RAF265

RAF265, also known as CHIR-265, is a novel and orally available small molecule inhibitor of multiple intracellular kinases, including BRAFV600E, BRAF (wild-type), c-RAF, vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), colony-stimulating factor (CSF) 1R, RET, c-KIT, SRC and STE20, with half maximal inhibitory concentration IC50 ranging from less than 20 nmo/L to more than 100 nmol/L, in which it exhibits the highest potency for BRAFV600E and VEGFR2 with half maximal effective concentration EC50 of 0.14 μM and 0.19 μM respectively. RAF265 has been found to inhibit proliferation of melanoma and colorectal cancer cell lines with active BRAF mutations and time- and dose-dependently suppress tumor regression in BRAFV600E melanoma and colorectal cancer xenograft models.

References:
[1]Huang T1, Karsy M, Zhuge J, Zhong M, Liu D. B-Raf and the inhibitors: from bench to bedside. J Hematol Oncol. 2013 Apr 25;6:30. doi: 10.1186/1756-8722-6-30.
[2]Garcia-Gomez A1, Ocio EM, Pandiella A, San Miguel JF, Garayoa M. RAF265, a dual BRAF and VEGFR2 inhibitor, prevents osteoclast formation and resorption. Therapeutic implications. Invest New Drugs. 2013 Feb;31(1):200-5. doi: 10.1007/s10637-012-9845-3. Epub 2012 Jul 7.
[3]Su Y1, Vilgelm AE, Kelley MC, Hawkins OE, Liu Y, Boyd KL, Kantrow S, Splittgerber RC, Short SP, Sobolik T, Zaja-Milatovic S, Dahlman KB, Amiri KI, Jiang A, Lu P, Shyr Y, Stuart DD, Levy S, Sosman JA, Richmond A. RAF265 inhibits the growth of advanced human melanoma tumors. Clin Cancer Res. 2012 Apr 15;18(8):2184-98. doi: 10.1158/1078-0432.CCR-11-1122. Epub 2012 Feb 20.

Discovery of RAF265: A Potent mut-B-RAF Inhibitor for the Treatment of Metastatic Melanoma.[Pubmed:26396681]

ACS Med Chem Lett. 2015 Aug 3;6(9):961-5.

Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.

Preclinical analysis of the anti-tumor and anti-metastatic effects of Raf265 on colon cancer cells and CD26(+) cancer stem cells in colorectal carcinoma.[Pubmed:25884645]

Mol Cancer. 2015 Apr 11;14:80.

BACKGROUND: In colorectal carcinoma (CRC), activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition, the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore, we hypothesized that an ATP-competitive pan-Raf inhibitor, RAF265, is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS: HT29 and HCT116 cells were treated with various concentrations of RAF265 to study the anti-proliferative and apoptotic effects of RAF265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with RAF265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS: RAF265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition, anti-migratory and invasive effect was found with RAF265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally, the anti-tumor and anti-metastatic effect of RAF265 in combination with 5FU was also demonstrated. CONCLUSIONS: This preclinical study demonstrates the anti-tumor and anti-metastatic activity of RAF265 in CRC, providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC.

The combination of RAF265, SB590885, ZSTK474 on thyroid cancer cell lines deeply impact on proliferation and MAPK and PI3K/Akt signaling pathways.[Pubmed:24821574]

Invest New Drugs. 2014 Aug;32(4):626-35.

Papillary thyroid cancer (PTC) is the most frequent thyroid cancer entity, accounting for 88 % of cases. It may metastasize and loose iodine uptake capability, preventing any radioiodine or surgical treatment. The main gene altered in PTC is BRAF, which is found altered in over 50 % of cases. Moreover MAPK and PI3K/Akt pathways are greatly implicated in PTC development. Many target therapies for PTC are currently under investigation, unfortunately without the expected results. Aim of this study was to characterized the preclinical effectiveness of novel promising drugs, RAF265, SB590885 and ZSTK474 in 3 thyroid cancer cell lines (BCPAP, K1, 8505C). RAF265 and SB590885 target differentially BRAF, while ZSTK474 acts on PI3K. IC50 demonstrated high drug activities ranging from 0.1 to 6.2 muM, depending on drugs and cell type, while combination index revealed an interesting synergistic effect of combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474) in almost all cell lines. Moreover this synergistic effect was particularly evident by Western blot, whereas dual MAPK and PI3K/Akt inhibition was detected. In addition, treating cells with SB590885 induced marked morphological changes, leading to massive vacuolization. This suggests an activation of apoptotic process, as underlined by Annexin V flow cytometry analysis. Also cell cycle was altered in treated cells, without evidence of a common pattern, but rather with a more specific effect relying on single drug or combination regimen used. Since beneficial effects of in vitro combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474), it is recommended additional investigation. These data suggest the potential use of combination regimen in in vivo experiment or afterwards in human PTC.

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells.[Pubmed:26081844]

J Cell Mol Med. 2015 Sep;19(9):2244-52.

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.