title: Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAFV600EConformation.
abstract: The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK, or splicing variants. RAF kinases signal as homo- and heterodimers, and another complication associated with small molecule BRAFV600Einhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called "transactivation" or "paradoxical activation." Here, we used BRAFV600Eand vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600Eboth in vitro and in cells but promotes an inactive dimeric BRAFV600Econformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over an active dimeric BRAFV600Econformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy.
authors: Department of Biochemistry and Biophysics and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania , 421 Curie Blvd., Philadelphia, Pennsylvania 19104, United States.|Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Michael
authors: M
authors: Grasso
authors: Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Michelle A
authors: MA
authors: Estrada
authors: Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Christian
authors: C
authors: Ventocilla
authors: Wistar Institute, Philadelphia , 3601 Spruce Street, Philadelphia, Pennsylvania 19104, United States.
authors: Minu
authors: M
authors: Samanta
authors: Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Jasna
authors: J
authors: Maksimoska
authors: Wistar Institute, Philadelphia , 3601 Spruce Street, Philadelphia, Pennsylvania 19104, United States.
authors: Jessie
authors: J
authors: Villanueva
authors: Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Jeffrey D
authors: JD
authors: Winkler
authors: Department of Biochemistry and Biophysics and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania , 421 Curie Blvd., Philadelphia, Pennsylvania 19104, United States.|Department of Chemistry, University of Pennsylvania , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
authors: Ronen
authors: R
authors: Marmorstein
mesh_terms: D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D008545:Melanoma / Q000473:pathology; D015394:Molecular Structure; D011487:Protein Conformation; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012996:Solutions; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D012996:Solutions; D013449:Sulfonamides; D000077484:Vemurafenib; D048493:Proto-Oncogene Proteins B-raf