Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity Sam Gil ette Jun Kor kolas s',Katrin Spre ng, M.SmalleyDaniel Fong Contributed by Joseph Schle inger,Decembe r 15,2007 (sent for re r15.2007) is the most freg ent o tein kin malignant melanoma.Here ethe vali in kinas rticula f the To identif with an ICso cted lib ntially in ccncdataoncntntiold kina the K pho three kina The e-Raf.melanom ograft model In p for 16 f th cribe d her aled the ing of dole gh lies in animal mod to he po of the binding ) 200 cancer cell signaling melanoma phosphorylation I protein kinases hi the -am ! the BRAF tent bin )Overlap of this stn rity and de his caffold c ented a general fra ue to ion and sev 5.6 :J.T.and J.T. k.J.T.J.T.L.5.-H G of this strategy to d wer a C.S in the B-Ra e p part t of in nd .S.are Plexxik this oth itad in the ar k of bi the (1 nt o highly sp org/cgi/content/full 2008 by The National Academy of Sciences of the USA /cgi/doi/10.1073/pnas711741105 PNAS February 26.2008 1vol.1051n0.813041-304 Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity James Tsai*, John T. Lee†, Weiru Wang*, Jiazhong Zhang*, Hanna Cho*, Shumeye Mamo*, Ryan Bremer*, Sam Gillette*, Jun Kong†, Nikolas K. Haass†, Katrin Sproesser†, Ling Li†, Keiran S. M. Smalley†, Daniel Fong*, Yong-Liang Zhu*, Adhirai Marimuthu*, Hoa Nguyen*, Billy Lam*, Jennifer Liu*, Ivana Cheung*, Julie Rice*, Yoshihisa Suzuki*, Catherine Luu*, Calvin Settachatgul*, Rafe Shellooe*, John Cantwell*, Sung-Hou Kim‡, Joseph Schlessinger§¶, Kam Y. J. Zhang*, Brian L. West*, Ben Powell*, Gaston Habets*, Chao Zhang*, Prabha N. Ibrahim*, Peter Hirth*, Dean R. Artis*, Meenhard Herlyn†¶, and Gideon Bollag*¶ *Plexxikon, Inc., 91 Bolivar Drive, Berkeley, CA 94710; †Department of Molecular and Cellular Oncogenesis, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104; ‡220 Calvin Laboratory, University of California, Berkeley, CA 94720; and §Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520 Contributed by Joseph Schlessinger, December 15, 2007 (sent for review October 15, 2007) BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting ‘‘active’’ protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhib￾its the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors. cancer
cell signaling
melanoma
phosphorylation
protein kinases Oncogenic mutations in the BRAF gene (1) correlate with increased severity and decreased response to chemotherapy in a wide variety of human tumors (2–4). Hence, direct therapeutic inhibition of oncogenic B-Raf kinase activity affords an avenue to treat these tumors. The therapeutic approach of targeting onco￾genic kinase activity has proved very valuable in oncology (5, 6). Recently, we have described the technique termed scaffold-based drug discovery, a strategy for identifying small molecule inhibitors of cyclic nucleotide phosphodiesterases (7). Here, we describe an expansion of this strategy to discover a scaffold targeting protein kinases, and we report the elaboration of this scaffold into the potent and selective B-RafV600E inhibitor PLX4720. Because a majority of all melanomas harbor an activating mis￾sense mutation (V600E) in the B-Raf oncogene (1), targeted inhibition of the V600E gene product is a particularly rational therapeutic goal in this otherwise therapy-resistant tumor type. Previous generations of B-Raf inhibitors possess Raf inhibitory activity at low nanomolar concentrations (8–13); however, the relative therapeutic efficacy of such inhibitors has been hampered by the lack of bioavailability or by the number of nonspecific targets that are also affected (14, 15). The development of highly specific and effectual inhibitors of the BRAFV600E gene product would provide insight into the true therapeutic relevance of this target in malignant melanoma. Here, we demonstrate the preclinical vali￾dation of such a compound, PLX4720; this B-RafV600E-selective inhibitor displays striking antimelanoma activity in both cell- and animal-based model systems. Results and Discussion Scaffold- and Structure-Based Discovery of the Inhibitors. To identify protein kinase scaffolds, a selected library of 20,000 compounds ranging in molecular mass between 150 and 350 daltons was screened at a concentration of 200
M through multiple divergent but structurally characterized kinases, and the screening data were further analyzed to select compounds for cocrystallography. For instance, out of the library, 238 compounds inhibited the activity of the three kinases, Pim-1, p38, and CSK, by at least 30% at the 200
M concentration. These 238 compounds were subjected to coc￾rystallographic analysis in at least one of these three kinases. From this experiment,
100 structures showing bound compound were solved. In particular, Pim-1 provided a robust system for cocrys￾tallizing small low affinity compounds (16), and one of these costructures revealed the binding of 7-azaindole to the ATP￾binding site. However, although the heterocycle occupied roughly the position of the adenine ring, multiple binding modes were observed (differing across the four protein monomers in the asymmetric unit), consistent with the weak affinity (IC50
200
M for Pim-1). Subsequently, a group of mono-substituted 7-azaindoles with increased affinity was synthesized, including the 3-aminophe￾nyl analog (IC50 100
M for Pim-1) that bound to Pim-1 with a single, consistent binding mode (Fig. 1A). Overlap of this structure with those of many different kinase family members suggested that this scaffold candidate represented a general framework capable of presenting two hydrogen bonding interactions with the kinase hinge region and several putative sites of substitution (the 2, 3, 4, 5, and 6 positions; Fig. 1) for the optimization of potency and selectivity. Author contributions: J.T. and J.T.L. contributed equally to this work; J.T., J.T.L., S.-H.K., J.S., K.Y.J.Z., B.L.W., G.H., C.Z., P.N.I., P.H., D.R.A., M.H., and G.B. designed research; J.T., J.T.L., W.W., J.Z., H.C., S.M., R.B., S.G., J.K., N.K.H., K.S., L.L., K.S.M.S., D.F., Y.-L.Z., A.M., H.N., B.L., J.L., I.C., J.R., Y.S., C.L., C.S., R.S., J.C., and B.P. performed research; J.T., J.T.L., W.W., J.Z., H.C., S.M., R.B., S.G., J.K., N.K.H., K.S., L.L., K.S.M.S., D.F., Y.-L.Z., A.M., H.N., B.L., J.L., I.C., J.R., Y.S., C.L., R.S., J.C., S.-H.K., J.S., K.Y.J.Z., B.L.W., G.H., C.Z., P.N.I., P.H., D.R.A., M.H., and G.B. analyzed data; and J.T., J.T.L., S.-H.K., J.S., K.Y.J.Z., B.L.W., G.H., P.N.I., D.R.A., M.H., and G.B. wrote the paper. Conflict of interest statement: S.-H.K. and J.S. are Plexxikon founders and share holders. Freely available online through the PNAS open access option. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 3C4C, 3C4D, 3C4E, and 3C4F). ¶To whom correspondence may be addressed. E-mail: joseph.schlessinger@yale.edu, herlynm@wistar.org, or gbollag@plexxikon.com. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0711741105/DC1. © 2008 by The National Academy of Sciences of the USA www.pnas.orgcgidoi10.1073pnas.0711741105 PNAS
February 26, 2008
vol. 105
no. 8
3041–3046 MEDICAL SCIENCES Downloaded at Univ of Sci & Tech of China on December 23, 2019