A Novel Probe for Spliceosomal Proteins that Induces Autophagy and Death of Melanoma Cells Reveals New Targets for Melanoma Drug Discovery

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155–14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599–608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155–14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155–14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 2155–14. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155–14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155–14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.