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.

BRAF V600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting ''active'' protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf V600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf V600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf V600E -bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf V600E -positive cells. In B-Raf V600E -dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf V600E -driven tumors.cancer ͉ cell signaling ͉ melanoma ͉ phosphorylation ͉ protein kinases O ncogenic mutations in the BRAF gene (1) correlate with increased severity and decreased response to chemotherapy in a wide variety of human tumors (2-4). Hence, direct therapeutic inhibition of oncogenic B-Raf kinase activity affords an avenue to treat these tumors. The therapeutic approach of targeting oncogenic kinase activity has proved very valuable in oncology (5, 6). Recently, we have described the technique termed scaffold-based drug discovery, a strategy for identifying small molecule inhibitors of cyclic nucleotide phosphodiesterases (7). Here, we describe an expansion of this strategy to discover a scaffold targeting protein kinases, and we report the elaboration of this scaffold into the potent and selective B-Raf V600E inhibitor PLX4720. Because a majority of all melanomas harbor an activating missense mutation (V600E) in the B-Raf oncogene (1), targeted inhibition of the V600E gene product is a particularly rational therapeutic goal in this otherwise therapy-resistant tumor type. Previous generations of B-Raf inhibitors possess Raf inhibitory activity at low nanomolar concentrations (8-13); however, the relative therapeutic efficacy of such inhibitors has been hampered by the lack of bioavailability or by the number of nonspecific targets that are also affected (14, 15). The development of highly specific and effectual inhibitors of the BRAF V600E gene product would provide insight into the true therapeutic rele...

Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

PLX3397 was well tolerated and readily crossed the blood-tumor barrier but showed no efficacy. Additional studies are ongoing, testing combination strategies and potential biomarkers to identify patients with greater likelihood of response.

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.