Structural Investigation of B‑Raf Paradox Breaker and Inducer Inhibitors Rohit Arora, † Michela Di Michele, ‡,§ Elisabeth Stes, ‡,§ Elien Vandermarliere, ‡,§ Lennart Martens, ‡,§ Kris Gevaert, ‡,§ Erika Van Heerde, ∥ Joannes T. M. Linders, ∥ Dirk Brehmer, ∥ Edgar Jacoby, ∥ and Pascal Bonnet* ,† † Institut de Chimie Organique et Analytique, UMR CNRS-Universite ́ d’Orle ́ ans 7311, Universite ́ d’Orle ́ ans BP 6759, Orle ́ ans 45067 Cedex 2, France ‡ Department of Medical Protein Research, VIB, Ghent B-9000, Belgium § Department of Biochemistry, Ghent University, Ghent B-9000, Belgium ∥ Janssen Research and Development, a division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, Beerse 2340, Belgium * S Supporting Information ABSTRACT: The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to hetero- dimerization with C-Raf causing a paradoxical hyperactiva- tion of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf V600E activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conforma- tional, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function. ■ INTRODUCTION B-Raf is a member of the Raf family of serine/threonine protein kinases. It plays a vital role in the Ras−Raf−MEK−ERK signal transduction pathway (also known as the mitogen-activated protein kinase or MAPK signaling cascade) and participates in cell proliferation and cell survival. 1 In the MAPK pathway, active Ras induces conformational changes in B-Raf upon binding. This promotes changes in its phosphorylation status triggering kinase activity. Phosphorylation of B-Raf activates the MEK protein, which in its turn activates the downstream ERK protein kinase that phosphorylates several substrates. 2,3 The B-Raf kinase is frequently mutated in melanomas, and the V600E missense mutation is most commonly observed. This mutation causes B-Raf to signal independently from upstream regulation. Uncontrolled amplification of downstream signaling is linked with transformation, increase of proliferation and finally tumorigenesis. 4 In order to target the B-Raf V600E mutant, ATP-competitive B-Raf inhibitors such as vemurafenib 5 (1), PLX4720 6 (2), sorafenib, 7 and dabrafenib 8 have been developed, which effectively block the MAPK signaling pathway and decrease tumor growth in cells expressing B-Raf V600E . How- ever, while these inhibitors effectively inhibit ERK signaling in tumor cells expressing B-Raf V600E , they have unexpected agonistic functions in cells expressing wild-type B-Raf. In particular, inhibitors binding to the wild-type B-Raf monomer were shown to induce homo/heterodimerization with a second Raf protomer (B-Raf/B-Raf, B-Raf/C-Raf) in the presence of GTP loaded KRAS at the membrane. As recently published, the dimerized B-Raf has lower affinity for the inhibitors compared with ATP with still high kinase activity activating the down- stream MAPK pathway. Because Ras-GTP levels are relatively much lower in B-Raf V600E cells, dimer-mediated activation of the MAPK pathway is disfavored. 9 Consequently, in cells expressing wild-type B-Raf, binding of these inhibitors paradoxically activates the wild-type B-Raf kinase domain by inducing conformational changes and dimerization and triggering C-Raf activation, leading to MEK/ERK phosphorylation and eventually causing enhanced cell growth (Figure 1). Therefore, the ATP-competitive inhibitors can either inhibit or activate the MAPK pathway, depending on wild-type or V600E B-Raf expression in tumor cell lines. 10−12 It has been reported that the combination of a MEK inhibitor, PD184352, with the B-Raf inhibitor 1 can help to overcome the paradoxical activation of the MAPK pathway. 13 Similar studies Received: October 29, 2014 Article pubs.acs.org/jmc © XXXX American Chemical Society A DOI: 10.1021/jm501667n J. Med. Chem. XXXX, XXX, XXX−XXX