Ellinger, B.

Gribbon, P.

Labes, Antje

Landskron, J.

Prashar, A.

Silber, Johanna

Tasken, K.

Wrigley, S.

OceanRep

A PENOTYPIC SCREENING TOOLBOX PERMITS THE IDENTIFICATION OF NOVEL COMPOUNDS WITH ANTI-CANCER PROPERTIES DERIVED FROM MARINE FUNGI Ellinger, B.1; Landskron, J.2; Prashar, A.3; Silber, J.4; Wrigley, S.3; Tasken, K.2; Labes, A.4; Gribbon, P.1  1European ScreeningPort GmbH, Schnackenburgallee 114, 22525 Hamburg, Germany. 2Biotechnology Centre of Oslo, Oslo, Norway. 3Hypha Discovery Ltd., Uxbridge, United Kingdom. 4Kieler Wirkstoff-Zentrum am GEOMAR, Kiel, Germany. INTRODUCTION BEAD TECHNOLOGY RESULTS OF THE CYTOTOXICITY PROFILING AND FURTHER ASSAYS ASSAY PRINCIPLE IMAGE BASED CYTOTOXICITY PROFILING European ScreeningPort gratefully acknowledges Boris Pinchuk and Janina Rahlff for technical assistance, Hamilton for providing the Biolevitator™,  Solentim Ltd and CENiBRA for providing the CellMetric™.  The MARINE FUNGI research project received funding from the EU Seventh Framework Program (FP7/2007-2013 under grant agreement number 265926). ACKNOWLEDGEMENTS compound known drug DMSO only emtpy well BioLevitator™; 3D cell culture on Microcarriers Schematic representation of the incubation chamber with magnetic separator Tissue origin No. of cell lines Leukemia 6 Non-Small Cell Lung 9 Colon 7 CNS 6 Melanoma 9 Ovarian 7 Renal 8 Prostate 2 Breast 6 Cell seeding and incubation for 24h Compound addition Detection of viability at day 1, set to 0% growth Detection of viability 48h incubation in the presence of compound Day 0 Day 1 Day 2 Day 3 0 20 40 60 80 100 0 20 40 60 80 100 80 6040 20 0 Confluence (%) -10 -9 -8 -7 -6 -5-100-75-50-250255075100 2  50 -25 -5  -75 -10  7  100 -5 -6 -7 -8 -9 -10 Cell growth (%) -8 -7 -6 -5 -4 -3 -2-100-75-50-2502550751001250 25 50 -25 -50 -75 -10  75 10  125 -2 -3 -4 -5 -6 -7 -8 Cell growth (%) Log10 [Cisplatin] (M) Log10 [Paclitaxel] (M) Source Extracts Organisms No. hits Hit Rate (%) Mediterranean sponge fungi 754 206 78 10.3 Chilean macro-algal fungi 125 125 48 38.4 Indonesian coral fungi 331 105 47 16.5 Totals 1210 436 173 14.3 Structure of the Marine Fungi consortium Summary of the collected fungi, their natural products and the corresponding bioactivity The ‚Marine Fungi‘ project, an inter-national FP7 program, aims at identification of novel compounds with anticancer properties from marine fungi. The projects spans        s from the isolation and characterisation of the marine fungi to fermentation, activity guided purification and screening of extracts and compounds. The 3 most interesting natural products are now being analysed in vivo. Schematic representation of the 3D Dextran Microcarriers M14 cells (melanoma) on Dextran Microcarriers -90 -30 Average growth, 3D 30 90 150 -90 -30 30 90 150 Average growth, 2D -60 -60 0 60 120 180 0 60 120 180 Direct comparison of 258 compounds screened against A549 cells in 2D and 3D (R2=0.95) Next to conven-tional plate based assays dextran microcarriers were evaluated. 56 Hits from the screen were followed up in dose response. No significant differences were observed. 3D data confirm 2D experiments.  Brightfield images of A549 cells after 3, 24, 48 and 72h 100   40 20 0 Confluence (%) 0 20 40 60 80 Time (h) Brightfield images of A549 cells after 3, 24, 48 and 72h Comparison of cell growth using CTG (grey) or image based (black) analyses. Comparison of cell growth using CTG (grey) or image based (black) analyses. Marine Fungi cytotoxicity assay principle based on the NIH procedure. By detecting viability at day 1 cytostatic and cytotoxic compounds can be identified 50 0 -50 Cell growth (%) Cyto-static effect Cyto-toxic effect GI50 TGI LC50 Log10 [compound] (M) Tissue specific composition of the NCI60 cell line panel. Schematic representation of dose-response curves obtained with the characteristic cellular parameters. 0 5 10 15 20 25 30 35 40 < 5 5 - 10 15 - 20 20 - 25 25 -30 30 -35 35 -40 40 - 45 45 - 50 50 - 55 55 -60 60 - 70 > 70 Frequency Binned LC-50 / TGI / GI-50 (mM) > 70 60 55 50 45 40 35 30 < 10 10 15 20 25 0 5 10 15 20 2  30 35 40 Frequency 1 2 3 Structures of myco-phenolic acid (1), penitrem A (2), rugulosin (3) -10 -9 -8 -7 -6 -5 -4-100-75-50-250255075100125Cell growth (%) -7  -50 -25  2  5  7  100 12  -4 -6 -8 -9 -7 -5 -10 Log10 [Secalonic acid] (M) -10  Normalised Cell Growth dose response curves for secalonic acid in 786-0, MCF-7, M14 and HL-60 cell lines showing TGI values between 8 and 30 µM -10 -9 -8 -7 -6 -5 -4-100-75-50-250255075100125-4 -6 -8 -10 Log10 [Bisdethiobis (methylthio)gliotoxin] (M) -5 -9 -7 Cell growth (%) -10  -75 -50 -25 0 25 50 75 10  125 Normalised Cell Growth dose response curves for bisdethiobis- (methylthio)-gliotoxin in 786-0, MCF-7, M14 and HL-60 cell lines Frequency distribution of GI50 (white), TGI (grey) and LC50 (black) values (µM) for 37 compounds against the same 4 cell lines Binned LC50 / TGI / GI50 (µM) 50 45 15 40 35 30 25 20 - - - - - - - - - - - 55 60 70 -12 -10 -8 -6 -40200040006000800010000Log10 [Valinomycin] (M)Area (µm2)Area (µm2) -4 -6 -8 -10 Log10 [Valinomycin] (M) -12 0 2000 4000 6000 8000 10000 Mitotox assay based on image analysis. Read out is the area of viable Mitochondria. IC50 of Valinomycin varies between 13 and 19 nM. The principle of the NCI60 human tumour cell line anticancer drug screen was developed 15 years ago. We adapted the assay for HTS purposes by changing the read out, from dye based to Lumines-cence, miniaturising it, from 96 to 384 well plates, and reducing the volume per well from 200 µl to 20 µl. The wealth of data available for the cell lines from the NCI60 panel, like COMPARE or the COSMIC database, is an invaluable source of information for the screening process. Parallel to the NIH procedure assays for improved automatisation, using the BioLevitator™ (Hamilton Bonaduz AG), or alternative read outs, using the CellMetric™ (Solentim Ltd), were evaluated. The screening process is based on a triage of assays. Starting with 4 very sensitive cell lines from the NCI60 to identify also weak anticancer activity, the compounds then undergo a full screening in the NCI60 panel to obtain tissue specific data and an understanding of the mode of action via COMPARE. Most of the marine natural products exhibit an activity in the low micromolar range. Further assays include apoptosis and necrosis assays as well as pathway analysis using PhosphoFlow analysis. ADMET assays like plasma protein binding or Cytochome P450 inhibition help to select the most interesting compounds for in-vivo studies. A research article describing the identification of the first 37 identified natural products has been submitted and will appear in the journal for ASSAY and Drug Development Technologies. 

A phenotypic screening approach to identify anti-cancer compounds derived from marine fungi

A phenotypic screening approach to identify anti-cancer compounds derived from marine fungi

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