Melanoma, a highly aggressive form of skin cancer, is primarily driven by two key oncogenic signaling pathways: MAPK/ERK and PI3K/AKT. In a discovery program aiming to identify natural products that inhibit one or both pathways, an in-house library of 2′576 plant extracts was screened using a high-content screening assay with melanoma cells expressing ERK/AKT activity biosensors to quantify inhibition at the single-cell level. The ethyl acetate extract from the leaves of Mammea americana was found to inhibit both pathways in the patient-derived cell line MM121224 and was selected for HPLC-based activity profiling. Scale-up isolation of the compounds eluting in the active window of the chromatogram afforded ten previously undescribed betulinic acid analogues (1–10), along with nine known coumarins (11–19). The isolated compounds were individually evaluated for their inhibitory activity on ERK and AKT in MM121224 cells. Interestingly, none of the betulinic acid derivatives 1–10 showed activity in the assay. In contrast, the isolated coumarins were shown to inhibit both pathways, with the most potent theraphin B (11) exhibiting an IC50 value of 37.0 ± 0.4 μM against the AKT pathway, while also demonstrating weaker activity against the ERK pathway.. Read more