This was explained by numerous contacts to Arg509 of the monomer and to His510 of the BRAF protein. As described above, truncation studies were included in the peptide library design since optimization of molecular excess weight is a key parameter defining drug-likeness and oral bioavailability in beyond rule of 5 (bRo5) space20, 23 (For a direct comparison of the peptides in the truncation series see Supplementary Table 1). inhibitors through macrocyclic drug discovery. TABLE OF CONTENTS GRAPHIC INTRODUCTION The Ras/Raf/MEK/ERK pathway entails the transduction of extracellular growth signals to the nucleus to regulate events in cell proliferation and differentiation. This pathway is frequently affected in tumor formation through the overexpression of growth factor receptors and activating mutations in Ras and Raf kinase are common events. Considerable efforts in drug discovery have been invested and have in recent years paid some dividends. In particular Raf kinases (ARAF, BRAF and Raf-1/C are known users) are considered as attractive therapeutic targets 1, 2. Of these, BRAF is the major activating kinase for MEK/ERK and as a result is probably the most frequently mutated kinase in cancers including melanoma, hairy cell leukemia and colorectal carcinomas among other tumor types 3, 4. A breakthrough in the treatment of Lithospermoside malignant melanomas has been achieved in the approval of vemurafenib, a BRAF inhibitor in the beginning producing dramatic responses in treated patients and which targets a constitutively active BRAF mutant (V600E). These drugs target the transmission transduction pathways stimulated by binding of growth factors to their receptors that then result in activation of Ras proteins. Oncogenic Ras signaling occurs in about 30% of all human cancers and triggers homo-or hetero-dimerization of Raf-kinases that is critical for several aspects of transmission propagation through downstream MEK and ERK kinases5, 6. Despite intense efforts, pharmacologic inhibition of RAS proteins themselves and inhibition of their downstream effector kinases has so far been unsuccessful in treating RAS-driven tumors. Despite the dramatic initial response rates of vemurafenib in mutant melanoma patients, drug resistance and secondary neoplasms emerge in treated patients thereby dampening Lithospermoside the initial enthusiasm for this approach 7, 8. Further investigation into the mechanisms driving these clinical complications has provided considerable TEF2 insights and decided that a major Lithospermoside cause results from paradoxical MEK/ERK signaling by the same mechanisms precluding the use of these drugs in Ras-driven tumors. These studies have exhibited that while vemurafenib inhibits BRAFV600E very potently, in the context of WT BRAF (in both homodimers and BRAF/C-Raf heterodimers) and activating Ras mutations, prospects to kinase activation of the other partner in the dimer thereby stimulating the downstream pathway rather than inhibiting it 9C11. Allosteric transactivation of a catalytically qualified RAF protomer by a drug-bound BRAF molecule requires an intact dimer interface12. This resistance pathway therefore requires further efforts to complement inhibition of the mutant V600E kinase with other ways of inhibiting downstream signaling. Despite clinical success, the emergence of resistants tumors necessitates continued investigation and drug discovery efforts round the Ras/Raf/MEK/ERK pathway. Combination of MEK inhibitors with approved BRAF drugs has been shown to be an effective strategy and has resulted in the recent approval of trametinib to treat BRAF mutated melanomas13. While a significant improvement, MEK inhibitors have some toxicity issues and thus further improvements are required. ATP-competitive Raf inhibitors that induce paradoxical ERK activation must not be used to treat mutant tumors12, 14. A recent preclinical study has shown that targeting the complete Raf node phenocopies the growth inhibiting effects of removing the oncogenic driver, mutant Ras15. A new class of inhibitors that take the dynamic interplay of Raf-isoforms by dimerization and feedback loops into consideration would therefore be beneficial and this requires a detailed understanding of BRAF and its homo and heterodimerization and effects on downstream signaling. In this study, based on elucidation of the residues in the DIF Lithospermoside important for dimer formation, the requirement for transactivation for an intact dimer interface12, 16 and published structural information on the BRAF dimer17, peptides were designed that successfully bind to BRAF and furthermore act to abrogate downstream signaling of ERK. These can be classified as type IV kinase inhibitors i.e. those that bind and inhibit allosterically at sites distant from the catalytic cleft18. The structure activity relationship of DIF peptides has been defined through computational analysis, alanine scanning and testing of these in an intrinsic tryptophan fluorescence (ITF) assay measuring direct binding to BRAF. Based on activities of the linear peptides and the observed loop structure at the dimer interface, highly potent cyclic peptides that mimic and stabilize the bioactive conformation have been generated. Macrocyclic drug discovery has in recent years become an area of interest especially in targeting protein-protein interactions 19C22. Macrocycles (MCs) typically go beyond the rule of 5 for orally available drugs especially with regard to allowing high MW compounds20, 23. This enables more extensive.