Screening and Isolation of Natural Products Targeting Oncogenic Signaling in Human Melanoma Cells

Melanoma is the deadliest form of skin cancer with a dramatically increasing incidence rate over the last decades. Despite the advent of targeted therapy and immunotherapy in 2011, the 5-year survival rate of advanced metastatic melanoma remained in the subsequent years at only 29.8%. Hence, there is an urgent need for new drugs, which could be used in single or combination therapies. Given that melanoma is linked to frequent driver mutations in the MAPK/ERK and the PI3K/AKT pathway, new compounds targeting these pathways are of interest. In this thesis, the unique chemical space of natural products was used to find new compounds inhibiting oncogenic ERK and AKT activity. In a first step, a high-content screening (HCS) pipeline was set up. It consisted of a state of the art phenotypic cell-based assay using genetically encoded kinase translocation reporters (KTR) to measure ERK and AKT activity. These biosensors were expressed in the well-established A2058 melanoma cell line and the patient-derived cell line MM121224. Both cell lines bear a different set of oncogenic mutations, which lead to aberrant signaling in the MAPK/ERK and the PI3K/AKT pathway. Additionally, the pipeline was optimized for semi-automated image acquisition and analysis. The developed pipeline enabled to screen an in-house library of 2,576 plant extracts, what resulted in 140 active plant extracts. Thereof, 139 extracts inhibited AKT activity while only nine extracts inhibited ERK activity. Based on the potency in the HCS and the taxonomic classification, 44 extracts were selected for further investigation through HPLC based activity profiling. The profiling then allowed to localize the activity within an extract, and to dereplicate compounds potentially leading to the activity. The results suggested Piper nigrum fruits for further investigation. In a large scale extract of P. nigrum five piperamides were isolated, which exhibited moderate inhibition of AKT activity on MM121224 cells. Additionally, active thymol derivatives were isolated in another thesis. Both findings confirmed that the established pipeline efficiently leads form crude extracts to single active compounds inhibiting oncogenic signaling in human melanoma cells. These results were published in the Journal of Natural Products. The screen further revealed Ericameria nauseosa (Pall. ex Pursh) G.L.Nesom & G.I.Baird having interesting activity on AKT. Through guidance of HPLC-based activity profiling 11 flavones, four flavanones, a labdane, a clerodane and a seco-caryophyllen were isolated from a larger scale extract. Thereby, (2S,5S,9R,10S,13S)-2-hydroxygrindelic acid and (2R,5R,8S,9R,10S,13*)-2-hydroxy-clerod-3-en-18-al-15-oic acid were identified as new natural products. The latter is also the first detected clerodane in the genus Ericameria. Activity testing of the isolated compounds revealed that flavonoids were responsible for the activity of E. nauseosa and that terpenoids are inactive. The variety of isolated flavonoids gave a preliminary structure-activity relationship. Flavones were shown to be more active than the flavanones. Additionally, flavones with two substituents on the B ring correlated also with higher potency. The most active flavonoid was 5,7-dihydroxy-3,3',4'-trimethoxyflavone, with an IC50 value of 14.7 ± 1.4 µM on AKT activity in MM121224 cells. In a next step, the combination of HPLC-based activity profiling with UHPLC-HRMS/MS based annotation and molecular networking was examined to further improve the prediction of active compounds within complex extracts. Therefore, eight plant extracts from the previous extract library screen were taken. The combination of the two approaches led to the precise prediction that brevipolides from Hyptis brevipes, and methoxylated flavonoids from three different extracts of Hyptis and Artemisia spp. are the active compounds. The prediction was proven by isolation and activity testing of five brevipolides, and seven methoxylated flavonoids. Among these, brevipolide A and 6 methoxytricin were the most potent compounds from each chemical class and displayed AKT activity inhibition on MM121224 cells with IC50 values of 17.6 ± 1.6 µM and 4.9 ± 0.2 µM, respectively. The highly confident predictions of active compounds by combining the two approaches facilitated and accelerated the prioritization of extracts for targeted larger scale isolation. In conclusion, the herein developed screening pipeline led to the selection of promising active extracts. In the scope of this thesis, large-scale extracts of P. nigrum, E. nauseosa, and H. brevipes were examined. They afforded piperamides, brevipolides and flavonoids inhibiting AKT activity in human melanoma cells. None of the isolated compounds inhibited ERK activity. Two compounds from the two latter scaffolds reached IC50 values below 20 µM and are therefore the most promising ones for further investigation