Chemical Probe-Based Approach Clarifies Binding Of Receptor Tyrosine Kinases (Rtks) To Lenvatinib In Preclinical Models

Background: Lenvatinib mesilate (lenvatinib) is the selective inhibitor of VEGFR1-3, and other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib inhibits angiogenesis through the inhibition of VEGFR2 and FGFR1 and tumor proliferation through the inhibition of FGFR1 and RET in preclinical thyroid cancer models. Chemical probe-based binding protein isolation is a powerful tool to demonstrate the direct compound-protein interactions even in complicated biological materials such as intact cells and their robust protein extracts. The purpose of this study is to assess the interaction of lenvatinib with RTKs by using lenvatinib derivative probe as an analysis of mode of action in preclinical models. Methods: Lenvatinib chemical probe was designed based on a result of co-crystal structure of lenvatinib and VEGFR2. A linker moiety was introduced onto free space of quinoline core (position-7) and its inhibitory activities against RTKs were confirmed by cell-free enzyme assay system. Lenvatinib chemical probe was immobilized onto the monolithic affinity matrices and the binding proteins were isolated from the extracts of the human umbilical vein endothelial cells (HUVEC), the human differentiated thyroid cancer cell lines, RO82-W-1 and TPC-1. SDS-PAGE and western blotting (WB) with drug competition assay were performed to assess the binding RTKs of lenvatinib. Results: The chemical probe showed acceptable inhibitory activities against RTKs for affinity isolation. In the isolated protein sample from HUVEC, VEGFR2 and FGFR1 were detected by WB and each protein band was clearly reduced by lenvatinib competition in a dose-dependent manner. It indicates lenvatinib specifically interacts with VEGFR2 and FGFR1 even in such complicated protein mixture. In the sample from RO82-W-1, WB analysis showed that lenvatinib chemical probe efficiently isolated PDGFRα and FGFR1. Those protein bands were dramatically decreased by drug competition, similar to the experiment with HUVEC. The chemical probe-based approach was subjected to the protein extract from TPC-1, which expresses aberrant RET fusion kinase derived from CCDC6-RET gene rearrangement. WB analysis showed that CCDC6-RET was detected as binding protein of lenvatinib probe. Conclusions: The chemical probe-based approach proved the interaction of lenvatinib with VEGFR2, FGFR1, PDGFRα and RET fusion kinase in the complicated biological materials from HUVEC and thyroid cancer cell lines. These results supported that lenvatinib is likely to possess a unique anti-tumor activity, in addition to the anti-angiogenic activity, through the interaction with these RTKs. Citation Format: Takayuki Kimura, Noboru Yamamoto, Hiroshi Kamiyama, Megumi Ikemori Kawada, Akihiko Yamamoto, Yoshihiko Kotake, Yasutaka Takase, Yasuhiro Funahashi, Yoshiya Oda. Chemical probe-based approach clarifies binding of receptor tyrosine kinases (RTKs) to lenvatinib in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2015-1377