Hunting for Natural Products Targeting Oncogenic Signaling in Human Melanoma Cells

The scaffold diversity of natural products has given rise to the development of many drugs. Around one-third of all therapeutic agents approved between 1981 and 2019 are natural products or inspired by them. This is not surprising, as nature provides a vast reservoir of highly structurally diverse and intrinsically complex molecules, often designed to interact with biological targets. In the field of cancer treatment, 50% of the 247 drugs approved over these 38 years, are natural products or derived from natural products. A prominent example is the taxol, isolated from the Pacific yew. Taxol targets tubulin, thereby hindering cell division. Although taxol has saved and prolonged many lives, this chemotherapeutic agent not only targets cancer cells, but also fast-dividing healthy cells, which is why patients suffer heavily from side effects. Therefore, the aim of current cancer treatments (i.e. targeted therapies) is specific targeting of cancer cells to minimize side effects and improve the efficacy of the therapy. This can be done by taking in account the genetics of the individual patient and thus targeting specific molecular targets of the cancer.
Melanoma is a highly aggressive cancer, accounting for most skin cancer deaths. Moreover, it is particularly heterogeneous and harbors the highest mutation rate of all cancers. The ERK1/2 and AKT/PI3K pathways are frequently harboring activating mutations and are therefore promising targets for targeted therapies. Mutated BRAF is found in over 50% of metastatic cutaneous melanomas and is driving aberrant ERK1/2 pathway signaling. First in line treatment of metastatic BRAF mutated melanomas are targeted therapies with BRAF and MEK inhibitors. Even though these drugs revolutionized the treatment outcome of melanoma patients, they are only delaying drug resistance. As a consequence, there is a need of novel compounds targeting different nodes of pathological signaling to break cancer drug resistance. The aim of this work was to find novel small molecules targeting aberrant proliferative ERK1/2 and PI3K/AKT signaling in melanoma cancer cells, with focus on natural products. This was done in two approaches: 1) by performing a medium-throughput High-Content Screen (HCS) of plant crude extracts, followed by isolation and elucidation of active compounds from promising extracts and 2) by performing a high-throughput HCS of pure compounds from natural and synthetic origin. The bioassay used in both approaches is based on a so-called Kinase Translocation Reporter (KTR), which reports on the activity of the downstream kinases ERK and AKT in living cells.
After setting up and optimizing the medium-throughput HCS at the University of Basel, an in-house library containing 2’576 crude plant extracts was screened on two melanoma cell lines. This resulted in 140 hits, which were prioritized based on potency and taxonomic diversity of the corresponding plants. The resulting set of 44 extracts was submitted to HPLC-based activity profiling to track the activity in these complex samples. This allowed the dereplication of well-known compounds prior to scale-up isolation of active compounds. This thesis focusses on the further investigation of the active extracts of Arnica montana L. roots (Asteraceae) and the aerial parts of Artemisia argyi H.Lév. & Vaniot (Asteraceae). Scale-up isolation from the petroleum ether and ethyl acetate extracts of A. montana led to the identification of seven known thymol derivatives with micromolar activity on both pathways. For the most active compound, IC50 values of 4.7 ± 0.6 and 11.8 ± 0.5 μM on ERK1/2 and AKT were determined. The establishment of the screening pipeline and the results of the isolation from A. montana have been published in the Journal of Natural Products.
The investigation of the A. argyi ethyl acetate extract led to the isolation of 21 compounds, including one novel dimerosesquiterpene and four new sesquiterpene lactones. The isolated compounds consist of eleven guaianolides, two eudesmanolides, two dimerosesquiterpenes, one true disesquiterpene, one fatty acid derivative and four flavonoids. Additionally, we observed the methanolysis of one of the eudesmanolides, which resulted in the isolation of a new eudesmanolide derivative. A total of nine of the isolated sesquiterpene lactones exhibited micromolar activity (< 20 μM) on the PI3K/AKT pathway while being toxic at higher concentrations. The most active sesquiterpene lactone displayed an IC50 value of 8.9 ± 0.9 μM. Among the 13 known sesquiterpene lactones seven still lacked the description of their absolute configuration, which was complemented by this work using ECD and X-ray crystallography. These results are summarized in two submitted manuscripts: One focusing on the isolation and activity of the sesquiterpene lactones and the second introducing the combination of HPLC-based activity profiling with UHPLC-HRMS/MS metabolite annotation molecular networking (not in the scope of this thesis), including the activity of the isolated flavonoids.
The established pipeline of the HCS was adapted for high-throughput screening at the FMP Berlin and was applied to screen 25’696 compounds from different libraries on four melanoma cell lines. These libraries were comprised of pure natural products and synthetic compounds, including a set of FDA-approved drugs and pharmacologically active compounds. Screening compounds with natural and synthetic origins allowed the identification of novel chemical entities, inhibiting the ERK1/2 and PI3K/AKT pathways (drug discovery). On the other hand, screening FDA-approved and pharmacologically active compounds could provide additional insight into the complex biology of cancer cell lines with different mutational landscapes, as well as potentially lead to repurposing of drugs that were previously not known to affect ERK1/2 and PI3K/AKT signaling. After successful assay transfer and optimization, the assay pipeline was validated by a pilot screen. The primary screen then led to a total of 453 hits, which were tested in a subsequent counter screen. A total of 97 hits from the counter screen were used for IC50 curve validation, leading to a final hit list of 28 compounds (with the purpose of drug discovery). These final hits consist of 9 natural products and 19 synthetic compounds. With exception of one compound, which was active in the nanomolar range, the compounds displayed activity in the lower micromolar range. The investigation of the FDA-approved drugs and pharmacologically active compounds is still on-going. For reasons of confidentiality, the structures of the final hits were not disclosed.
The results of this thesis demonstrate the robustness of the established screening platform by successfully applying it in a medium-throughput and high-throughput HCS. The two screens identified promising compounds of both natural and synthetic origin, thus showcasing its applicability throughout the chemical space.