Abstract C255: Discovery of TAK-632: A selective kinase inhibitor of pan-RAF with potent antitumor activity against BRAF and NRAS mutant melanomas.
Authors: Okaniwa Masanori , Hirose Masaaki , Arita Takeo , Yabuki Masato , Nakamura Akito , Takagi Terufumi , Kawamoto Tomohiro , Uchiyama Noriko , Sumita Akihiko , Tsutsumi Shunichirou , Tottori Tsuneaki , Inui Yoshitaka , Sang Bi-Ching , Yano Jason , Aertgeerts Kathleen , Yoshida Sei , Ishikawa Tomoyasu 
Published: August 2014

The RAF family kinases play critical roles in cancer progression. Recently, BRAF selective inhibitors have shown significant clinical efficacy in melanoma patients bearing oncogenic BRAFV600E mutation. However, several studies reported that RAF inhibitors instinctively transactivate RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF(wt)) and activate RAS dependent MAPK signaling. Along with this mechanism, it has been reported that selective BRAF inhibitors have not shown potent anti-proliferative activity against cancer cell lines such as NRAS mutant melanoma in which RAS dependent MAPK signaling is activated (Hong Yang et al., Cancer Res., 2010, 70, 5518-5527). However, our initial investigation using fibroblast CsFb (BRAFwt) cells indicated that phosphorylation of MEK and ERK was inhibited by some DFG-out inhibitors, but not by DFG-in inhibitors. These results led to the hypothesis: continuous inhibition of pan-RAF (BRAF and CRAF) with DFG-out type inhibitors could suppress the feedback activation.

Here we report the discovery and characterization of pan-RAF inhibitor TAK-632. We designed novel 1,3-benzothiazole class derivatives using knowledge of structure-activity relationships gained from studies of our thiazolo[5,4-b]pyridine class RAF/VEGFR2 inhibitor (Masanori Okaniwa et al., J. Med. Chem., 2012, 55, 3452-3478). To enrich RAF kinase selectivity vs. VEGFR2, we utilized the cocrystal structures of our lead compound with both BRAF and VEGFR2. Eventually, we designed and selected 7-cyano derivative TAK-632 as a development candidate. Cocrystal structure analysis of BRAF bearing TAK-632 revealed that accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF inhibition and selectivity vs. VEGFR2.

Reflecting its potent pan-RAF inhibition (IC50: BRAFV600E 2.4 nM, CRAF 1.4 nM) and slow dissociation (koff) profile measured by surface plasmon resonance (SPR) spectroscopy, TAK-632 demonstrated significant cellular activity against mutated BRAF or mutated NRAS cancer cell lines. Furthermore, in both A375 (BRAFV600E) and HMVII (NRASQ61K) xenograft models in rats, TAK-632 demonstrated regressive antitumor activity by twice daily, 14-day repetitive administration without significant body weight loss.

In conclusion, these results raise the possibility of using slow off-rate pan-RAF inhibitors such as TAK-632 for the treatment of human cancers harboring either BRAFV600E or NRAS mutant.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C255.

Citation Format: Masanori Okaniwa, Masaaki Hirose, Takeo Arita, Masato Yabuki, Akito Nakamura, Terufumi Takagi, Tomohiro Kawamoto, Noriko Uchiyama, Akihiko Sumita, Shunichirou Tsutsumi, Tsuneaki Tottori, Yoshitaka Inui, Bi-Ching Sang, Jason Yano, Kathleen Aertgeerts, Sei Yoshida, Tomoyasu Ishikawa. Discovery of TAK-632: A selective kinase inhibitor of pan-RAF with potent antitumor activity against BRAF and NRAS mutant melanomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C255.