Identification of a Novel Family of BRAF Inhibitors

The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAF) accounts for over 90% of BRAF-mediated cancers. Several BRAF inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAF inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAF over BRAF and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing the mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogues that inhibit BRAF in vitro with IC50 values in the 80−200 nM range under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed. ■ INTRODUCTION RAF family kinases are central players in the highly conserved MAPK signaling pathway (RAS-RAF-MEK-ERK), which relay 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 on the kinase activation loop, leading to the subsequent activation of ERK1/ 2. The three isoforms of the RAF kinases, ARAF, BRAF, and CRAF, share a common three-domain structure containing the conserved regions CR1, CR2, and CR3. The regulation of RAF kinase activity is complex and involves a variety of different kinases and scaffolding proteins. However, compared to ARAF and CRAF, BRAF requires fewer regulation events for its activation. Due to this property, BRAF has significantly higher basal activity than the other RAF family isoforms and has also been found to be a major activator of MEK1/2. BRAF is an important oncogene that is mutated in about a half of malignant melanomas and at a lower frequency in a wide range of other human cancers, such as thyroid, colon, ovarian, lung, and breast cancers. Oncogenic mutations occur within the kinase domain, with a single valine to glutamate substitution at residues 600 (BRAF) accounting for over 90% of these mutations. Because of these findings, BRAF, and BRAF in particular, has emerged as an attractive anticancer drug target. A number of inhibitors against BRAF have been developed so far and more are at various stages of preclinical and clinical development. For example, vemurafenib (PLX4032), an azaindole compound and orally available ATP competitive BRAF inhibitor that shows selectivity for BRAF, has received FDA approval for the treatment of late stage metastatic melanoma. Encouragingly, in phase III clinical trials, PLX4032 produced 2 complete responses and 24 partial responses out of 32 patients, extending life in many cases by more than 6 months, prior to the eventual development of drug resistance through reactivation of the MAPK pathway or through the activation of alternative compensatory pathways, involving receptor tyrosine kinases (RTKs), PI3K-AKT, and other pathways. However, about half of the patients had the drug dose reduced, and nearly two-thirds had to have their treatments temporarily stopped because of side effects. In addition, approximately one-quarter of patients developed cancerous or precancerous nonmelanoma skin lesions. Given these limitations of vemurafenib, and some other drugs that are being evaluated to treat metastatic melanoma, it would be useful to have additional BRAF inhibitor drug options for some patients. Here, we report the development of an ELISA-based highthroughput assay to screen a combined diverse library of over 30,000 organic compounds for BRAF inhibition. This screen, the structure determination of BRAF bound to one of the identified inhibitors, and the follow-up structure-based medicinal chemistry efforts resulted in the identification of a family of related compounds containing a quinolol or naphthol backbone that selectively inhibit BRAF over BRAF in vitro, display IC50 values in the 80−200 nM range under saturation ATP concentrations, and inhibit MAPK signaling in melanoma cells. ■ RESULTS Identification of a Novel Family of BRAF Inhibitors. In order to screen for BRAF inhibitors in a high-throughput Received: February 11, 2012 Published: April 26, 2012 Article