Chemical Biology of Microbial Anticancer Natural Products

Filamentous fungi and other microorganisms have amazing abilities to synthesize structural complex, diverse and unique small organic molecules. Many are bioactive and numerous compounds such as mycotoxins, antifungal, and anticancer agents have been reported in the literature within the last more than 100 years. New natural products (NPs) are continually discovered and with the increase in selective biological assays, previously described compounds often also display novel bioactivities, justifying their presence in novel screening efforts. Screening and discovery of compounds with activity towards chronic lymphocytic leukemia (CLL) cells is crucial since CLL is considered as an incurable disease. To discover novel agents that targets CLL cells is complicated. CLL cells rapidly undergo apoptosis in vitro when they are removed from their natural microenvironment, even though they are long-living cells in vivo. Fortunately viability of CLL cells can be maintained in vitro by co-cultivation with stromal cells mimicking in vivo conditions. This has led to the development of a co-culture assay that is ideally suited for screening of NPs. The main goal of this study has been to discover fungal NPs with activity towards CLL cells in vitro. We based our screening on a combined analytical and bio-guided approach of LC-DADHRMS based dereplication, explorative solid-phase-extraction (E-SPE), and a co-culture platform of CLL and stromal cells. The activity was tracked to single compounds in seven of the most active extracts in a screening setup including 289 fungal extracts. The novel ophiobolin U was isolated together with the known ophiobolin C, H, K as well as 6-epiophiobolin G, K and N from three fungal strains in the Aspergillus section Usti, and further ophiobolins were bought from commercial sources. Ophiobolin A, B, C and K induced apoptosis in CLL cells with LC50 values of values of 1, 2, 8, and 4 nM, respectively, with a possible narrow therapeutic window. The remaining ophiobolins were inactive. Eight other bioactive extracts were addressed and the compounds responsible for the activity towards CLL cells were identified from six of the extracts. The known compounds: penicillic acid, viridicatumtoxin, calbistrin A, brefeldin A, emestrin A, and neosolaniol monoacetate all displayed activity towards CLL cells though they all showed general cytotoxic in the assay. In the remaining two extracts the bioactive compounds were tentatively identified as cycloaspeptide E and a compound belonging to the statin family of compounds though these results are inconclusive.
A second aim of this PhD project has been to discover novel bioactive NPs using a targetguided approach based on MS and NMR. In one project a target-guided approach based on MS lead to isolation of two new cytochalasins, sclerotionigrin A and B and the known proxiphomin from the fungal species Aspergillus sclerotieoniger. The compounds are moderately cytotoxic towards CLL. In a second approach three novel bioactive micropeptins produced by cyanobacteria were discovered through target-guided isolation based on NMR. The micropeptins displayed inhibitory activity towards serine proteases: chymotrypsin and elastase with IC50 values between 5.9 and 28.0 μM.
In conclusion, this PhD study adds to the knowledge of bioactive NPs produced by filamentous fungi, and in particular activity towards CLL cells. The results obtained here have been based on the use of a combined bio-guided and analytical dereplication approach. This PhD study also includes a review of 50 compounds or compound families with anticancer activity primarily produced by Aspergillus, Penicillium and Talaromyces.