conclusion: The insulin/PI3K/Akt pathway has been previously shown by us and others to be critical for the survival of renal cell carcinoma. By activating PKCs, EA indirectly induces an “insulin resistant” phenotype which appears to be selectively lethal for RCC tumor cells. Thus, EA represents a novel potential therapeutic lead for renal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 959. doi:10.1158/1538-7445.AM2011-959

SummaryThe natural product englerin A (EA) binds to and activates protein kinase C-θ (PKCθ). EA-dependent activation of PKCθ induces an insulin-resistant phenotype, limiting the access of tumor cells to glucose. At the same time, EA causes PKCθ-mediated phosphorylation and activation of the transcription factor heat shock factor 1, an inducer of glucose dependence. By promoting glucose addiction, while simultaneously starving cells of glucose, EA proves to be synthetically lethal to highly glycolytic tumors.