BRAF, a serine/threonine kinase, regulates the mitogen-activated protein kinase (RAS/RAF/MEK/ERK) pathway, which is involved in the transduction of mitogenic signals from the cell membrane to the nucleus. BRAF is known to be mutated in 70% of malignant melanomas. The most common BRAF mutation is V600E, and it increases the kinase activity that constitutively stimulates ERK signaling, cell proliferation and survival. The mutation is an early event for the development of melanoma and it is present in 80% of primary melanomas.1, 2 Recently, it has been reported that mutant BRAF protein is a client of Hsp90, which is required for the folding and stability of mutant BRAF. Therefore, BRAF-mutated cells are more sensitive to the Hsp90 inhibitor, compared with BRAF wild-type cells.3 On the basis of the above-mentioned background, we established the screening system using BRAF wild-type and BRAF mutant cells to identify the BRAF pathway inhibitors from microbial metabolites.

First, we observed that ERK1/2 in wild-type BRAF cells was activated by the addition of serum or growth factors, and that ERK1/2 in BRAF-mutated cells is constitutively active (data not shown) by western blotting experiments. Next, we examined the effects of some known inhibitors—actinomycin D (RNA synthesis inhibitor), cycloheximide (protein synthesis inhibitor), paclitaxel (tubulin inhibitor), leptomycin B (CRM1 inhibitor), tunicamycin (N-linked glycosylation inhibitor), staurosporine (protein kinase inhibitor), trichostatin A (histone deacetylase inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), LY294002 (PI3 kinase inhibitor), U0126 (MEK inhibitor), geldanamycin (Hsp90 inhibitor), RK-682 (tyrosine phosphatase inhibitor), tautomycin (protein phosphatase 1 and 2A inhibitor) and phoslactomycin C (protein phosphatase 2A inhibitor)—on the cell-based assay.4, 5 We found that U0126 and geldanamycin preferentially decreased the cell viability of BRAF mutant WM266-4 cells (Table 1). The selectivity ratio of CHL-1/WM266-4 was more than 10-fold. The selectivity ratios of other inhibitors were smaller than 1.0. These results suggest that this screening system is suitable for the detection of BRAF-signaling inhibitors from microbial metabolites.

Table 1 Effect of test compounds on BRAF mutated and wild-type melanoma cell lines
Full size table

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