The Crystal Structure of BRAF in Complex with an Organoruthenium Inhibitor Reveals a Mechanism for Inhibition of an Active Form of BRAF Kinase

Abstract

Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in ∼70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF V600E , have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF V600E oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF V600E oncogene selective BRAF inhibitors for therapeutic application.RAF 1 kinases were originally identified as cellular homologues of v-raf oncogenes acquired by retroviruses and contain three members: CRAF (RAF-1 or c-RAF-1), BRAF, and ARAF (1-3). RAF family kinases are central players in the highly conserved mitogen-activated protein kinase (MAPK) signaling pathway (RAS-RAF-MEK-ERK) which relays signals from the extracellular space through receptor tyrosine kinases (RTKs) to the nucleus to promote the expression of genes involved in cell proliferation and survival. RAF kinases function by specifically phosphorylating MEK1/2 within the kinase activation loop leading to the subsequent activation of MEK1/2, which in turn activates ERK1/2. Activated ERK1/2 translocates into the nucleus and activates transcription factors to promote cellular outcomes, including survival, growth, proliferation, and differentiation (4). RAF family kinases are † This work was supported by Grant CA 114046 from the National Institutes of Health.* To whom correspondence should be addressed. Telephone: (215) 898-5006. Fax: (215) 898-0381. marmor@wistar.org. ‖ Current address: Department of Chemistry, Philipps-Universität Marburg, Marburg, Germany 1 Abbreviations: CI, chemical ionization; c-KIT, proto-oncogene receptor tyrosine kinase; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; ERK, extracellular signal-regulated kinase; ES, electrospray; FDA, Food and Drug Administration; GSK3β, glycogen synthase kinase 3 β isoform; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; MEK, dual-specificity mitogenactivated protein kinase kinase; PDB, Protein Data Bank; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; RAF, RAF proto-oncogene serine/threonine-protein kinase (sub...

Inhibitory activities and possible anticancer targets of Ru(II)-based complexes using computational docking method

“…While DNA-Gyrase, TrXR, and HP-NCP are either predicted as an average or one of the best by each of the two methods. These are in good agreement with the experimental findings as CatB [14], HP-NCP [23] and Kinase [22] have been suggested as possible targets of Ru(II)-based complexes. Also, the prediction of TrXR as less preferred target compared to CatB by both Glide and Autodock docking agree well with experimental report especially for rapta complexes [14].…”

“…The remaining two of this member are: CRAF (RAF-1 or c-RAF-1) and ARAF. RAF kinases function by the activation of ERK1/2 as a subsequent effect of their specific phosphorylation of MEK1/2 within the kinase activation loop which leads to its activation [22]. The activated ERK1/2 translocates into the nucleus and activates transcription factors to promote cellular outcomes, including survival, growth, proliferation, and differentiation.…”

“…The activated ERK1/2 translocates into the nucleus and activates transcription factors to promote cellular outcomes, including survival, growth, proliferation, and differentiation. BRAF KINASE represents an excellent target for anticancer drugs development because it differs significantly from other two members (CRAF and ARAF) as a major activator of MEK1/2 and requires fewer regulatory events for activation [22]. It has been found out that mutations notably V600E which is reported to account for 90% of cancer-associated BRAF mutations render the BRAF kinase constitutively active [22].…”

“…BRAF KINASE represents an excellent target for anticancer drugs development because it differs significantly from other two members (CRAF and ARAF) as a major activator of MEK1/2 and requires fewer regulatory events for activation [22]. It has been found out that mutations notably V600E which is reported to account for 90% of cancer-associated BRAF mutations render the BRAF kinase constitutively active [22]. NCP has been pointed out as a basic repeating element of histone-packaged DNA comprising chromatin.…”

The phosphorylation specificity of B-RAFWT, B-RAFD594V, B-RAFV600E and B-RAFK601E kinases: An in silico study

“…The crystal structures of the inactive B-RAF WT and B-RAF V600E kinase binding domains in a complex with the inhibitor Sorafenib (BAY-439006) have been published [5] and recently the structures of the active B-RAF form were solved as well [12][13][14], adding to the understanding of these type of kinases. However, a significant portion of the activation segment, which plays an essential role in phosphorylation and kinase activation, was not solved in any of the available structures.…”