The urgent need for novel substances for the treatment of severe human diseases such as cancer, combined with the recognition that marine organisms provide a rich potential source of such substances, support the intensive exploration of new substances from marine organisms [1
]. Oceans are sources of a large group of structurally unique natural products that are mainly accumulated in marine macroorganisms such as invertebrates (e.g., sponges, soft corals, tunicates) and algae. Several of these secondary metabolites have pronounced pharmacological activities [2
]. Microorganisms such as fungi and bacteria are truly prolific producers of bioactive molecules and an increasing number of studies support the hypothesis that many compounds originally thought to be produced by macroorganisms actually come from associated microbes [3
]. To adapt to, and survive in, the marine ecosystem, characterized by very special conditions that differ from those found in other habitats, marine microorganisms sometimes produce structurally unique bioactive secondary metabolites not found in terrestrial organisms [6
Although intensive research is necessary to unravel the important resources of the ocean, marine biotechnology has now started to develop integrated strategies. During past decades, one of the most serious bottlenecks in developing natural products from marine sources has been the availability of biomass and/or of optimised cultivation conditions to gain sufficient amounts of substances for preclinical and clinical studies. The concentrations of the compounds of interest are often minute, sometimes accounting for less than 10−6
percent of the wet weight of the macroorganism [7
]. Exploitation is further complicated by the fact that most of these metabolites possess highly complex structures, making an economical production via chemical synthesis difficult. Many unsuccessful attempts have been made to extract these substances in sufficient amounts from invertebrates and algae including harvesting from appropriate sites, aquaculture and even cell culture of the respective organisms [8
]. Prominent examples are the bryostatins, the halichondrins and other antitumoral or anti-inflammatory active substances from marine invertebrates. In these cases, the content in the animals was very low (less than 1 g per ton of biomass) and it was not possible to harvest such large amounts of organisms from nature without destroying their habitats, nor was it possible to cultivate the organisms or cell cultures thereof in sufficient scale and appropriate time [9
In contrast, the focus on microbes associated with marine macroorganisms is a highly sustainable approach aiming to conserve natural habitats, since only small pieces of tissue of macroorganisms are required. These microbes are considered to be an excellent source of secondary metabolites involved in the interspecies communication because they presumably evolved for specific functions such as protecting the host and/or the producer against competitors and/or diseases. From a biotechnological point of view, many of these compounds have pharmaceutical properties, often antibiotic or cytotoxic, that may be useful as lead structures for the development of new drugs [10
]. Therefore, the use of marine fungi, which can easily be grown in the laboratory and at large scale, and that are prolific producers of bioactive compounds, including anti-tumoral substances, provides a solution for the supply issue.
In all cases studied, marine fungi have been revealed to produce natural products (NP) for a variety of ecological purposes. Fungal natural products are often produced in response to a plethora of environmental cues, which might be also small molecules [11
]. Despite their potential for secondary metabolite production, marine fungi are still poorly characterised and underutilised for biotechnological application [12
]. A literature survey covering more than 23,000 bioactive microbial products, i.e., antifungal, antibacterial, antiviral, cytotoxic and immunosuppressive agents, shows that the producing organisms are mainly from the fungal kingdom. Hence, fungi represent one of the most promising sources of bioactive compounds [13
]. Prominent examples of compounds isolated from marine fungi comprise ulocladol, halimide, avrainvillamide, pestalone, and the halovirs A-E [15
]. However, the number of available strains from marine sources is limited and the knowledge of marine fungi in general is scarce. For example, there is a deficit in systematic research of the potential of marine fungi in different application fields and the availability of new structures of bioactive compounds, especially targeted against cancer, is still low [18
Within the comprehensive screening approach of the EU FP7 project MARINE FUNGI, the potential of secondary metabolites from fungi associated with marine macroorganisms was evaluated to provide lead compounds for the development of cancer treatments. Extracts from fermentations of fungi isolated from Mediterranean sponges, Indonesian corals and Chilean macroalgae were screened against tumour cell lines [19
Secondary metabolite biosynthesis has long been known to depend on environmental cues, including carbon and nitrogen sources, ambient temperature, light and pH [20
]. As a consequence, changes to culture conditions can largely modify the spectrum and amounts of secondary metabolites produced by fungi [21
]. A number of stimuli, such as culture media and cultivation condition changes, which are known to induce the production of secondary metabolites [22
], were employed to induce variation of the metabolite pattern of the isolated marine fungi.
In our investigation, extracts from fermentations of the sponge-associated strain MF458, subsequently identified as Tolypocladium geodes
, were found to have potent anti-tumour effects derived predominantly from anti-proliferative rather than overtly cytotoxic profiles. As small-scale assay-guided fractionation did not reveal the obvious presence of compounds known to have anti-tumour properties, we commissioned larger scale fermentations. Extracts of these fermentations were rich in diverse secondary metabolites. The strain was cultivated in a variety of media in order to characterise the metabolite spectrum of this strain and its susceptibility to stimulation by changing cultivation conditions [21
spp. have attracted significant attention as producers of bioactive secondary metabolites. Efrapeptins, pyridoxatin and terricolin have previously been reported as products of terrestrial isolates of T. geodes
, while production of the medicinally-significant cyclosporins is usually associated with other Tolypocladium
]. Metabolites from marine Tolypocladium
spp., such as the new efrapeptin J, have also been reported [24
4.1. Isolation and Taxonomy of MF458
Strain MF458 was isolated by Dr. Karsten Schaumann from a sponge sample. DNA extraction, amplification of the internal transcribed spacer region (ITS) and the 18S rRNA gene, as well as sequencing were performed as described by [30
] with slight modifications, centrifugation of the DNA at 8000× g
and 35 cycles of DNA amplification. The DNA sequences were deposited in GenBank under the accession number KY696657, for the 18S rRNA sequence (MF458_18S), and KY696658, for the ITS region (MF458_ITS1). Cryopreserved stock cultures of strain MF458 were kept at −100 °C using the Microbank system (Microbank system, MAST DIAGNOSTIKA, Reinfeld, Germany). Closest relatives were identified by sequence comparison with the NCBI Genbank database using BLAST (Basic Local Alignment Search Tool) [54
]. Sequence similarity values were determined with the “bl2seq” tool of the NCBI database [55
4.2. Fermentation and Crude Extract Production
The fungal strain was cultivated in the following media: casamino acids glucose medium [56
], casamino acids medium at half and double concentration, casamino acids supplemented with B12 (5 g/L), GM medium (as modified by [57
]), casamino acids supplemented with molasses (80 g/L) and modified Wickerham medium [58
]. Cryopreserved stock cultures were activated on WSP agar (3% NaCl) at 28 °C in the dark for 7 days. Main cultures were done in 2 L EM flasks (containing 1000 mL medium) using the different conditions. Flasks were inoculated using agar pieces from precultures (Ø = 2.6 cm) and incubated at 22 °C in the dark, at 120 rpm or using standing conditions. Crystalline cellulose (10 g/L), paper (40 g/L), gelatine (18 g/L), CaCO3
(15 g/L), and agar (3 g/L) were added as pellet enhancers.
For isolation of compounds, fermentation was done in multiple shake flasks containing casamino acids medium, a temperature of 22 °C and standing conditions.
The cultivation was subsequently scaled up in stirred tank reactors in 10 L (Braun Biostat MD, glass tank), containing 8 L casamino acid medium supplemented with 10 g/L crystalline cellulose (Carl Roth, Karlsruhe, Germany). Precultures were established in the same medium and cultivated for 20 days at 22 °C, 120 rpm. The inoculation volume was 100 mL (1.25%). The fermenter was run at 25 °C with pH, oxygen, and stirring speed being measured and oxygen being controlled to a minimum of 30% air saturation (flow up to 400 L/min, stirrer speed up to 200 rpm). Foam formation was stopped by addition of antifoam (Sigma, Taufkirchen, Germany). A second fermentation run was performed using the same conditions but controlling the pH at 4.3 using 3 M NaOH.
All samples and final harvests of the fermentations were extracted using liquid-liquid extraction. 2 volumes of ethyl acetate were added to one volume of culture, homogenized and separated by means of separating funnels. The organic phase was washed using distilled water and dried in vacuo at 40 °C. Extracts were stored at −20 °C until use.
4.3. Anti-Tumour Assay-Guidance for Purification and Pure Compound Evaluation
Crude extracts and HPLC fractions were profiled at three concentrations against MCF-7, M14 and 786-0 cell lines in a 96-well microtitre assay format using the neutral red cell viability protocol to identify those containing potentially useful anticancer components [19
]. The 100-fold concentrated marine fungal fermentation extracts and HPLC fractions concentrated to dryness and re-dissolved in DMSO-methanol (3:1) were tested for anti-tumour activity at three different dilutions: (A) 1/200, (B) 1/1000, and (C) 1/5000, to facilitate ranking of activities detected based on potency. This resulted, after two or three purification steps, in the isolation of single active compounds. These pure compounds were then characterized in a wider range of cell lines from the NCI60 panel.
4.4. Purification and Identification of Compounds 1–8
The ethyl acetate extract of a 10 L fermentation in multiple shake flasks was dissolved in DMSO-methanol (3:1) and chromatographed in multiple injections on a Waters Xbridge phenyl column (19 × 100 mm) eluted with a linear water-acetonitrile gradient in the presence of 0.1% formic acid, increasing from 10% to 100% acetonitrile over a period of 8 min, holding at 100% acetonitrile for a further 5 min before returning to the starting composition in 1 min and re-equilibrating for another 6 min, at a flow rate of 17 mL/min. After the first minute, eluate fractions were collected every 35 s (24 fractions in total). These fractions were concentrated to dryness, redissolved in DMSO-methanol (3:1) and aliquots diluted 1/10 for assay against tumour cell lines as described above. Based on the assay results and HPLC-MS analysis of the chemical contents of the active fractions, fractions were combined and further purified as follows. The fractions (5–9) eluting between 3 and 5 min were concentrated and further purified by chromatography on a Waters Symmetry Shield RP8 column (19 × 100 mm) eluted isocratically with 25% aqueous acetonitrile containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 17 mL/min with UV detection at 352 nm. The peaks eluting between 8–10 and 11–13 min were separately collected and rotary evaporated to remove acetonitrile before applying to Mega Bond Elut Plexa solid phase extraction (SPE) columns (Agilent Technologies, Santa Clara, CA, USA, 500 mg columns) that had been pre-conditioned with methanol followed by water, for de-salting. The columns were then washed with several column volumes of water and eluted with acetonitrile, and the acetonitrile eluates concentrated to dryness to yield compounds 5 (17 mg) and 6 (24 mg).
The Xbridge phenyl column fractions (10–13) eluting between 5 and 7 min were combined and further purified by chromatography on a Symmetry Shield RP8 column (19 × 100 mm) eluted with a linear gradient increasing from 35% to 65% acetonitrile in water in the presence of 10 mM ammonium formate and 0.1% formic acid over a period of 15 min at a flow rate of 17 mL/min with UV detection at 254 nm. Eluate collected from 6 to 8 min was concentrated to dryness and purified by chromatography on the Symmetry Shield RP8 column eluted isocratically with 42% aqueous acetonitrile containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 17 mL/min with UV detection at 230 nm. The peak eluting between 5 and 6 min was collected, de-salted by SPE as above and concentrated to dryness to yield 2 (1.3 mg). The peak eluting between 8 and 11 min was collected, concentrated to dryness and purified by chromatography on the Symmetry Shield RP8 column eluted isocratically with 35% aqueous acetonitrile containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 17 mL/min with UV detection at 250 nm. The peak eluting between 21 and 23 min was collected, de-salted by SPE as above and concentrated to dryness to yield 3 (15 mg). The peak eluting between 11 and 14 min was concentrated to dryness and purified by chromatography on the Symmetry Shield RP8 column eluted isocratically with 45% aqueous acetonitrile containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 17 mL/min with UV detection at 250 nm. The peak eluting between 7 and 8 min was collected, de-salted by SPE as above and concentrated to dryness to yield 7 (9 mg). Eluate collected from 14 to 15.5 min was concentrated to dryness and purified by chromatography on the Symmetry Shield RP8 column eluted isocratically with 55% aqueous acetonitrile containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 17 mL/min with UV detection at 300 nm. The peak eluting between 12 and 13 min was collected, de-salted by SPE as above and concentrated to dryness to yield 8 (5 mg). Eluate eluting between 17 and 18 min yielded 1 (2.4 mg).
4.5. Assessment of Compound Cytotoxicity
Cell lines used were part of the NCI60 panel and maintained using RPMI-1640 medium (PAA, Hesse, Germany) containing 2 mM glutamine, 100 U/mL penicillin G, 100 mg/mL streptomycin, and 10% fetal calf serum (FCS). At about 80% confluence, cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS), trypsinised, suspended and counted in RPMI-1640 medium before seeding into 384-well plates. Cells were seeded at 20 µL per well in different densities (see Appendix A Table A4
for complete overview) in white, 384-well, PS, Cellstar plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated at 37 °C in the presence of 5% carbon dioxide. At 24 h post seeding, baseline growth was assessed using a control plate and CellTiter-Glo reagent (CTG reagent, Promega Inc., Madison, WI, USA). Briefly, 20 µL of CTG detection mix was added, and plates were analysed on an EnVision Multimode reader (PerkinElmer, Waltham, MA, USA) after 10 min incubation in the dark. In parallel assay plates were dosed with compounds in 11 point dose-response curves and analysed after 48 h incubation at 37 °C in the presence of 5% carbon dioxide using CellTiter-Glo as described. Raw data were normalized to percent of cell growth by using the baseline growth and the corresponding high control (C), containing only the solvent DMSO (Carl Roth, Karlsruhe, Germany). The measured luminescence signal of a certain sample (S) was converted into percent of cell growth compared to the average signal of the baseline control (B). In case of a sample signal higher than the average baseline the following formula was used: percent effect = (S − B)/(C − B) × 100. In case of a sample signal lower than the average baseline the following formula was used: percent effect = (S − B)/B × 100. This relative growth was used to calculate cell viability and proliferation parameters (GI50, LC50, and TGI) as described elsewhere [59
]. Briefly the GI50 value corresponds to the concentration were growth is reduced to 50%, the TGI corresponds to full growth inhibition (100%) and the LC50 corresponds to 50% cell death compared to the baseline measurement. Data analysis was performed on a single plate level using ActivityBase software (IDBS Ltd, Guildford, UK). All data were recorded 3 times or more and average and estimated standard deviation of the corresponding proliferation parameters (GI50, LC50, and TGI) were calculated using Microsoft Excel (Microsoft Inc., Redmond, WA, USA). Values are given without estimated standard deviation, in case they were identical, resulting in a standard deviation of 0, or when only one of the three replicates was reaching the specific proliferation parameter. For hit compounds, a counter assay was used to determine the influence of compounds on CTG performance.
HPLC-mass spectrometry (HPLC-MS) to support compound purification and characterisation was conducted on a system comprising a 2795 Alliance HT Separations Module (Waters, Milford, MA, USA), a 2996 Photodiode Array Detector (Waters, Milford, MA, USA) an Acquity SQ detector (Waters, Wexford, Ireland) and a PL-ELS 2100Ice Evaporative Light Scattering Detector (Polymer Laboratories, Church Stretton, UK). The standard analytical HPLC method employed a SymmetryShield RP8 column (3.5 µm; 4.6 × 75 mm) eluted with a linear gradient of 10%–95% MeCN in water, containing 10 mM ammonium formate + 0.1% formic acid, at a flow rate of 1 mL/min, held at 95% MeCN for 1.0 min before returning to initial conditions over 0.5 min; total run time 12 min. Putative molecular weights, UV-Visible maxima and producing organism taxonomic data were used to search two natural products databases to identify known compounds: Antibase and the Chapman and Hall Dictionary of Natural Products. NMR spectra were recorded on a DRX500 spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively, Bruker, Karlsruhe, Germany) using the signals of the residual solvent protons and the solvent carbons as internal references (δH 3.31 and δC 49.15 ppm for CH3OH-d4). High-resolution mass spectra were obtained on a micrOTOF II spectrometer (Bruker Daltonics, Bremen, Germany) using an ESI ion source in negative mode. UV spectra were obtained with a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The optical rotation was determined using an AA10 Automatic Polarimeter (Optical Activity Ltd., Huntingdon, UK). IR spectra were measured using a Spectrum 100 FTIR spectrometer (Perkin Elmer, Bridgeport, CT, USA) by diffuse reflectance using a thin film.
Analytical HPLC-UV/MS to support fermentation development was conducted on a VWR-Hitachi LaChrom Elite system (pump L-2130, diode array detector L-2450, autosampler L-2200 and column oven L-2300) (VWR, Darmstadt, Germany) with a Phenomenex Onxy Monolithic column (C18, 100 × 3.00 mm) (Phenomenex Inc., Aschaffenburg, Germany) applying a gradient of 0.1% formic acid in H2O (A) and 0.1% formic acid in acetonitrile (B): 0 min 5% B, 4 min 60% B, 6 min 100% B; flow 2 mL·min−1. Coupling of the HPLC system to a Bruker esquire4000 ESI-ion trap (Bruker Daltonics, Bremen, Germany) allowed mass detection. Samples were solved in MeOH and filtered (0.2 µm) before application to the column.
In summary this report shows that the marine isolate of Tolypocladium geodes, analysed as part of the MARINE FUNGI FP7 program, is a very effective secondary metabolite producer. It therefore shows that the investigation of new marine isolates of well-known terrestrial fungal species opens up the possibility to discover new compounds with relevant biological activities. The reported malettinin E and also the acyl tetramate tolypocladenol C were not previously described from terrestrial isolates. The assay-guided purification approach, which was used to characterise the extracts, resulted in the characterisation of molecules with potent anti-tumour effects and a predominantly anti-proliferative activity profile. The most potent activities found were due to compounds known to have anti-tumour effects, namely efrapeptin D (2) and pyridoxatin (3), but also new active molecules were discovered. The acyltetramates tolypocladenol A1/A2 (8) as well as the novel tolypocladenol C (7) only had very moderate anti-tumour potency, but the malettinins B and E (5 and 6) show more potent effects and are being evaluated further.
In analogy to reported biosynthesis pathways it can be assumed that the marine species of Tolypocladium expresses at least 5 biosynthetic pathways under conditions investigated to date. The compounds of most interest as potential anti-tumour leads were the malettinins, and production of these was significantly improved by using tailored culture conditions. The impact of media and cultivation conditions on the secondary metabolite profile increases the relevance of MF458 as a production host and shows that marine isolates, even of well-studied terrestrial species, are definitely worth exploring as serious sources for new and biologically active natural products.