Scientific approach

MARINE FUNGI covered two approaches to gain effective producer strains, which led to the stage of in vivo proof of concept building the basis for clinical trials.
First, a molecular based approach using promising strains of marine fungi that are already known to produce cytotoxic compounds and which are taken from the culture collection of the Kieler Wirkstoff-Zentrum at GEOMAR was applied. Second, a culture based approach was used to increase the number of fungi isolated from marine macrobes and from different habitats of geographically distant regions.

In the first approach, three fungi were selected and sequenced to establish genome sequences and genetic systems. These three candidate strains were selected due to their production of new compounds active against cancer cell lines. Three different next generation sequencing methods were used. Currently, all three genomes are searched for biosynthesis genes, allowing the discovery of new compounds as well as the underlying regulation patterns for the already identified hit-structures. Using random (UV) and targeted (molecular genetic tools) mutagenesis, the strains are optimised producer of anti-cancer compounds. The first experiments in order to gain proteomic information on one of the strains and its mutants were conducted.

Within the second approach, new marine fungal strains were isolated from key environments, such as Mediterranean hard rock habitats colonised by sponges, Indonesian coral reef habitats and Chilean macroalgal forests. Approximately 600 new fungal isolates were obtained and are included into the existing culture collection in order to ensure their long term availability. Taxonomic classification was done by molecular and microscopic methods. All these new fungal strains were identified and cultivated in a screening approach.

Culture conditions for these new isolates are optimised for the production of new anti-cancer metabolites. Nearly 2500 extracts and more than 250 pure compounds were screened in the preliminary panel of cancer cell lines. Active extracts of these cultures were fractionated for chemical identification using state of the art technologies. Promising compounds were tested in the full panel of cancer cell lines and various additional bioassays available in different laboratories of the consortium. Out of a selection of more than 50 compounds that were screened in the full panel a selection of six compounds is now ready for liability testing. The aim is to select lead structures for further optimisation and tests of in vivo efficacy of the chosen compounds with respect to anti-cancer activity.

The culture conditions for the fungal strains are improved regarding the product profile and the amount of secondary metabolites produced. Therefore three candidate strains were taken into controlled cultivation processes in submerged culture fermentation to establish conditions ready to be used in industrial scale-up. The gained data of various fermentation techniques and the subsequent downstream are compiled into a process concept for these compounds.

Progress

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From sea to culture

Key environments

Within the second approach, new marine fungal strains were isolated from key environments, such as Mediterranean hard rock habitats colonised by sponges, Indonesian coral reef habitats and Chilean macroalgal forests. Approximately 600 new fungal isolates were obtained and are included into the existing culture collection in order to ensure their long term availability. Taxonomic classification was done by molecular and microscopic methods. All these new fungal strains are currently identified and cultivated in a screening approach.

Culture conditions for these new isolates are optimised for the production of new anti-cancer metabolites. Nearly 2500 extracts and more than 250 pure compounds have already been screened in the preliminary panel of cancer cell lines. Active extracts of these cultures are fractionated for chemical identification using state of the art technologies. Promising compounds are tested in the full panel of cancer cell lines and various additional bioassays available in different laboratories of the consortium. Out of a selection of more than 50 compounds that were screened in the full panel a selection of six compounds is now ready for liability testing. The aim is to select lead structures for further optimisation and tests of in vivo efficacy of the chosen compounds with respect to anti-cancer activity.
The culture conditions for the fungal strains will be improved regarding the product profile and the amount of secondary metabolites produced. Therefore three candidate strains were taken into controlled cultivation processes in submerged culture fermentation to establish conditions ready to be used in industrial scale-up. The gained data of various fermentation techniques and the subsequent downstream will be compiled into a process concept for these compounds.