Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214

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.


Introduction
During prior investigations into Australian marine-derived fungi, we reported on the gastrointestinal tract (GIT) of fresh market-purchased fish (Mugil mullet) as a rich source of taxonomically and chemically diverse fungi. We went on to report on the discovery of rare lipodepsipeptide scopularides from Scopulariopsis spp. CMB-F458 and CMB-F115, and Beauveria sp. CMB-F585 [1]; unprecedented hydrazine N-amino-L-proline methyl ester and associated Schiff base artifact prolinimines from Evlachovaea sp. CMB-F563 [2,3]; and N-benzoyl and N-cinnamoyl phenylpropanoid piperazine chrysosporazines from Chrysosporium spp. CMB-F214 and CMB-F294, respectively [4,5]. The chrysosporazines were particular noteworthy, being non-cytotoxic to human carcinoma cells but exhibiting promising inhibitory activity against the multidrug resistance efflux pump P-glycoprotein (P-gp). For example, P-gp-overexpressing human colon carcinoma (SW620 Ad300) cells pre-treated with chrysosporazine F (2.5 µM) acquired a gain in sensitivity (GS 14) against the anticancer agent doxorubicin, >2-fold that of the positive control verapamil (GS 6.1), making chrysosporazine F one of the more potent P-gp inhibitors reported to date [5]. We now report a precursor-directed biosynthesis strategy where supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible new natural products-namely azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), chrysosporazines N-P (7-9) and spirochrysosporazine A (10)-along with the known chrysosporazines A-D (11-14). By contrast, a CMB-F214 M2 solid agar culture without precursor supplementation produced a new natural product, chrysosporazine Q (15). Structures were assigned to 1-15 ( Figure 1) on the basis of detailed spectroscopic and chemical analyses, and biosynthetic considerations. Access to 1-15 facilitated a structure-activity relationship analysis on the P-gp inhibitory properties of this novel class of phenylpropanoid piperazines.

Results and Discussion
UPLC-DAD (210 nm) analysis of an M1 agar plate cultivation of CMB-F214 revea the known chrysosporazines A-D (11-14) accompanied by a suite of earlier-eluting ve minor co-metabolites 1-6 ( Figure 2A). A microbioreactor (MATRIX) cultivation profili analysis employing 12 media compositions under solid phase as well as shaken and sta broth failed to deliver cultivation conditions that significantly improved the yields of 1 however, it did reveal that M2 solid-phase agar cultivations produced the new natu product 15 ( Figure 2B and Figures S1-S2). Notwithstanding low yields for 1-6, a tra amount of pure 4 recovered from a large-scale CMB-F214 rice cultivation possessed a m lecular formula (C28H27N3O5) suggestive of an aza analogue of the co-metabolite chry
HRESI(+)MS measurement on 10 returned a molecular formula (C 20 H 22 N 2 O 5 , ∆mmu +0.1) suggestive of truncated analogue of chrysosporazine Q (15). Analysis of the 1D NMR (DMSO-d 6 ) data for 10 (Tables 3 and 4 and Table S8, Figures S47-S52) revealed major and minor acetamide rotamers (ratio 1:0.6), with resonances attributed to C-1 to C-9 in common with 15. Consideration of diagnostic 2D NMR correlations allowed assembly of the planar structure for 10, with a ROESY correlation between N-COCH 3 and H-2 confirming an E configuration about the major acetamide rotamer (Figure 7). ROESY correlations between H-2 and H α -3, and between H β -3 and H-7, established the relative configuration about C-2/C-4, which together with biosynthetic considerations permitted assignment of the structure for spirochrysosporazine A (10) as shown. A plausible biosynthesis pathway leading to 10 could proceed via enzyme-mediated (stereospecific) oxidative aromatic ring contraction and sequential lactonisation and lactamisation of 15 (Figure 8).
Significantly, 1-10 and 15 reversed doxorubicin resistance in SW620 Ad300 carcinoma cells, with 1 and 2 inducing a gain in sensitivity (GS) comparable to, and 3-5 >2.5-fold that of the positive control verapamil (Table 5, Figure 9). A structure-activity relationship analysis based on these results suggests the methylenedioxy ring, C-3/C-3″ cyclisation and C-2' substitution are key determinants for improved P-gp inhibition. Table 5. Effect of metabolites 1-15 on inhibition of P-gp-mediated resistance to doxorubicin in human colon (SW620 Ad300) carcinoma cells, and cytotoxicity against doxorubicin-susceptible human colon (SW620) carcinoma cells. cultures. b FR: fold resistance was determined by dividing the IC50 value for doxorubicin for P-gp-overexpressing cancer cells by the IC50 value for doxorubicin for sensitive cancer cells. c GS: Gain in sensitivity was the ratio of IC50 value of doxorubicin against SW620 Ad300 without testing compound to IC50 value of doxorubicin against SW620 Ad300 with testing compound. --: not calculated.   a MTT assay showing data as means of SEM of two independent cultures. b FR: fold resistance was determined by dividing the IC50 value for doxorubicin for P-gp-overexpressing cancer cells by the IC50 value for doxorubicin for sensitive cancer cells. c GS: Gain in sensitivity was the ratio of IC50 value of doxorubicin against SW620 Ad300 without testing compound to IC50 value of doxorubicin against SW620 Ad300 with testing compound. -: not calculated.

Chrysosporium sp. CMB-F214 Collection, Isolation and Taxonomy
The fungus Chrysosporium sp. CMB-F214 was isolated from the gastrointestinal tract of a specimen of Mugil mullet fish, on an M1 agar plate in the presence of 3.3% artificial sea salt (M1S), and incubated at 26.5 • C for eight days. Genomic DNA for CMB-F214 was extracted from its mycelia using the DNeasy Plant Mini Kit (Qiagen, Brisbane, Australia) as per the manufacturer's protocol and as previously described [4]. A BLAST analysis (NCBI database) on the amplified ITS gene sequence (GenBank accession no. MN249497) revealed 99% homology with Chrysosporium lobatum.

Chrysosporium sp. CMB-F214 Media MATRIX Study
From an agar plate culture of the fungus Chrysosporium sp. CMB-F214, spores were transferred into 24-well microbioreactors (MBRs) charged with a range of different culture media (×12), and in solid (2.5 mL), broth static (1.5 mL) and broth shaken (1.5 mL) formats. MBRs were incubated at 26.5 • C for 8 days, with 190 rpm for shaken broth. After incubation, individual wells were extracted in situ with EtOAc (2 mL, each), filtered and dried under N 2 . The resulting extracts were dissolved in MeOH (100 µL for solid and 50 µL for broth, each containing trace levels of an internal calibrant) and analysed by Ultra-High Performance Liquid Chromatography-diode array detector (UPLC-DAD) and Ultra-High Performance Liquid Chromatography-quadrupole time of flight (UPLC-QTOF), with the resulting chromatograms compared at the same scale ( Figures S1 and S2)

Analytical Precursor-Directed (Nicotinate) Feeding Study
Sodium salts of nicotinic acid were prepared by dissolving in equal amounts of saturated NaHCO 3 solution and sterile H 2 O to provide the required concentrations (pH~7). A 24-well MBR was used to obtain miniaturised cultures where M1 broth media (1400 µL) was mixed with the corresponding sodium salt concentration (100 µL) to provide final concentrations of 2 and 4 mg/mL. A single colony of Chrysosporium sp. CMB-F214