GST-MEK-His protein substrate for the B-Raf inhibitor screen was overexpressed at 37 C in BL21 (Gold) cells. exclusive cytotoxicity against B-RafV600E harboring cancer cells. Introduction The evolutionarily conserved Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway, relaying the proliferative signals generated by cell surface receptors and cytoplasmic signaling components into the nucleus, is a critical regulator of many cellular processes involved in cell proliferation, differentiation, and survival.1C3 Aberrant activation of the MAPK pathway represents a common indicator for a wide variety of proliferative diseases and especially cancer.4, 5 Moreover, accumulating data demonstrates that high-frequency activating mutation6C8 or amplification9C11 at several levels in this pathway greatly prompt multiple tumor oncogenic processes, such as cell immortalization, angiogenesis, invasive growth, metastases and drug resistance.12C14 Therefore, the Ras/Raf/MEK/ERK signaling cascade has become one of the primary candidates for novel molecular targeted cancer therapies.15C17 The Raf serine/threonine kinase family was initially identified as a cellular homolog of the retroviral oncogene v-Raf and consists of three isoforms named Raf-1 (C-Raf), A-Raf and B-Raf.18C20 Distinct from other Raf paralogs, B-Raf requires fewer post-translational modifications for optimal activation and possesses substantially greater basal activity.21, 22 In addition, B-Raf has also been shown to be the major Raf effector in the MAPK signaling cascade. 23C26 All of these unique properties in cellular physiology provide a molecular shortcut for B-Raf activation and greatly substantiate the potential of to be a oncogene.27 The importance of B-Raf in oncogenesis is highlighted by the finding that it is mutated in approximately 7% of human cancers, with the highest incidence in malignant melanoma (60%C70%), thyroid (30%C50%), ovarian (~30%) and colorectal (5%C20%) carcinomas, and with a relatively lower frequency (1%C3%) in a wide variety of other cancers. 7, 28 Among more than 75 identified B-Raf mutations in human cancers, a Glu for Val substitution at residue 600 in the kinase domain accounts for over 90% of B-Raf MK8722 mutations, which harbors 500-fold higher intrinsic kinase activity. 29 The B-RafV600E could effectively circumvent the requirement of FAAP95 phosphorylation-dependent regulation by Ras, and result in constitutive activation of the MAPK signaling pathway, even in the absence of extracellular mitogenic stimuli, facilitating the malignant transformation.30, 31 The presence of B-RafV600E in certain cancer cohorts also correlates with the increased risk of MK8722 deterioration and poor prognosis.32, 33 Taken together, these findings strongly suggested MK8722 that B-Raf is a promising therapeutic target in melanoma and other carcinomas that depend upon B-Raf signaling. Given the significance of B-Raf in tumorigenesis and progression, the inhibitors targeting B-Raf and especially the oncogenic forms of B-Raf, have increasingly come into the limelight of the drug discovery arena. To date, a number of oncogenic B-Raf inhibitors have been reported in various developmental stages. 34C39 Some of them have entered clinical trials in recent years and exhibit encouraging clinical efficiency.40C42 Among them, Vemurafenib, which was approved by the FDA and EMA (European Medicines Agency) for the treatment of unresectable or metastatic melanoma, is characterized as a selective B-RafV600E inhibitor.43 It demonstrated complete or partial tumor regression in the majority of patients harboring the B-RafV600E mutation and with an overall increased median survival time after the treatment, which validates the effectiveness of targeting the oncogenic B-Raf in cancer therapy.40, 44 However, the emerging data also indicates that some mutant B-Raf tumors have intrinsic resistances to B-Raf inhibitors accessed clinically to date, and the acquired resistance occurred through a variety of mechanisms.45C47 Furthermore, a series of side effects have also been observed, in particular the induction of keratoacanthoma was frequently observed in Vemurafenib treated patients.44 Therefore, there is clearly an unmet therapeutic need to develop novel, potent and specific B-Raf inhibitors possessing different properties to benefits the patients with B-RafV600E-driven cancers. Structure based virtual screening (SBVS) has become a powerful tool in the medicinal chemists toolkit, and the incorporation of the characteristic information from the known active ligands into the virtual screening campaign could further improve the hit enrichment as well as the potential for scaffold hopping.48,.