plx4032 was synthesized using the general procedures previously described.6 expression and purification of b - raf , structure determination , protein kinase activity measurements , and xenograft studies were carried out as previously described.6 clinical methods have also been recently described.5 melanoma patients were selected for study using previously described taqman methodology.8 semi - quantitative immunohistochemistry for perk and ki67 was performed on 5 m - thick formalin - fixed paraffin - embedded tumor biopsies following h&e staining to determine pathologic diagnosis and tissue morphology and integrity .
the degree of phospho - erk staining in the nucleus and cytoplasm was interpreted semiquantitatively by assessing the intensity and extent of staining on the slides . for ki67 staining ,
 b - raf is the most frequently mutated protein kinase in human cancers.1 the finding that oncogenic mutations in braf are common in melanoma2 followed by the demonstration that these tumors are dependent on the raf / mek / erk pathway3 offered hope that inhibition of b - raf kinase activity could benefit melanoma patients . herein , we describe the structure - guided discovery of plx4032 ( rg7204 ) , a potent inhibitor of oncogenic b - raf kinase activity . preclinical experiments demonstrated that plx4032 selectively blocked the raf / mek / erk pathway in braf mutant cells and caused regression of braf mutant xenografts.4 toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity , enabling phase 1 clinical trials using a crystalline formulation of plx4032.5 in a subset of melanoma patients , pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment . 
 this analysis revealed substantial inhibition of erk phosphorylation , yet clinical evaluation did not show tumor regressions . at higher drug exposures afforded by a new amorphous drug 
 formulation,4,5 greater than 80% inhibition of erk phosphorylation in the tumors of patients correlated with clinical response . 
 indeed , the phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 these data demonstrate that braf - mutant melanomas are highly dependent on b - raf kinase activity . 
METHODS SUMMARY
Supplementary Material