Chemical analysis of Chrysosporium sp. CMB-F214, yielded five new piperazines, chrysosporazines A–E (1–5), with structures assigned by spectroscopic and X-ray analyses and biosynthetic considerations. The chrysosporazines 2–5 exist as an equilibrium of major and minor N-acyl rotamers, while 1–3 incorporate an unprecedented hexahydro-6H-pyrazino­[1,2-b]­isoquinolin-6-one scaffold. The noncytotoxic chrysosporazines reverse doxorubicin drug resistance in P-glycoprotein overexpressing colon carcinoma cells (SW620 Ad300), with 2 delivering a comparable gain in sensitivity to the positive control, verapamil.

This review presents an account of the microbial biodiscovery methodology developed and applied in our laboratory at The University of Queensland, Institute for Molecular Bioscience, with examples drawn from our experiences studying natural products produced by Australian marine-derived (and terrestrial) fungi and bacteria.

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized Dysidea sp. (CMB-01171) for chemical investigation. Although the published natural products literature on Australian Dysidea sponges extends back over half a century and suffers from the perception of being near exhausted, fractionation of Dysidea sp. (CMB-01171) led to the discovery of a family of 10 new biosynthetically and chemically related sesquiterpenes. Detailed spectroscopic analysis guided structure elucidation identified dysidealactams A−F (1−6), dysidealactones A and B (7 and 8), and two solvolysis artifacts, 9 and 10. The dysidealactams A−D (1−4) incorporate a rare glycinyl-lactam functionality, while dysidealactam E ( 5) is particularly noteworthy in incorporating an unprecedented glycinyl-imide moiety. In addition to expanding knowledge of Dysidea natural products, this study demonstrates the value of applying GNPS molecular networking to map chemical diversity and prioritize the selection of marine sponge extracts for more detailed chemical analysis.

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F−M (1− 8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure−activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 μM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A–D (11–14) in addition to a suite of very minor aza analogues 1–6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1–6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N–P (7–9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1–15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1–5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2′ substituted analogues 3–5.